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WO2019178207A1 - Agents ciblant la phosphatidylsérine et utilisations de ceux-ci pour des thérapies adoptives à base de cellules t - Google Patents

Agents ciblant la phosphatidylsérine et utilisations de ceux-ci pour des thérapies adoptives à base de cellules t Download PDF

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Publication number
WO2019178207A1
WO2019178207A1 PCT/US2019/022020 US2019022020W WO2019178207A1 WO 2019178207 A1 WO2019178207 A1 WO 2019178207A1 US 2019022020 W US2019022020 W US 2019022020W WO 2019178207 A1 WO2019178207 A1 WO 2019178207A1
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antigen
cell
certain embodiments
seq
cells
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PCT/US2019/022020
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English (en)
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Jedd D. Wolchok
Taha MERGHOUB
Daniel HIRSCHHORN
Sara SCHAD
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Memorial Sloan-Kettering Cancer Center
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Publication of WO2019178207A1 publication Critical patent/WO2019178207A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4244—Enzymes
    • A61K40/4245—Tyrosinase or tyrosinase related proteinases [TRP-1 or TRP-2]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3053—Skin, nerves, brain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/876—Skin, melanoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the presently disclosed subject matter provides methods and compositions for enhancing the immune response toward cancers and pathogens. It relates to
  • PS phosphatidyl serine
  • T cells and other immune cells may be modified to target tumor antigens through the introduction of genetic material coding for artificial or synthetic receptors for antigen, termed Chimeric Antigen Receptors (CARs), specific to selected antigens.
  • CARs Chimeric Antigen Receptors
  • Targeted T cell therapy using CARs has shown clinical success in treating hematologic malignancies.
  • Innate immune cells can impair the function and activation of anti-tumor T cells. Accordingly, there is a need for novel therapeutic strategies capable of inducing potent tumor eradication with minimal toxicity and immunogenicity.
  • the presently disclosed subject matter provides various methods, including methods of reducing tumor burden in a subject, methods of treating and/or preventing a neoplasm, and methods of lengthening survival of a subject having a neoplasm.
  • each of the various methods comprises administering to the subject: (a) an effective amount of immunoresponsive cells or a pharmaceutical composition comprising thereof, wherein the immunoresponsive cells comprises an antigen recognizing receptor that binds to an antigen, and (b) an effective amount of a phosphatidyl serine (PS) blocker.
  • an effective amount of immunoresponsive cells or a pharmaceutical composition comprising thereof wherein the immunoresponsive cells comprises an antigen recognizing receptor that binds to an antigen
  • PS phosphatidyl serine
  • the method reduces the number of tumor cells. In certain embodiments, the method reduces tumor size. In certain embodiments, the method eradicates the tumor in the subject.
  • the antigen recognizing receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In certain embodiments, the antigen recognizing receptor is a CAR.
  • the antigen recognizing receptor is exogenous or endogenous. In certain embodiments, the antigen recognizing receptor is recombinantly expressed. In certain embodiments, the antigen recognizing receptor is expressed from a vector.
  • the immunoresponsive cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a Natural Killer T (NKT) cell, a human embryonic stem cell, and a pluripotent stem cell from which lymphoid cells may be differentiated. In certain embodiments, the immunoresponsive cell is a T cell. The immunoresponsive cell can be autologous or non-autologous.
  • the antigen is a tumor antigen or a pathogen antigen. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the tumor antigen is selected from the group consisting of melanoma differentiation antigens (MDAs), CD 19, MUC16, MUC1, CA1X, CEA, CD8, CD7, CD 10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, Fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-l3R-a2, K-light chain, KDR, LeY, Ll cell adhesion molecule, MAGE-A1, Mesothelin, ERBB2, MAGE A3, p53, MART l,GP 100, Proteinase3 (PR), CD 19, M
  • the MDA is Trpl. In certain embodiments, the tumor antigen is CD19.
  • the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.
  • the PS blocker is capable of inhibiting the interaction between PS and another molecule, the biological activity of PS and/or the transportation of PS.
  • the PS blocker is an antibody targeting PS (referred to as “PS-targeting antibody”).
  • the neoplasm is selected from the group consisting of melanoma, glioblastoma multiforme, anaplastic astrocytoma, ependymoma, meningioma, oligodendroglioma, blood cancer, B cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, and non-Hodgkin’s lymphoma.
  • the neoplasm is melanoma.
  • the neoplasm is lymphoblastic leukemia (ALL).
  • Figures 1 depicts the transduction rate of the TA99 CAR (i.e., Trpl targeting CAR, upper panels) and the cytolytic effect of TA99 CARs towards B16 melanoma tumor cells in vitro.
  • TA99 CAR i.e., Trpl targeting CAR, upper panels
  • Figure 2 depicts that TA99 CAR T cell treatment delayed tumor growth of B16 melanoma in vivo. Mice were preconditioned with cyclophosphamide.
  • Figure 3 depicts that the combination of TA99 CAR and PS-targeting antibody (mchlNl 1) delayed tumor growth and increased overall survival compared to TA99 CAR alone.
  • Figure 4 depicts that PS-targeting antibody directly enhanced cytolytic effects of CAR T cells.
  • Figure 5 depicts that mchlNl 1 enhanced survival of mice treated with CDl928z CAR T cells in a mouse model of EL4-CD19 thymoma.
  • the presently disclosed subject matter provides cells, including genetically modified immunoresponsive cells (e.g., T cells (e.g., CTL cells) or K cells) comprising a combination of an antigen recognizing receptor (e.g., a TCR or a CAR).
  • T cells e.g., CTL cells
  • K cells comprising a combination of an antigen recognizing receptor (e.g., a TCR or a CAR).
  • an antigen recognizing receptor e.g., a TCR or a CAR
  • a phosphatidyl serine (PS) blocker with genetically modified immunoresponsive cells (e.g., T cells (e.g., CTL cells) or K cells) comprising an antigen recognizing receptor (e.g., a TCR or a CAR) for inducing and/or enhancing an immune response to a target antigen, methods of reducing tumor burden in a subject, methods of lengthening survival of a subject having a neoplasm, and/or methods of treating and/or preventing a neoplasm or other diseases/disorders.
  • PS phosphatidyl serine
  • a phosphatidyl serine (PS) blocker enhances the anti-tumor effect of an immunoresponsive cell comprising an antigen recognizing receptor (e.g., a TCR or a CAR) (e.g., a CAR- expressing T cell). It was observed that the combination of a phosphatidylserine (PS) blocker and a CAR (e.g., a CAR targeting Trpl or a CAR targeting CD 19) led to increased anti-tumor effects, e.g., the anti-tumor activity for treating melanoma.
  • an antigen recognizing receptor e.g., a TCR or a CAR
  • a CAR e.g., a CAR targeting Trpl or a CAR targeting CD 19
  • the term“about” or“approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.
  • “about” can mean within 3 or more than 3 standard deviations, per the practice in the art
  • “about” can mean a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value.
  • the term can mean within an order of magnitude, e.g., within 5-fold or within 2-fold, of a value.
  • activates an immunoresponsive cell is meant induction of signal transduction or changes in protein expression in the cell resulting in initiation of an immune response. For example, when CD3 Chains cluster in response to ligand binding and
  • immunoreceptor tyrosine-based inhibition motifs a signal transduction cascade is produced.
  • ITAMs immunoreceptor tyrosine-based inhibition motifs
  • a formation of an immunological synapse occurs that includes clustering of many molecules near the bound receptor (e.g. CD4 or CD8, O ⁇ 3g/d/e/z, etc.).
  • This clustering of membrane bound signaling molecules allows for ITAM motifs contained within the CD3 chains to become phosphorylated.
  • This phosphorylation in turn initiates a T cell activation pathway ultimately activating transcription factors, such as NF-kB and AP-l.
  • transcription factors induce global gene expression of the T cell to increase IL-2 production for proliferation and expression of master regulator T cell proteins in order to initiate a T cell mediated immune response.
  • stimulates an immunoresponsive cell is meant a signal that results in a robust and sustained immune response. In various embodiments, this occurs after immune cell (e.g., T-cell) activation or concomitantly mediated through receptors including, but not limited to, CD28, CD137 (4-1BB), 0X40, CD40 and ICOS.
  • immune cell e.g., T-cell
  • receptors including, but not limited to, CD28, CD137 (4-1BB), 0X40, CD40 and ICOS.
  • Receiving multiple stimulatory signals can be important to mount a robust and long-term T cell mediated immune response. T cells can quickly become inhibited and unresponsive to antigen. While the effects of these co-stimulatory signals may vary, they generally result in increased gene expression in order to generate long lived, proliferative, and anti- apoptotic T cells that robustly respond to antigen for complete and sustained eradication.
  • the term“antibody” means not only intact antibody molecules, but also fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. Accordingly, as used herein, the term“antibody” means not only intact immunoglobulin molecules but also the well-known active fragments F(ab') 2 , and Fab. F(ab') 2 , and Fab fragments that lack the Fe fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et ak, J. Nucl. Med. 24:316-325 (1983).
  • antibodies include whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab’, single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies.
  • an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant (C H ) region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant C L region.
  • the light chain constant region is comprised of one domain, C L .
  • the V H and V L regions can be further sub-divided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g ., effector cells) and the first component (Cl q) of the classical complement system.
  • CDRs are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of
  • antibodies comprise three heavy chain and three light chain CDRs or CDR regions in the variable region.
  • CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope.
  • the CDRs regions are delineated using the Kabat system (Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, ET.S.
  • variable fragment As used herein, the term“single-chain variable fragment” or“scFv” is a fusion protein of the variable regions of the heavy (V H ) and light chains (V L ) of an
  • V H immunoglobulin covalently linked to form a V H : V L heterodimer.
  • the V H and V L are either joined directly or joined by a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids), which connects the N-terminus of the V H with the C-terminus of the V L , or the C- terminus of the V H with the N-terminus of the V L .
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility.
  • the linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain.
  • the linker comprises amino acids having the sequence set forth in SEQ ID NO: 11 as provided below.
  • nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 11 is set forth in SEQ ID NO:26, which is provided below:
  • polypeptide antibodies can be expressed from a nucleic acid including V H - and V L -encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879- 5883, 1988). See, also , U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754.
  • Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-5l; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol2009 l83(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3): 173- 84; Moosmayer et al., Ther Immunol 1995 2(10:31-40).
  • Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003
  • affinity is meant a measure of binding strength
  • affinity can depend on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and/or on the distribution of charged and hydrophobic groups.
  • affinity also includes“avidity”, which refers to the strength of the antigen-antibody bond after formation of reversible complexes.
  • Methods for calculating the affinity of an antibody for an antigen are known in the art, including, but not limited to, various antigen-binding experiments, e.g., functional assays (e.g., flow cytometry assay).
  • chimeric antigen receptor or“CAR” as used herein refers to a molecule comprising an extracellular antigen-binding domain that is fused to an intracellular signaling domain that is capable of activating or stimulating an
  • the extracellular antigen-binding domain of a CAR comprises an scFv.
  • the scFv can be derived from fusing the variable heavy and light regions of an antibody.
  • the scFv may be derived from Fab’s (instead of from an antibody, e.g., obtained from Fab libraries).
  • the scFv is fused to the
  • the CAR is selected to have high binding affinity or avidity for the antigen.
  • nucleic acid molecules include any nucleic acid molecule that encodes a polypeptide of interest or a fragment thereof. Such nucleic acid molecules need not be 100% homologous or identical with an endogenous nucleic acid sequence, but may exhibit substantial identity. Polynucleotides having“substantial identity” or“substantial homology” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant a pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, e.g., less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, e.g., at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, at least about 37° C, or at least about 42° C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In certain embodiments, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In certain embodiments, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg/ml denatured salmon sperm DNA (ssDNA).
  • SDS sodium dodecyl sulfate
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. ETseful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps can be less than about 30 mM NaCl and 3 mM trisodium citrate, e.g., less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, of at least about 42° C, or of at least about 68° C.
  • wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In certain embodiments, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In certain embodiments, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Rogness (Proc. Natl. Acad.
  • substantially identical or“substantially homologous” is meant a polypeptide or nucleic acid molecule exhibiting at least about 50% homologous or identical to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences described herein
  • nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
  • such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous or identical to the sequence of the amino acid or nucleic acid used for comparison.
  • Sequence identity can be measured by using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-lOO indicating a closely related sequence.
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Bio
  • the term“cross-compete” or“compete” refers to the situation where binding of an extracellular antigen-binding domain of a presently disclosed CAR to a given antigen or a given polypeptide, i.e., an MDA (e.g, Trpl), decreases or reduces binding of a reference antibody or an antigen-binding portion thereof, e.g. , that comprises the VH and VL CDR1, CDR2, and CDR3 sequences or VH and VL sequences disclosed in Table 1, to the same antigen, i.e., an MDA (e.g, Trpl).
  • an MDA e.g, Trpl
  • cross-compete or“compete” also refers to the situation where binding of a reference antibody or an antigen-binding portion thereof to a given antigen or a given polypeptide, i.e., an MDA (e.g, Trpl), decreases or reduces binding of an extracellular antigen-binding domain of a presently disclosed CAR to the same antigen.
  • MDA e.g, Trpl
  • the“cross-competing” or“competing” extracellular antigen-binding domain binds to the same or substantially the same epitope, an overlapping epitope, or an adjacent epitope on an MDA (e.g, Trpl) as the reference antibody or antigen-binding portion thereof.
  • analog is meant a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.
  • ligand refers to a molecule that binds to a receptor. In certain embodiments, the ligand binds to a receptor on another cell, allowing for cell-to- cell recognition and/or interaction.
  • disease is meant any condition, disease or disorder that damages or interferes with the normal function of a cell, tissue, or organ, e.g., neoplasia, and pathogen infection of cell.
  • an“effective amount” is meant an amount sufficient to have a therapeutic effect.
  • an“effective amount” is an amount sufficient to arrest, ameliorate, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion, or migration) of a neoplasia.
  • endogenous is meant a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue.
  • exogenous is meant a nucleic acid molecule or polypeptide that is not endogenously present in a cell.
  • the term“exogenous” would therefore encompass any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and over-expressed nucleic acid molecules and polypeptides.
  • exogenous nucleic acid is meant a nucleic acid not present in a native wild-type cell; for example, an exogenous nucleic acid may vary from an endogenous counterpart by sequence, by position/location, or both.
  • an exogenous nucleic acid may have the same or different sequence relative to its native endogenous counterpart; it may be introduced by genetic engineering into the cell itself or a progenitor thereof, and may optionally be linked to alternative control sequences, such as a non-native promoter or secretory sequence.
  • a“heterologous nucleic acid molecule or polypeptide” is meant a nucleic acid molecule (e.g., a cDNA, DNA or RNA molecule) or polypeptide that is not normally present in a cell or sample obtained from a cell.
  • This nucleic acid may be from another organism, or it may be, for example, an mRNA molecule that is not normally expressed in a cell or sample.
  • immunoresponsive cell is meant a cell that functions in an immune response or a progenitor, or progeny thereof.
  • modulate is meant positively or negatively alter.
  • exemplary modulations include a about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100% change.
  • By“increase” is meant to alter positively by at least about 5%.
  • An alteration may be by about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.
  • By“reduce” is meant to alter negatively by at least about 5%.
  • An alteration may be by about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even by about 100%.
  • isolated cell is meant a cell that is separated from the molecular and/or cellular components that naturally accompany the cell.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.“Isolate” denotes a degree of separation from original source or
  • nucleic acid or peptide is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • purified can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
  • antigen-binding domain refers to a domain capable of specifically binding a particular antigenic determinant or set of antigenic determinants present on a cell.
  • Linker shall mean a functional group (e.g., chemical or polypeptide) that covalently attaches two or more polypeptides or nucleic acids so that they are connected to one another.
  • a“peptide linker” refers to one or more amino acids used to couple two proteins together (e.g., to couple VH and VL domains).
  • the linker comprises a sequence set forth in
  • Neoplasm is meant a disease characterized by the pathological proliferation of a cell or tissue and its subsequent migration to or invasion of other tissues or organs. Neoplasia growth is typically uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells.
  • Neoplasia can affect a variety of cell types, tissues, or organs, including but not limited to an organ selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestines, kidney, liver, lung, lymph node, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and vagina, or a tissue or cell type thereof.
  • Neoplasia include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumor of the plasma cells).
  • receptor is meant a polypeptide, or portion thereof, present on a cell membrane that selectively binds one or more ligand.
  • a T cell that recognizes a tumor can expresses a receptor (e.g., a TCR or CAR) that binds to a tumor antigen.
  • a receptor e.g., a TCR or CAR
  • By“reference” or“control” is meant a standard of comparison. For example, the level of scFv-antigen binding by a cell expressing a CAR and an scFv may be compared to the level of scFv-antigen binding in a corresponding cell expressing CAR alone.
  • By“secreted” is meant a polypeptide that is released from a cell via the secretory pathway through the endoplasmic reticulum, Golgi apparatus, and as a vesicle that transiently fuses at the cell plasma membrane, releasing the proteins outside of the cell.
  • polypeptide or fragment thereof that recognizes and binds to a biological molecule of interest (e.g., a polypeptide), but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a presently disclosed polypeptide.
  • tumor antigen refers to an antigen (e.g., a polypeptide) that is uniquely or differentially expressed on a tumor cell compared to a normal or non- IS neoplastic cell.
  • antigen e.g., a polypeptide
  • treatment refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology.
  • Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • a treatment can prevent deterioration due to a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment may prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder.
  • An“individual” or“subject” herein is a vertebrate, such as a human or non-human animal, for example, a mammal.
  • Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents and pets.
  • Non-limiting examples of non human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.
  • immunocompromised refers to a subject who has an immunodeficiency.
  • the subject is very vulnerable to opportunistic infections, infections caused by organisms that usually do not cause disease in a person with a healthy immune system, but can affect people with a poorly functioning or suppressed immune system.
  • Other aspects of the presently disclosed subject matter are described in the following disclosure and are within the ambit of the presently disclosed subject matter.
  • the present disclosure provides use of an antigen recognizing receptor that binds to an antigen of interest in combination with a PS blocker for treatment.
  • the antigen recognizing receptor can be a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).
  • the antigen recognizing receptor is a chimeric antigen receptor (CAR).
  • the antigen recognizing receptor can bind to a tumor antigen or a pathogen antigen.
  • TCR T-cell receptor
  • the antigen recognizing receptor is a TCR.
  • a TCR is a disulfide-linked heterodimeric protein consisting of two variable chains expressed as part of a complex with the invariant CD3 chain molecules.
  • a TCR is found on the surface of T cells, and is responsible for recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • a TCR comprises an alpha chain and a beta chain (encoded by TRA and TRB, respectively).
  • a TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).
  • Each chain of a TCR is composed of two extracellular domains: Variable (V) region and a Constant (C) region.
  • the Constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail.
  • the Variable region binds to the peptide/MHC complex.
  • the variable domain of both chains each has three complementarity determining regions (CDRs).
  • a TCR can form a receptor complex with three dimeric signaling modules CD35/e, CD3y/e and CD247 z/z or z/h.
  • a TCR complex engages with its antigen and MHC (peptide/MHC)
  • MHC peptide/MHC
  • the antigen recognizing receptor is a recombinant TCR.
  • the antigen recognizing receptor is a non-naturally occurring TCR.
  • the non-naturally occurring TCR differs from any naturally occurring TCR by at least one amino acid residue.
  • the non-naturally occurring TCR differs from any naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about
  • the non-naturally occurring TCR is modified from a naturally occurring TCR by at least one amino acid residue. In certain embodiments, the non-naturally occurring TCR is modified from a naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about
  • the antigen recognizing receptor is a CAR.
  • CARs are engineered receptors, which graft or confer a specificity of interest onto an immune effector cell.
  • CARs can be used to graft the specificity of a monoclonal antibody onto a T cell; with transfer of their coding sequence facilitated by retroviral vectors.
  • the CAR binds to a tumor antigen.
  • Any tumor antigen (antigenic peptide) can be used in the tumor-related embodiments described herein.
  • Sources of antigen include, but are not limited to, cancer proteins.
  • the antigen can be expressed as a peptide or as an intact protein or portion thereof.
  • the intact protein or a portion thereof can be native or mutagenized.
  • tumor antigens include melanoma differentiation antigens (MDAs), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD8, CD7, CD 10, CD 19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,
  • Interleukin- 13 receptor subunit alpha-2 ( ⁇ L-l3Ra2), k-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), Ll cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGEA3, p53, MARTl,GPl00, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, NKG2D ligands, cancer-testis antigen NY-ES0-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), and Wilms tumor protein (WT-l), BCMA,
  • the tumor antigen that the CAR binds to is an MDA.
  • Genes that encode melanoma antigens recognized by tumor-infiltrating lymphocytes (TIL) have been identified (Rosenberg. Immunol. Today (1997); 18: 175). With the exception of melanocytes and retina, normal tissues do not express these antigens, and no expression of these genes has been observed in cancers other than melanoma. Hence, these antigens represent molecules associated with the melanocyte lineage and are called melanoma differentiation antigens (MDA).
  • MDA melanoma differentiation antigens
  • TIL targeting MDA were associated with in vivo tumor regression when adoptively transferred to patients with metastatic melanoma (Rosenberg, et al. N. Engl. ./. Med. (1988);319: 1676).
  • the MDA is selected from the group consisting of Tyrosinase related protein 1 (“TRP1”), tyrosinase, Melan-A, gplOO, and TRP2.
  • TRP1 Tyrosinase related protein 1
  • tyrosinase tyrosinase
  • Melan-A gplOO
  • TRP2 TRP2
  • Tyrosinase is also known as OCA1 or SKC35.
  • Melan-A is also known as MART-l.
  • gplOO is also known as D10H12S53E, Dl2S53Eh, gp87, Pmell7, or Si.
  • TRP2 is also known as TRP-2, tyrosinase-related protein-2, Tyrp2, Tyrp-2, or DCT.
  • the MDA is Trpl.
  • TRP1 also known as TRP, TRP-l, CAS2, CATB, GP75, OCA3, TRP1, TYRP, TYRP1, b-PROTEIN encodes a
  • Trpl is involved in stabilizing and modulating tyrosinase protein, and affects melanosome structure and melanocyte proliferation. Defects in this gene can cause rufous oculocutaneous albinism and oculocutaneous albinism type III (OCA3).
  • the tumor antigen to which the CAR binds is CD 19.
  • the CAR binds to a pathogen antigen, e.g., for use in treating and/or preventing a pathogen infection or other infectious disease, for example, in an immunocompromised subject.
  • pathogen includes a virus, bacteria, fungi, parasite and protozoa capable of causing disease.
  • viruses include, Retroviridae (e.g. human
  • immunodeficiency viruses such as HIV-l (also referred to as HDTV-III, LAVE or HTLV-IIELAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g.
  • Flaviridae e.g. dengue viruses, encephalitis viruses, yellow fever viruses
  • Coronoviridae e.g. coronaviruses
  • Rhabdoviridae e.g. vesicular stomatitis viruses, rabies viruses
  • Fdoviridae e.g. ebola viruses
  • Paramyxoviridae e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus
  • Orthomyxoviridae e.g. influenza viruses
  • Bungaviridae e.g. Hantaan viruses, bunga viruses, phleboviruses and Naira viruses
  • Arena viridae hemorrhagic fever viruses
  • Reoviridae e.g. reoviruses, orbiviurses and rotaviruses
  • Birnaviridae Hepadnaviridae Hepatitis B virus
  • Parvovirida parvoviruses
  • papilloma viruses, polyoma viruses papilloma viruses, polyoma viruses
  • Adenoviridae most adenoviruses
  • Herpesviridae herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus
  • Poxviridae variola viruses, vaccinia viruses, pox viruses
  • Iridoviridae e.g. African swine fever virus
  • Non-limiting examples of bacteria include Pasteur ella, Staphylococci ,
  • infectious bacteria include but are not limited to, Helicobacter pyloris , Borelia burgdorferi , Legionella pneumophilia , Mycobacteria sps (e.g. M. tuberculosis , M. avium , M. intr acellular e, M. kansaii , M.
  • Streptococcus (viridans group), Streptococcus faecalis , Streptococcus bovis ,
  • Streptococcus (anaerobic sps.), Streptococcus pneumoniae , pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae , Bacillus antracis , corynebacterium diphtheriae , corynebacterium sp ., Erysipelothrix rhusiopathiae , Clostridium perfringers , Clostridium tetani , Enterobacter aerogenes, Klebsiella pneumoniae , Pasturella multocida , Bacteroides sp ., Fusobacterium nucleatum , Streptobacillus moniliformis , Treponema palladium, Treponema per pneumonia , Leptospira , Rickettsia , and Actinomyces israelii.
  • the pathogen antigen is a viral antigen present in
  • Cytomegalovirus a viral antigen present in Epstein Barr Virus (EBV), a viral antigen present in Human Immunodeficiency Virus (HIV), or a viral antigen present in influenza virus.
  • CARs are engineered receptors, which graft or confer a specificity of interest onto an immune effector cell.
  • CARs can be used to graft the specificity of a monoclonal antibody onto a T cell; with transfer of their coding sequence facilitated by retroviral vectors.
  • CARs There are three generations of CARs.“First generation” CARs are typically composed of an extracellular antigen-binding domain (e.g., an scFv), which is fused to a transmembrane domain, which is fused to cytoplasmic/intracellular signaling domain.
  • an extracellular antigen-binding domain e.g., an scFv
  • “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4 + and CD8 + T cells through their CD3z chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation.“Second generation” CARs add intracellular signaling domains from various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, 0X40) to the cytoplasmic tail of the CAR to provide additional signals to the T cell.“Second generation” CARs comprise those that provide both co- stimulation (e.g., CD28 or 4-1BB) and activation (O ⁇ 3z). “Third generation” CARs comprise those that provide multiple co-stimulation (e.g., CD28 and 4- 1BB) and activation (O ⁇ 3z).
  • the antigen recognizing receptor is a second-generation CAR.
  • the extracellular antigen-binding domain of the CAR (embodied, for example, an scFv or an analog thereof) binds to an antigen with a dissociation constant (K d ) of about 2 x 10 7 M or less. In certain embodiments, the K d is about 2 x 10 7 M or less.
  • the K d is about 2 x 10 7 M or less, about 1 x 10 7 M or less, about 9 x 10 8 M or less, about 1 x 10 8 M or less, about 9 x 10 9 M or less, about 5 x 10 9 M or less, about 4 x 10 9 M or less, about 3 x 10 9 M or less, about 2 x 10 9 M or less, or about 1 x 10 9 M or less.
  • the K d is from about 3 x 10 9 M or less.
  • the K d is from about 1 x 10 9 M to about 3 x 10 7 M.
  • the K d is from about 1.5 x 10 9 M to about 3 x 10 7 M.
  • the K d is from about 1.5 x 10 9 M to about 2.7 x 10 7 M.
  • Binding of the extracellular antigen-binding domain can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS analysis e.g., FACS analysis
  • bioassay e.g., growth inhibition
  • Western Blot assay Western Blot assay.
  • Each of these assays generally detect the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g ., an antibody, or an scFv) specific for the complex of interest.
  • a labeled reagent e.g ., an antibody, or an scFv
  • the scFv can be radioactively labeled and used in a radioimmunoassay (RIA) (see
  • the radioactive isotope can be detected by such means as the use of a g counter or a scintillation counter or by autoradiography.
  • the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker.
  • fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g. , EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g, ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g, YFP, Citrine, Venus, and YPet).
  • a CAR comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to an antigen, e.g., a tumor antigen (e.g., an MDA (e.g., Trp-l) or CD19) or a pathogen antigen.
  • the extracellular antigen-binding domain is an scFv.
  • the extracellular antigen-binding domain is a Fab, which is optionally crosslinked.
  • the extracellular antigen-binding domain is a F(ab) 2.
  • any of the foregoing molecules may be comprised in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain.
  • the extracellular antigen-binding domain specifically binds to an antigen.
  • the extracellular antigen-binding domain is an scFv.
  • the scFv is a human scFv.
  • the scFv is a humanized scFv.
  • the scFv is a murine scFv.
  • the extracellular antigen-binding domain is a Fab, which is optionally crosslinked.
  • the extracellular antigen-binding domain is a F(ab) 2.
  • any of the foregoing molecules may be comprised in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain.
  • the scFv is identified by screening scFv phage library with an antigen-Fc fusion protein.
  • the extracellular antigen-binding domain of a presently disclosed CAR is a murine scFv or a human scFv.
  • the extracellular antigen-binding domain comprises a murine scFv that binds specifically to an MDA (e.g., Trpl).
  • the extracellular antigen-binding domain comprises a human scFv that binds specifically to an MDA (e.g., Trpl).
  • the scFv is identified by screening scFv phage library with MDA-Fc fusion proteins.
  • the extracellular antigen-binding domain specifically binds to an MDA polypeptide (e.g., a Trpl polypeptide).
  • the Trpl polypeptide is a human Trpl polypeptide.
  • the human Trpl polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the sequence having a NCBI Reference No: NP_00054l .1 (SEQ ID NO: 10, provided below), or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the human Trpl polypeptide has an amino acid sequence that is a consecutive portion of SEQ ID NO: 10 which is at least 20, or at least 30, or at least 40, or at least 50, or at least 100, or at least 200, or at least 300, or at least 400, or at least 500, and up to 537 amino acids in length.
  • the human Trpl polypeptide has an amino acid sequence of amino acids 1 to 537, 1 to 50, 50 to 100, 100 to 200, 200 to 300,
  • the Trpl polypeptide is a mouse/murine Trpl
  • the mouse Trpl polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the sequence having a NCBI Reference No: NR_001268944.1 (SEQ ID No: 37, provided below), or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the mouse Trpl polypeptide has an amino acid sequence that is a consecutive portion of SEQ ID NO: 37 which is at least 20, or at least 30, or at least 40, or at least 50, or at least 100, or at least 200, or at least 300, or at least 400, or at least 500, and up to 537 amino acids in length.
  • the mouse Trpl polypeptide has an amino acid sequence of amino acids 1 to 537, 1 to 50, 50 to 100, 100 to 200, 200 to 300, 300 to 400, or 400 to 537 of SEQ ID NO: 37.
  • the extracellular antigen-binding domain specifically binds to a human Trpl polypeptide as well as a mouse Trpl polypeptide.
  • the extracellular antigen-binding domain is an scFv.
  • the scFv is a murine scFv.
  • the scFv is a human scFv.
  • the scFv is a humanized scFv.
  • the scFv comprises, consists essentially or has the amino acid sequence set forth in SEQ ID NO: 9, which is described in the following Table 1.
  • the scFv is derived from the TA99 antibody disclosed in International Patent Publication No. WO96/40249, the content of which is herein incorporated by reference in its entirety.
  • the extracellular antigen-binding domain is an scFv, which comprises a heavy chain variable region (V H ) comprising the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region (V L ) comprising the amino acid sequence set forth in SEQ ID NO:8, optionally with a linker sequence, for example a linker peptide, between the heavy chain variable region and the light chain variable region.
  • the linker comprises the amino acid sequence set forth in SEQ ID NO: 11.
  • the extracellular anti gen -binding domain is an scFv-Fc fusion protein or full-length human IgG with VH and VL regions or CDRs selected from Table 1.
  • the extracellular antigen-binding domain comprises a VH comprising an amino acid sequence that is at least about 80% (e.g, at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the sequence set forth in SEQ ID NO: 7, as shown in Table 1.
  • the extracellular antigen-binding domain comprises a VH comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous to the sequence set forth in SEQ ID NO: 7.
  • the extracellular antigen-binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:7.
  • the extracellular antigen-binding domain comprises a VL comprising an amino acid sequence that is at least about 80% (e.g, at least about 85%, at least about 90%, or at least about 95%) homologous to the sequence set forth in SEQ ID NO: 8, as shown in Table 1.
  • the extracellular antigen-binding domain comprises a VL comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous to the sequence set forth in SEQ ID NO: 8.
  • the extracellular antigen-binding domain comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 8.
  • the extracellular antigen-binding domain comprises a VH comprising an amino acid sequence that is at least about 80% (e.g, at least about 85%, at least about 90%, or at least about 95%) homologous to the sequence set forth in SEQ ID NO: 7, and a VL comprising an amino acid sequence that is at least about 80% (e.g, at least about 85%, at least about 90%, or at least about 95%) homologous to the sequence set forth in SEQ ID NO: 8.
  • the extracellular antigen-binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 8.
  • the extracellular antigen-binding domain comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or a conservative modification thereof, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2 or a conservative modification thereof, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3 or conservative modifications thereof, as shown in Table 1.
  • the extracellular antigen-binding domain comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3.
  • the extracellular antigen-binding domain comprises a VL CDR1 comprising the sequence amino acid set forth in SEQ ID NO:4 or a conservative modification thereof, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 or a conservative modification thereof, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof, as shown in Table 1.
  • the extracellular antigen-binding domain comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6.
  • the extracellular antigen-binding domain comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or a conservative modification thereof, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2 or a conservative modification thereof, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3 or conservative modifications thereof, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 or a conservative modification thereof, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 or a conservative modification thereof, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof.
  • the extracellular antigen-binding domain comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6.
  • Table 1
  • the CAR is a CAR targeting an MDA (e.g., Trpl) disclosed in International Patent Application No. PCT/US17/057098, which is incorporated by reference herein in its entirety.
  • the extracellular antigen-binding domain comprises a murine scFv that binds specifically to a CD 19 polypeptide.
  • the extracellular antigen-binding domain comprises a human scFv that binds specifically to a CD 19 polypeptide.
  • the extracellular antigen-binding domain is a murine scFv that binds to a human CD 19 polypeptide.
  • the extracellular antigen-binding domain is a murine scFv that binds to a murine CD 19 polypeptide.
  • the scFv comprises, consists essentially or has the amino acid sequence set forth in SEQ ID NO: 46, which is described in the following Table 2.
  • the extracellular antigen binding domain is an scFv-Fc fusion protein or full-length human IgG with V H and V L regions or CDRs selected from Table 2.
  • the extracellular antigen-binding domain is an scFv, which comprises the amino acid sequence set forth in SEQ ID NO: 46 and specifically binds to a murine CD 19 polypeptide.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 46 is set forth in SEQ ID NO: 47.
  • the scFv comprises a heavy chain variable region (V H ) comprising the amino acid sequence set forth in SEQ ID NO: 44.
  • the murine scFv comprises a light chain variable region (V L ) comprising the amino acid sequence set forth in SEQ ID NO: 45.
  • the scFv comprises a V H comprising the amino acid sequence set forth in SEQ ID NO: 44 and a V L comprising the amino acid sequence set forth in SEQ ID NO:
  • the linker comprises the amino acid sequence set forth in SEQ ID NO: 11.
  • the extracellular antigen-binding domain comprises a V H comprising an amino acid sequence that is at least about 80% ( e.g ., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 44.
  • the extracellular antigen-binding domain comprises a V H comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to SEQ ID NO: 44.
  • the extracellular antigen-binding domain comprises a V H comprising the amino sequence set forth in SEQ ID NO: 44.
  • the extracellular antigen-binding domain comprises a V L comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 45.
  • the extracellular antigen-binding domain comprises a VL comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous to SEQ ID NO: 45.
  • the extracellular antigen-binding domain comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 45.
  • the extracellular antigen binding domain comprises a VH comprising an amino acid sequence that is at least about 80% (e.g, at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 44, and a VL comprising an amino acid sequence that is at least about 80% (e.g, at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 45.
  • the extracellular antigen-binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 44 and a VL comprising the amino acid sequence set forth in SEQ ID NO:
  • the extracellular antigen-binding domain comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, or a conservative modification thereof, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39 or a conservative modification thereof, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, a conservative modification thereof.
  • the extracellular antigen-binding domain comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40.
  • the extracellular antigen-binding domain comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41 or a conservative modification thereof, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42 or a conservative modification thereof, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43 or a conservative modification thereof.
  • the extracellular antigen-binding domain comprises a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43.
  • the extracellular antigen-binding domain comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38 or a conservative modification thereof, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39 or a conservative modification thereof, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, a conservative modification thereof, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41 or a conservative modification thereof, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42 or a conservative modification thereof, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43 or a conservative modification thereof.
  • the extracellular antigen-binding domain comprises a VH CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 38, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42 and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43.
  • a conservative modification refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the presently disclosed CAR (e.g ., the extracellular antigen-binding domain of the CAR) comprising the amino acid sequence.
  • Conservative modifications can include amino acid substitutions, additions and deletions. Modifications can be introduced into the scFv of the presently disclosed CAR by standard techniques known in the art, such as site- directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties such as charge and polarity.
  • amino acids can be classified by charge: positively-charged amino acids include lysine, arginine, histidine, negatively-charged amino acids include aspartic acid, glutamic acid, neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine.
  • one or more amino acid residues within a CDR region can be replaced with other amino acid residues from the same group and the altered antibody can be tested for retained function (i.e., the functions set forth in (c) through (1) above) using the functional assays described herein.
  • the altered antibody can be tested for retained function (i.e., the functions set forth in (c) through (1) above) using the functional assays described herein.
  • no more than one, no more than two, no more than three, no more than four, no more than five residues within a specified sequence or a CDR region are altered.
  • the VH and/or VL amino acid sequences having at least about 80%, at least about 85%, at least about 90%, or at least about 95% e.g., about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%
  • the specified sequences e.g, SEQ ID NOs: 7 and/or 8
  • substitutions e.g, conservative substitutions
  • an MDA polypeptide e.g, a human MDA polypeptide (e.g., a human Trpl polypeptide), and/or a mouse MDA polypeptide (e.g., a mouse Trpl polypeptide)
  • the extracellular antigen-binding domain specifically binds to an MDA polypeptide (e.g, a human MDA polypeptide (e.g., a human Trpl polypeptide), and/or a mouse MDA polypeptide (e.g., a mouse Trpl polypeptide)) with a binding affinity (K d ) of about 3 x 10 9 or less.
  • the extracellular antigen binding domain binds to an MDA polypeptide (e.g, a human MDA polypeptide (e.g., a human Trpl polypeptide), or a mouse MDA polypeptide (e.g., a mouse Trpl
  • the extracellular antigen-binding domain binds to an MDA polypeptide (e.g, a human MDA polypeptide (e.g., a human Trpl polypeptide), and/or a mouse MDA polypeptide (e.g., a mouse Trpl polypeptide)) with a binding affinity (K d ) of from about 1.5 x 10 9 M to about 3 x 10 9 M.
  • K d binding affinity
  • a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NOs: 7 and/or 8.
  • substitutions, insertions, or deletions occur in regions outside the CDRs (e.g, in the FRs) of the extracellular antigen-binding domain.
  • the extracellular antigen-binding domain comprises VH and/or VL sequence selected from the group consisting of SEQ ID NOs: 7 and/or 8, including post- translational modifications of that sequence (SEQ ID NO: 7 and/or 8).
  • the VH and/or VL amino acid sequences having at least about 80%, at least about 85%, at least about 90%, or at least about 95% may contain substitutions (e.g, conservative substitutions), insertions, or deletions relative to the specified sequence(s), but retain the ability to bind to a target antigen (e.g., CD19).
  • a total of 1 to 10 amino acids are substituted, inserted and/or deleted in a specific sequence (e.g, SEQ ID NOs: 44 and/or 45).
  • substitutions, insertions, or deletions occur in regions outside the CDRs (e.g, in the FRs) of the extracellular antigen-binding domain.
  • the extracellular antigen-binding domain comprises VH and/or VL sequence selected from the group consisting of SEQ ID NOs: 44 and/or 45, including post-translational modifications of that sequence (SEQ ID NO: 44 and/or 45).
  • the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.
  • amino acids sequences of the presently disclosed subject matter can further be used as a“query sequence” to perform a search against public databases to, for example, identify related sequences.
  • search can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(l7):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the extracellular antigen-binding domain of a presently disclosed CAR cross-competes for binding to an MDA polypeptide (e.g, a human MDA polypeptide (e.g ., a human Trpl polypeptide), and/or a mouse MDA polypeptide (e.g., a mouse Trpl polypeptide)) with a reference antibody or an antigen-binding portion thereof comprising, e.g., the VH CDR1, CDR2, and CDR3 sequences and/or the VL CDR1, CDR2, and CDR3 sequences described in Table 1.
  • an MDA polypeptide e.g, a human MDA polypeptide (e.g ., a human Trpl polypeptide), and/or a mouse MDA polypeptide (e.g., a mouse Trpl polypeptide)
  • a reference antibody or an antigen-binding portion thereof comprising, e.g., the VH CDR1, CDR2, and CDR3 sequences and/
  • the extracellular antigen-binding domain of a presently disclosed CAR cross-competes for binding to an MDA polypeptide (e.g, a human MDA polypeptide (e.g., a human Trpl polypeptide), and/or a mouse MDA polypeptide (e.g., a mouse Trpl polypeptide)) with a reference antibody or an antigen-binding portion thereof comprising a VH CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 1; a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 2; a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 3; a VL CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 4; a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 5; and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 6.
  • the extracellular antigen-binding domain of a presently disclosed CAR cross-competes for binding to an MDA polypeptide (e.g, a human MDA polypeptide (e.g., a human Trpl polypeptide), and/or a mouse MDA polypeptide (e.g., a mouse Trpl polypeptide)) with a reference antibody or an antigen-binding portion thereof comprising, e.g., the VH and VL sequences described in Table 1.
  • an MDA polypeptide e.g, a human MDA polypeptide (e.g., a human Trpl polypeptide), and/or a mouse MDA polypeptide (e.g., a mouse Trpl polypeptide)
  • a reference antibody or an antigen-binding portion thereof comprising, e.g., the VH and VL sequences described in Table 1.
  • the extracellular antigen-binding domain of a presently disclosed CAR cross-competes for binding to an MDA polypeptide (e.g, a human MDA polypeptide (e.g., a human Trpl polypeptide) and/or a mouse MDA polypeptide (e.g., a mouse Trpl polypeptide)) with a reference antibody or an antigen binding portion thereof comprising a VH comprising amino acids having the sequence set forth in SEQ ID NO: 7, and a VL comprising amino acids having the sequence set forth in SEQ ID NO: 8.
  • an MDA polypeptide e.g, a human MDA polypeptide (e.g., a human Trpl polypeptide) and/or a mouse MDA polypeptide (e.g., a mouse Trpl polypeptide)
  • a reference antibody or an antigen binding portion thereof comprising a VH comprising amino acids having the sequence set forth in SEQ ID NO: 7
  • a VL comprising amino acids having the sequence
  • the extracellular antigen-binding domain binds to the same epitope on an MDA (e.g, a human MDA (e.g., human Trpl) and/or a mouse MDA (e.g., mouse Trpl)) as the reference antibody or antigen-binding portion thereof.
  • an MDA e.g, a human MDA (e.g., human Trpl) and/or a mouse MDA (e.g., mouse Trpl)
  • an MDA e.g., a human MDA (e.g., human Trpl) and/or a mouse MDA (e.g., mouse Trpl)
  • the extracellular antigen-binding domain of a presently disclosed CAR binds to the same epitope on an MDA (e.g, a human MDA (e.g., human Trpl) and/or a mouse MDA (e.g., mouse Trpl)) as a reference antibody or an antigen-binding portion thereof comprising, e.g., the VH CDR1, CDR2, and CDR3 sequences and the VL CDR1, CDR2, and CDR3 sequences described in Table 1.
  • MDA e.g, a human MDA (e.g., human Trpl) and/or a mouse MDA (e.g., mouse Trpl)
  • an antigen-binding portion thereof comprising, e.g., the VH CDR1, CDR2, and CDR3 sequences and the VL CDR1, CDR2, and CDR3 sequences described in Table 1.
  • the extracellular antigen- binding domain of a presently disclosed CAR binds to the same epitope on an MDA (e.g ., a human MDA (e.g., human Trpl, e.g., a human Trpl polypeptide) and/or a mouse MDA (e.g., mouse Trpl, e.g., a mouse Trpl polypeptide) as a reference antibody or an antigen binding portion thereof comprising a VH CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 1; a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 2; a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 3; a VL CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 4; a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 5; and a VL CDR3 comprising amino acids having the
  • the extracellular antigen binding domain of a presently disclosed CAR binds to the same or substantially the same epitope on an MDA (e.g, a human MDA (e.g., human Trpl) and/or a mouse MDA (e.g., mouse Trpl)) as a reference antibody or an antigen-binding portion thereof comprising the VH and VL sequences described in Table 1.
  • MDA e.g., a human MDA (e.g., human Trpl) and/or a mouse MDA (e.g., mouse Trpl)
  • a reference antibody or an antigen-binding portion thereof comprising the VH and VL sequences described in Table 1.
  • the extracellular antigen binding domain of a presently disclosed CAR binds to the same or substantially the same epitope on an MDA (e.g, a human MDA (e.g., human Trpl) and/or a mouse MDA (e.g., mouse Trpl, e.g., a mouse Trpl polypeptide) as a reference antibody or an antigen binding portion thereof comprising a VH comprising amino acids having the sequence set forth in SEQ ID NO: 7, and a VL comprising amino acids having the sequence set forth in SEQ ID NO: 8.
  • MDA e.g, a human MDA (e.g., human Trpl) and/or a mouse MDA (e.g., mouse Trpl, e.g., a mouse Trpl polypeptide) as a reference antibody or an antigen binding portion thereof comprising a VH comprising amino acids having the sequence set forth in SEQ ID NO: 7, and a VL comprising amino acids having the sequence set forth in SEQ
  • Extracellular antigen-binding domains that cross-compete or compete with the reference antibody or antigen-binding portions thereof for binding to an MDA
  • polypeptide e.g, a human MDA polypeptide (e.g., a human Trpl polypeptide) and/or a mouse MDA polypeptide (e.g., a mouse Trpl polypeptide)
  • a human MDA polypeptide e.g., a human Trpl polypeptide
  • a mouse MDA polypeptide e.g., a mouse Trpl polypeptide
  • RIAs radioimmunoassays
  • Biacore flow cytometry
  • Western blotting any other suitable quantitative or qualitative antibody-binding assays.
  • Competition ELISA is described in Morris,“Epitope Mapping of Protein Antigens by Competition ELISA”, The Protein Protocols Handbook (1996), pp 595-600, edited by J. Walker, which is incorporated by reference in its entirety.
  • the antibody-binding assay comprises measuring an initial binding of a reference antibody or an antigen binding portion thereof to an MDA polypeptide (e.g., a human MDA polypeptide (e.g., a human Trpl polypeptide) and/or a mouse MDA polypeptide (e.g., a mouse Trpl polypeptide)), admixing the reference antibody with a test extracellular antigen-binding domain, measuring a second binding of the reference antibody or antigen-binding portion thereof to the MDA polypeptide in the presence of the test extracellular antigen-binding domain, and comparing the initial binding with the second binding of the reference antibody, wherein a decreased second binding of the reference antibody or antigen binding portion thereof to the MDA polypeptide in comparison to the initial binding indicates that the test extracellular antigen-binding domain cross-competes with the reference antibody or antigen-binding portion thereof for binding to the MDA
  • an MDA polypeptide e.g., a human MDA polypeptide (e.g.
  • the polypeptide e.g ., one that recognizes the same or substantially the same epitope, an overlapping epitope, or an adjacent epitope.
  • the reference antibody or antigen-binding portion thereof is labeled, e.g. , with a fluorochrome, biotin, or peroxidase.
  • the MDA polypeptide is expressed in cells, e.g. , in a flow cytometry test.
  • the MDA polypeptide is immobilized onto a surface, including a Biacore ship (e.g, in a Biacore test), or other media suitable for surface plasmon resonance analysis.
  • the binding of the reference antibody or antigen-binding portion thereof in the presence of a completely irrelevant antibody (that does not bind to the MDA polypeptide) can serve as the control high value.
  • the control low value can be obtained by incubating a labeled reference antibody with an unlabeled reference antibody, where competition and reduced binding of the labeled reference antibody would occur.
  • a test extracellular antigen-binding domain that reduces the binding of the reference antibody or antigen-binding portion thereof to an MDA polypeptide by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% is considered to be an extracellular antigen-binding domain that cross-competes with the reference antibody or antigen-binding portion thereof for binding to the MDA polypeptide.
  • the assays are performed at room temperature.
  • the CDR3 domain independently from the CDR1 and/or CDR2 domain(s), alone can determine the binding specificity of an antibody or an antigen-binding portion thereof, for a cognate antigen and that multiple antibodies can predictably be generated having the same binding specificity based on a common CDR3 sequence. See, for example, Klimka et al, British J. of Cancer 83(2):252-260 (2000) (describing the production of a humanized anti-CD30 antibody using only the heavy chain variable domain CDR3 of murine anti-CD30 antibody Ki-4); Beiboer et al, J. Mol. Bioi.
  • the extracellular antigen-binding domain comprises a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 3 or a conservative modification thereof, and/or a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 6 or a conservative
  • the extracellular antigen-binding domain can comprise a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 2 or a conservative modification thereof, and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 5 or a conservative modification thereof.
  • the extracellular antigen-binding domain can further comprise a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 1 or a conservative modification thereof, and a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 4 or a conservative modification thereof.
  • the extracellular antigen-binding domain comprises a VH CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 1, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 2, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 3, a VL CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 4, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 5, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 6.
  • the extracellular antigen-binding domain comprises a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 40 or a conservative modification thereof, and/or a light chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 43 or a conservative modification thereof.
  • the extracellular antigen-binding domain can comprise a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 39 or a conservative modification thereof, and a light chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 42 or a conservative modification thereof.
  • the extracellular antigen-binding domain can further comprise a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 38 or a conservative modification thereof, and a light chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 41 or a conservative modification thereof.
  • the extracellular antigen-binding domain comprises a VH CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 38, a VH CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 39, a VH CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 40, a VL CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 41, a VL CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 42, and a VL CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 43.
  • an extracellular antigen-binding domain of a presently disclosed CAR can comprise a linker connecting the heavy chain variable region and light chain variable region of the extracellular antigen-binding domain.
  • the term“linker” refers to a functional group (e.g ., chemical or polypeptide) that covalently attaches two or more polypeptides or nucleic acids so that they are connected to one another.
  • a“peptide linker” refers to one or more amino acids used to couple two proteins together ( e.g ., to couple VH and VL domains).
  • the linker comprises amino acids having the sequence set forth in SEQ ID NO: 11.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11 is set forth in SEQ ID NO: 26.
  • the extracellular antigen-binding domain can comprise a leader or a signal peptide that directs the nascent protein into the endoplasmic reticulum.
  • Signal peptide or leader can be essential if the CAR is to be glycosylated and anchored in the cell membrane.
  • the signal sequence or leader can be a peptide sequence (about 5, about 10, about 15, about 20, about 25, or about 30 amino acids long) present at the N-terminus of newly synthesized proteins that directs their entry to the secretory pathway.
  • the signal peptide is covalently joined to the 5’ terminus of the extracellular antigen-binding domain.
  • the signal peptide comprises amino acids having the sequence set forth in SEQ ID NO: 12 as provided below.
  • SEQ ID NO: 29 The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 12 is set forth in SEQ ID NO: 29, which is provided below:
  • the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane.
  • the transmembrane domain of the CAR can comprise a CD8 polypeptide, a CD28 polypeptide, a O ⁇ 3z polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a synthetic peptide (not based on a protein associated with the immune response), or a combination thereof.
  • the transmembrane domain comprises a CD8
  • the CD8 polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to the sequence having a NCBI Reference No:
  • the CD8 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 13 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acids in length.
  • the CD8 polypeptide comprises or has an amino acid sequence of amino acids 1 to 235, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 235 of SEQ ID NO: 13.
  • the CAR of the presently disclosed comprises a transmembrane domain comprising a CD8 polypeptide that comprises an amino acid sequence of amino acids 137 to 209 of SEQ ID NO: 13.
  • the CD8 polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to the sequence having a NCBI Reference No:
  • AAA92533.1 (SEQ ID NO: 30) (homology herein may be determined using standard software such as BLAST or FASTA) as provided below, or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the CD8 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 30 which is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, and up to 247 amino acids in length.
  • the CD8 polypeptide comprises or has an amino acid sequence of amino acids 1 to 247, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 151 to 219, or 200 to 247 of SEQ ID NO: 30.
  • the CAR of the presently disclosed comprises a transmembrane domain comprising a CD8 polypeptide that comprises an amino acid sequence of amino acids 151 to 219 of SEQ ID NO: 30.
  • the CD8 polypeptide comprises or has the amino acid sequence set forth in SEQ ID NO: 32, which is provided below:
  • a“CD8 nucleic acid molecule” refers to a polynucleotide encoding a CD8 polypeptide.
  • the CD8 nucleic acid molecule encoding the CD8 polypeptide comprised in the transmembrane domain of the presently disclosed CAR comprises nucleic acids having the sequence set forth in SEQ ID NO:
  • the transmembrane domain of a presently disclosed CAR comprises a CD28 polypeptide.
  • the CD28 polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having a NCBI Reference No: P 10747 or NP 006130 (SEQ ID NO: 14), or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the CD28 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 14 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length.
  • the CD28 polypeptide has an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, or 200 to 220 of SEQ ID NO: 14.
  • SEQ ID NO: 14 is provided below:
  • a“CD28 nucleic acid molecule” refers to a polynucleotide encoding a CD28 polypeptide.
  • a CAR can also comprise a spacer region that links the extracellular antigen-binding domain to the transmembrane domain.
  • the spacer region can be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition.
  • the spacer region can be the hinge region from IgGl, or the CH 2 CH 3 region of immunoglobulin and portions of CD3.
  • a CAR can also comprise a spacer region that links the extracellular antigen-binding domain to the transmembrane domain.
  • the spacer region can be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition.
  • the spacer region can be the hinge region from IgGl, or the CH2CH3 region of immunoglobulin and portions of CD3, a portion of a CD28 polypeptide, a portion of a CD8 polypeptide, a variation of any of the foregoing which is at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous thereto, or a synthetic spacer sequence.
  • an intracellular domain of the CAR can comprise a CD3zeta polypeptide, which can activate or stimulate a cell (e.g. , a cell of the lymphoid lineage, e.g., a T cell).
  • CD3zeta comprises 3 IT AMs, and transmits an activation signal to the cell (e.g, a cell of the lymphoid lineage, e.g, a T cell) after antigen is bound.
  • the CD3zeta polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the sequence having a NCBI Reference No: NP_932l70 (SEQ ID NO: 15), or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the CD3zeta polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 15 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acids in length.
  • the CD3zeta polypeptide has an amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 100 to 150, or 150 to 164 of SEQ ID NO: 15.
  • the CD3zeta polypeptide comprises or has an amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 15.
  • SEQ ID NO: 15 is provided below:
  • the CD3zeta polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to the sequence having a NCBI Reference No:
  • the CD3zeta polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 16 which is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 90, or at least about 100, and up to 188 amino acids in length.
  • the CD3zeta polypeptide has an amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 142, 100 to 150, or 150 to 188 of SEQ ID NO: 16.
  • the CD3zeta polypeptide comprises or has an amino acid sequence of amino acids 52 to 142 of SEQ ID NO: 16.
  • SEQ ID NO: 16 is provided below:
  • the CD3zeta polypeptide comprises or has the amino acid sequence set forth in SEQ ID NO: 34, which is provided below:
  • a“CD3zeta nucleic acid molecule” refers to a polynucleotide encoding a CD3zeta polypeptide.
  • the CD3zeta nucleic acid molecule encoding the CD3zeta polypeptide comprised in the intracellular domain of a presently disclosed CAR comprises the nucleotide sequence set forth in SEQ ID NO: 31 as provided below.
  • an intracellular signaling domain of the CAR further comprises at least a co-stimulatory signaling region.
  • the co-stimulatory region comprises at least one co-stimulatory molecule, which can provide optimal lymphocyte activation.
  • co-stimulatory molecules refer to cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to antigen.
  • the at least one co- stimulatory signaling region can include a CD28 polypeptide, a 4-1BB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a DAP- 10 polypeptide, or a combination thereof.
  • the co-stimulatory molecule can bind to a co- stimulatory ligand, which is a protein expressed on cell surface that upon binding to its receptor produces a co- stimulatory response, i.e., an intracellular response that effects the stimulation provided when an antigen binds to its CAR molecule.
  • Co-stimulatory ligands include, but are not limited to CD80, CD86, CD70, OX40L, and 4-1BBL.
  • a 4-1BB ligand ⁇ i.e., 4-1BBL may bind to 4-1BB (also known as“CD137”) for providing an intracellular signal that in combination with a CAR signal induces an effector cell function of the CAR + T cell.
  • CARs comprising an intracellular signaling domain that comprises a co- stimulatory signaling region comprising 4-1BB, ICOS or DAP-10 are disclosed in U.S. 7,446,190, which is herein incorporated by reference in its entirety.
  • the CD28 polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the sequence having a NCBI Reference No: P10747 or NP 006130 (SEQ ID NO: 14), or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • the CD28 polypeptide has an amino acid sequence that is a consecutive portion of SEQ ID NO: 14 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length.
  • the CD28 polypeptide has an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, or 200 to 220 of SEQ ID NO: 14.
  • the CD28 polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the sequence having a NCBI Reference No:
  • the CD28 polypeptide has an amino acid sequence that is a consecutive portion of SEQ ID NO: 35 which is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to 218 amino acids in length.
  • the CD28 polypeptide has an amino acid sequence of amino acids 1 to 218, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 178 to 218, or 200 to 220 of SEQ ID NO: 35.
  • the co- stimulatory signaling region of a presently disclosed CAR comprises a CD28 polypeptide that comprises or has the amino acids 178 to 218 of SEQ ID NO: 35.
  • SEQ ID NO: 35 is provided below:
  • a“CD28 nucleic acid molecule” refers to a polynucleotide encoding a CD28 polypeptide.
  • a CD28 nucleic acid molecule that encodes a CD28 polypeptide comprised in the co-stimulatory signaling region of a presently disclosed CAR comprises or has a nucleotide sequence set forth in SEQ ID NO: 36, which is provided below.
  • the intracellular signaling domain of the CAR comprises a co- stimulatory signaling region that comprises two co-stimulatory molecules: CD28 and 4-1BB or CD28 and 0X40.
  • 4-1BB can act as a tumor necrosis factor (TNF) ligand and have stimulatory activity.
  • the 4-1BB polypeptide can have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous to the sequence having a NCBI Reference No: P41273 or NP_00l552 (SEQ ID NO: 17) or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • SEQ ID NO: 17 is provided below:
  • a“4-1BB nucleic acid molecule” refers to a polynucleotide encoding a 4-1BB polypeptide.
  • An 0X40 polypeptide can comprise or have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to the sequence having a NCBI Reference No: P43489 or NP 003318 (SEQ ID NO: 18), or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • SEQ ID NO: 18 is provided below:
  • an“0X40 nucleic acid molecule” refers to a polynucleotide encoding an 0X40 polypeptide.
  • An ICOS polypeptide can comprise or have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to the sequence having a NCBI Reference No: NP_036224 (SEQ ID NO: 19) or fragments thereof, and/or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.
  • SEQ ID NO: 19 is provided below:
  • an“ICOS nucleic acid molecule” refers to a polynucleotide encoding an ICOS polypeptide.
  • the CAR comprises, from 5’ to 3’, an extracellular antigen-binding region that comprises an scFv that specifically binds to a Trpl polypeptide, a transmembrane domain comprising a CD8 polypeptide, and an intracellular domain comprising a co-stimulatory signaling region that comprises a CD28 polypeptide and a CD3zeta polypeptide.
  • the CAR further comprises a signal peptide or a leader covalently joined to the 5’ terminus of the extracellular antigen binding domain.
  • the signal peptide comprises amino acids having the sequence set forth in SEQ ID NO: 12.
  • the scFv comprises the sequences provided in Table 1.
  • the CAR comprises, from 5’ to 3’, an extracellular antigen-binding region that comprises an scFv that specifically binds to a CD 19 polypeptide, a transmembrane domain comprising a CD8 polypeptide, and an intracellular domain comprising a co-stimulatory signaling region that comprises a CD28 polypeptide and a CD3zeta polypeptide.
  • the CAR further comprises a signal peptide or a leader covalently joined to the 5’ terminus of the extracellular antigen binding domain.
  • the scFv comprises the sequences provided in Table 2.
  • a presently disclosed CAR further comprises an inducible promoter, for expressing nucleic acid sequences in human cells.
  • Promoters for use in expressing CAR genes can be a constitutive promoter, such as ubiquitin C (UbiC) promoter.
  • the presently disclosed subject matter also provides isolated nucleic acid molecule encoding a presently disclosed CAR (e.g., an MDA-targeted CAR) or a functional portion thereof.
  • a presently disclosed CAR e.g., an MDA-targeted CAR
  • the term“functional portion” refers to any portion, part or fragment of a presently disclosed CAR (e.g., an MDA-targeted CAR), which portion, part or fragment retains the biological activity of the parent CAR).
  • functional portions encompass the portions, parts or fragments of a presently disclosed CAR that retains the ability to recognize a target cell, to treat a disease, e.g., melanoma, to a similar, same, or even a higher extent as the parent CAR.
  • an isolated nucleic acid molecule encoding a functional portion of a presently disclosed CAR can encode a protein comprising, e.g, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, and about 95%, or more of the parent CAR.
  • the isolated nucleic acid molecule encodes an MDA- targeted CAR comprising an scFv that specifically binds to an MDA polypeptide (e.g., a Trpl polypeptide), a transmembrane domain comprising a CD8 polypeptide, and an intracellular domain comprising a co-stimulatory signaling region comprising a CD28 polypeptide and a CD3zeta polypeptide.
  • an MDA polypeptide e.g., a Trpl polypeptide
  • a transmembrane domain comprising a CD8 polypeptide
  • an intracellular domain comprising a co-stimulatory signaling region comprising a CD28 polypeptide and a CD3zeta polypeptide.
  • the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 25, which is provided below.
  • the isolated nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 25 encodes a Trpl-targeted CAR comprising an scFv that comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7, a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8, and a linker comprising the amino acid sequence set forth in SEQ ID NO: 11 that is positioned between the heavy chain variable region and the light chain variable region, a transmembrane domain comprising a CD8 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 30, an intracellular domain comprising a co- stimulatory signaling region comprising a CD28 polypeptide comprising an amino acid sequence of amino acids 178 to 218 of SEQ ID NO: 35 and a CD3zeta polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 34.
  • a presently disclosed CAR targeting CD 19 comprises an scFv that comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 44, a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 45, and a linker comprising the amino acid sequence set forth in SEQ ID NO: 11 that is positioned between the heavy chain variable region and the light chain variable region, a transmembrane domain comprising a CD8 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 32, an intracellular domain comprising a co-stimulatory signaling region comprising a CD28 polypeptide comprising an amino acid sequence of amino acids 178 to 218 of SEQ ID NO: 35 and a CD3zeta polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 34.
  • the presently disclosed subject matter provides immunoresponsive cells comprising an antigen recognizing receptor (e.g., a TCR or a CAR) that binds to an antigen.
  • an antigen recognizing receptor e.g., a TCR or a CAR
  • the immunoresponsive cells comprising and/or expressing a CAR that comprises an extracellular antigen-binding domain, a
  • the immunoresponsive cells of the presently disclosed subject matter can be cells of the lymphoid lineage.
  • the lymphoid lineage comprising B, T and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like.
  • Non-limiting examples of immunoresponsive cells of the lymphoid lineage include T cells, Natural Killer (NK) cells, embryonic stem cells, and pluripotent stem cells (e.g., those from which lymphoid cells may be differentiated).
  • T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system.
  • the T cells of the presently disclosed subject matter can be any type of T cells, including, but not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g, TEM cells and TEMRA cells, Regulatory T cells (also known as suppressor T cells), Natural killer T cells, Mucosal associated invariant T cells, and gd T cells.
  • Cytotoxic T cells CTL or killer T cells
  • TTL or killer T cells are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells.
  • a patient’s own T cells may be genetically modified to target specific antigens through the introduction of an antigen- recognizing receptor, e.g., a CAR or a TCR.
  • the immunoresponsive cell is a T cell.
  • the T cell can be a CD4 + T cell or a CD8 + T cell.
  • the T cell is a CD4 + T cell.
  • the T cell is a CD8 + T cell.
  • Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation in order to perform their cytotoxic effect on target cells.
  • Types of human lymphocytes of the presently disclosed subject matter include, without limitation, peripheral donor lymphocytes, e.g., those disclosed in Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CARs), in Morgan, R.A., et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen recognizing T cell receptor complex comprising the a and b heterodimer), in Panelli, M.C., et al. 2000 J Immunol 164:495-504; Panelli, M.C., et al. 2000 J Immunol
  • TILs tumor infiltrating lymphocytes
  • AAPCs artificial antigen-presenting cells
  • immunoresponsive cells e.g, T cells
  • T cells can be autologous, non-autologous (e.g, allogeneic), or derived in vitro from engineered progenitor or stem cells.
  • the presently disclosed immunoresponsive cells are capable of modulating the tumor microenvironment.
  • Tumors have a microenvironment that is hostile to the host immune response involving a series of mechanisms by malignant cells to protect themselves from immune recognition and elimination. This“hostile tumor
  • microenvironment comprises a variety of immune suppressive factors including infiltrating regulatory CD4 + T cells (Tregs), myeloid derived suppressor cells (MDSCs), tumor associated macrophages (TAMs), immune suppressive cytokines including TGF-b, and expression of ligands targeted to immune suppressive receptors expressed by activated T cells (CTLA-4 and PD-l).
  • Tregs infiltrating regulatory CD4 + T cells
  • MDSCs myeloid derived suppressor cells
  • TAMs tumor associated macrophages
  • immune suppressive cytokines including TGF-b
  • CTL-4 and PD-l immune suppressive cytokines
  • Collectively these immune suppressive factors can induce either marked anergy or apoptosis of adoptively transferred CAR modified T cells upon encounter with targeted tumor cells.
  • the unpurified source of CTLs may be any known in the art, such as the bone marrow, fetal, neonate or adult or other hematopoietic cell source, e.g., fetal liver, peripheral blood or umbilical cord blood.
  • hematopoietic cell source e.g., fetal liver, peripheral blood or umbilical cord blood.
  • Various techniques can be employed to separate the cells. For instance, negative selection methods can remove non-CTLs initially.
  • mAbs are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation for both positive and negative selections.
  • a large proportion of terminally differentiated cells can be initially removed by a relatively crude separation.
  • magnetic bead separations can be used initially to remove large numbers of irrelevant cells.
  • at least about 80%, usually at least 70% of the total hematopoietic cells will be removed prior to cell isolation.
  • Procedures for separation include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that modify cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents joined to or used in conjunction with a mAh, including, but not limited to, complement and cytotoxins; and panning with antibody attached to a solid matrix, e.g. plate, chip, elutriation or any other convenient technique.
  • Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels.
  • the cells can be selected against dead cells, by employing dyes associated with dead cells such as propidium iodide (PI).
  • the cells are collected in a medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable, e.g., sterile, isotonic medium.
  • FCS fetal calf serum
  • BSA bovine serum albumin
  • an immunoresponsive cell e.g., a T cell or aNK cell
  • a retroviral vector either gamma-retroviral or lentiviral
  • a polynucleotide encoding an antigen recognizing receptor can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest.
  • Non-viral vectors may be used as well.
  • a retroviral vector is generally employed for transduction, however any other suitable viral vector or non-viral delivery system can be used.
  • elements that create polycistronic expression cassette include, but is not limited to, various viral and non-viral Internal Ribosome Entry Sites (IRES, e.g., FGF-l IRES, FGF-2 IRES, VEGF IRES, IGF- II IRES, NF-kB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, aphthovirus IRES, picomavirus IRES, poliovirus IRES and
  • IRS Internal Ribosome Entry Sites
  • encephalomyocarditis virus IRES encephalomyocarditis virus
  • cleavable linkers e.g., 2A peptides , e.g., P2A, T2A, E2A and F2A peptides.
  • cleavable linkers e.g., 2A peptides , e.g., P2A, T2A, E2A and F2A peptides.
  • retroviral vector and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells.
  • Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. ( 1988) I 1 roc. Natl.
  • Non-amphotropic particles are suitable too, e.g., particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art.
  • Possible methods of transduction also include direct co-culture of the cells with producer cells, e.g., by the method ofBregni, et al. (1992) Blood 80: 1418-1422, or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89: 1817.
  • transducing viral vectors can be used to modify an immunoresponsive cell.
  • the chosen vector exhibits high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71 :6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. EDS. A. 94: 10319, 1997).
  • viral vectors that can be used include, for example, adenoviral, lentiviral, and adena-associated viral vectors, vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1 :55-61, 1990; Sharp, The Lancet 337: 1277-1278, 1991; Cometta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:98
  • Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).
  • Non-viral approaches can also be employed for genetic modification of an immunoresponsive cell.
  • a nucleic acid molecule can be introduced into an immunoresponsive cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S. A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci.
  • Transplantation of normal genes into the affected tissues of a subject can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue or are injected systemically.
  • a cultivatable cell type ex vivo e.g., an autologous or heterologous primary cell or progeny thereof
  • Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (e.g. Zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR). Transient expression may be obtained by RNA electroporation.
  • transposases or targeted nucleases e.g. Zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR.
  • Transient expression may be obtained by RNA electroporation.
  • CRISPR Clustered regularly-interspaced short palindromic repeats
  • the system includes Cas9 (a protein able to modify DNA utilizing crRNA as its guide), CRISPR RNA (crRNA, contains the RNA used by Cas9 to guide it to the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form) forming an active complex with Cas9), trans-activating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9), and an optional section of DNA repair template (DNA that guides the cellular repair process allowing insertion of a specific DNA sequence).
  • Cas9 a protein able to modify DNA utilizing crRNA as its guide
  • CRISPR RNA CRISPR RNA
  • tracrRNA trans-activating crRNA
  • Cas9 DNA that guides the cellular repair process allowing insertion of a specific DNA sequence.
  • CRISPR/Cas9 often employs a plasmid to transfect the target cells.
  • the crRNA needs to be designed for each application as this is the sequence that Cas9 uses to identify and directly bind to the target DNA in a cell.
  • the repair template carrying CAR expression cassette need also be designed for each application, as it must overlap with the sequences on either side of the cut and code for the insertion sequence.
  • Multiple crRNA's and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA). This sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells.
  • a zinc-finger nuclease is an artificial restriction enzyme, which is generated by combining a zinc finger DNA-binding domain with a DNA-cleavage domain.
  • a zinc finger domain can be engineered to target specific DNA sequences which allows a zinc-finger nuclease to target desired sequences within genomes.
  • the DNA- binding domains of individual ZFNs typically contain a plurality of individual zinc finger repeats and can each recognize a plurality of basepairs.
  • the most common method to generate new zinc-finger domain is to combine smaller zinc-finger "modules" of known specificity.
  • the most common cleavage domain in ZFNs is the non-specific cleavage domain from the type IIs restriction endonuclease Fokl.
  • ZFNs can be used to insert the CAR expression cassette into genome.
  • the HR machinery searches for homology between the damaged chromosome and the homologous DNA template, and then copies the sequence of the template between the two broken ends of the
  • chromosome whereby the homologous DNA template is integrated into the genome.
  • Transcription activator-like effector nucleases are restriction enzymes that can be engineered to cut specific sequences of DNA. TALEN system operates on almost the same principle as ZFNs. They are generated by combining a transcription activator-like effectors DNA-binding domain with a DNA cleavage domain.
  • Transcription activator-like effectors are composed of 33-34 amino acid repeating motifs with two variable positions that have a strong recognition for specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be engineered to bind desired DNA sequence, and thereby guide the nuclease to cut at specific locations in genome.
  • cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element or intron (e.g. the elongation factor la
  • CMV human cytomegalovirus
  • SV40 simian virus 40
  • metallothionein promoters e.g. the metallothionein promoters
  • enhancer/promoter/intron structure For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid.
  • the enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers.
  • regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.
  • the resulting cells can be grown under conditions similar to those for unmodified cells, whereby the modified cells can be expanded and used for a variety of purposes.
  • PS phosphatidylserine
  • PS is a phospholipid that is exposed on surface of apoptotic cells, tumor cells and tumor endothelium. PS has been shown to promote immunosuppressive signals in the tumor microenvironment. PS binding to its receptors promote anti inflammatory and immunosuppressive signals in the tumor microenvironment (Graham et ah, Nature Reviews Cancer (2014); 14:769-785).
  • Antibodies that target PS have been shown to reactivate anti-tumor immunity by polarizing tumor associated macrophages into a pro-inflammatory Ml phenotype, reducing the number of MDSCs in tumors and promoting the maturation of dendritic cells into functional APCs.
  • a PS blocker is a molecule that is capable of inhibiting one or more of (a) the interaction between PS and another molecule (e.g., a binding partner of PS), (b) the biological activity of PS, and (c) the transportation of PS.
  • the PS blocker is an antibody that specifically targets PS (“a PS- targeting antibody”).
  • the PS-targeting antibody is mchlNl 1, which is a mouse chimeric monoclonal antibody that specifically targets PS.
  • the PS blocker is an antibody that specifically binds to a PS receptor.
  • the PS blocker is an antibody that specifically binds to PS carrier protein.
  • the PS blocker is an aptamer that specifically binds to PS.
  • the PS blocker is a small molecule that specifically binds to PS. 6. Polypeptides and Analogs
  • Trpl a Trpl, CD 19, CD8, 4- 1BB, CD28, and CD3z polypeptides or fragments thereof that are modified in ways that enhance their anti -neoplastic activity when expressed in an immunoresponsive cell.
  • the presently disclosed subject matter provides methods for optimizing an amino acid sequence or nucleic acid sequence by producing an alteration in the sequence. Such alterations may include certain mutations, deletions, insertions, or post-translational modifications.
  • the presently disclosed subject matter further includes analogs of any naturally-occurring polypeptide disclosed herein. Analogs can differ from a naturally- occurring polypeptide disclosed herein by amino acid sequence differences, by post- translational modifications, or by both.
  • Analogs can exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more homologous to all or part of a naturally-occurring amino, acid sequence of the presently disclosed subject matter.
  • the length of sequence comparison is at least 5, 10, 15 or 20 amino acid residues, e.g., at least 25, 50, or 75 amino acid residues, or more than 100 amino acid residues.
  • a BLAST program may be used, with a probability score between e 3 and e 100 indicating a closely related sequence.
  • Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes.
  • Analogs can also differ from the naturally-occurring polypeptides by alterations in primary sequence.
  • polypeptides In addition to full-length polypeptides, the presently disclosed subject matter also provides fragments of any one of the polypeptides or peptide domains disclosed herein.
  • a fragment means at least 5, 10, 13, or 15 amino acids. In certain embodiments, a fragment comprises at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids. In certain embodiments, a fragment comprises at least 60 to 80, 100, 200, 300 or more contiguous amino acids. Fragments can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).
  • Non-protein analogs have a chemical structure designed to mimic the functional activity of a protein disclosed herein. Such analogs may exceed the physiological activity of the original polypeptide.
  • Methods of analog design are well known in the art, and synthesis of analogs can be carried out according to such methods by modifying the chemical structures such that the resultant analogs increase the anti -neoplastic activity of the original polypeptide when expressed in an immunoresponsive cell. These chemical modifications include, but are not limited to, substituting alternative R groups and varying the degree of saturation at specific carbon atoms of a reference polypeptide.
  • the protein analogs are relatively resistant to in vivo degradation, resulting in a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below. 7. Administration
  • the PS blocker and the presently disclosed immunoresponsive cells or compositions comprising thereof can be provided systemically or directly to a subject for inducing and/or enhancing an immune response to an antigen and/or treating and/or preventing a neoplasm, pathogen infection, or infectious disease.
  • the PS blocker and CAR-modified immunoresponsive cells or
  • compositions comprising thereof are directly injected into an organ of interest (e.g., an organ affected by a neoplasia).
  • an organ of interest e.g., an organ affected by a neoplasia
  • the PS blocker and the presently disclosed immunoresponsive cells or compositions comprising thereof are provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the tumor vasculature).
  • Expansion and differentiation agents can be provided prior to, during or after administration of the cells or compositions to increase production of T cells, NK cells, or CTL cells in vitro or in vivo.
  • the PS blocker and CAR-modified immunoresponsive cells or compositions comprising thereof can be administered in any physiologically acceptable vehicle, normally intravascularly, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus). Usually, at least about 1 c 10 5 cells will be administered, eventually reaching about 1 x 10 10 or more.
  • the presently disclosed immunoresponsive cells can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of the presently disclosed immunoresponsive cells in a population using various well-known methods, such as fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • Suitable ranges of purity in populations comprising the presently disclosed immunoresponsive cells are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%. In certain embodiments, the purity is about 70% to about 75%, about 75% to about 80%, or about 80% to about 85%. In certain embodiments, the purity is about 85% to about 90%, about 90% to about 95%, and about 95% to about 100%.
  • Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage).
  • the cells can be introduced by injection, catheter, or the like.
  • compositions can be pharmaceutical compositions comprising the presently disclosed immunoresponsive cells or their progenitors and a pharmaceutically acceptable carrier.
  • Administration can be autologous or heterologous.
  • immunoresponsive cells, or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject.
  • Peripheral blood derived immunoresponsive cells or their progeny e.g., in vivo , ex vivo or in vitro derived
  • a therapeutic composition of the presently disclosed subject matter e.g., a pharmaceutical composition comprising a presently disclosed immunoresponsive cell
  • it can be formulated in a unit dosage injectable form (solution, suspension, emulsion).
  • the PS blocker and the presently disclosed immunoresponsive cells or compositions comprising thereof can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH.
  • sterile liquid preparations e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH.
  • Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues.
  • Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.
  • carriers can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.
  • Sterile injectable solutions can be prepared by incorporating the genetically modified immunoresponsive cells in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired.
  • Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
  • the compositions can also be lyophilized.
  • the compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
  • Standard texts such as“REMINGTON’S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.
  • compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • antimicrobial preservatives for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the presently disclosed subject matter, however, any vehicle, diluent, or additive used would have to be compatible with the genetically modified
  • immunoresponsive cells or their progenitors.
  • compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid.
  • the desired isotonicity of the compositions may be
  • sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • Sodium chloride can be particularly for buffers containing sodium ions.
  • Viscosity of the compositions can be maintained at the selected level using a pharmaceutically acceptable thickening agent.
  • a pharmaceutically acceptable thickening agent for example, methylcellulose is is readily and economically available and is easy to work with.
  • suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like.
  • concentration of the thickener can depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity.
  • liquid dosage form e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form.
  • the quantity of cells to be administered will vary for the subject being treated. In a one embodiment, between about 10 4 and about 10 10 , between about 10 5 and about 10 9 , or between about 10 6 and about 10 8 of the presently disclosed immunoresponsive cells are administered to a human subject. More effective cells may be administered in even smaller numbers. In certain embodiments, at least about 1 c 10 8 , about 2/ 10 8 , about 3 > ⁇ l0 8 , about 4x l0 8 , or about 5x l0 8 of the presently disclosed immunoresponsive cells are administered to a human subject. The precise determination of what would be considered an effective dose may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.
  • any additives in addition to the active cell(s) and/or agent(s) are present in an amount of 0.001 to 50% (weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, about 0.0001 to about 1 wt %, about 0.0001 to about 0.05 wt% or about 0.001 to about 20 wt %, about 0.01 to about 10 wt %, or about 0.05 to about 5 wt %.
  • any composition to be administered to an animal or human the followings can be determined: toxicity such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse; the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response.
  • toxicity such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse
  • the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation.
  • the presently disclosed subject matter provides methods for inducing and/or increasing an immune response in a subject in need thereof.
  • a PS blocker and an effective amount of the presently disclosed immunoresponsive cells and compositions comprising thereof can be used for treating and/or preventing a neoplasm in a subject.
  • a PS blocker and PS blocker and an effective amount of the presently disclosed immunoresponsive cells and compositions comprising thereof can be used for treating and/or preventing a neoplasm in a subject.
  • immunoresponsive cells and compositions comprising thereof can be used for prolonging the survival of a subject suffering from a neoplasm.
  • the PS blocker and an effective amount of the presently disclosed immunoresponsive cells and compositions comprising thereof can also be used for treating and/or preventing a pathogen infection or other infectious disease in a subject, such as an immunocompromised human subject.
  • Such methods comprise administering a PS blocker and an effective amount of the presently disclosed immunoresponsive cells or a composition (e.g., pharmaceutical composition) comprising thereof to achieve the desired effect, be it palliation of an existing condition or prevention of recurrence.
  • the amount administered is an amount effective in producing the desired effect.
  • An effective amount can be provided in one or a series of administrations.
  • An effective amount can be provided in a bolus or by continuous perfusion.
  • an“effective amount” is an amount sufficient to effect a beneficial or desired clinical result upon treatment.
  • An effective amount can be administered to a subject in one or more doses.
  • an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease.
  • the effective amount is generally determined by the physician on a case-by- case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the immunoresponsive cells administered.
  • cell doses in the range of about 10 6 -10 10 are typically infused.
  • T cells are induced that are specifically directed against the specific antigen.
  • the modified cells can be administered by any method known in the art including, but not limited to, intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal and directly to the thymus.
  • the presently disclosed subject matter provides methods for treating and/or preventing a neoplasm in a subject.
  • the method can comprise administering a PS-l blocker and an effective amount of the presently disclosed immunoresponsive cells or a composition comprising thereof to a subject having a neoplasm.
  • Non-limiting examples of neoplasia include blood cancers (e.g. leukemias, lymphomas, and myelomas), melanoma, glioblastoma multiforme, anaplastic astrocytoma, ependymoma, meningioma, and oligodendroglioma, ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, throat cancer, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate and small cell lung cancer).
  • blood cancers e.g. leukemias, lymphomas, and myelomas
  • melanoma e.g. leukemias, lymphomas, and myelomas
  • glioblastoma multiforme e.g.
  • Suitable carcinomas further include any known in the field of oncology, including, but not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neural ectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small and large cell lung adenocarcinomas, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchoalveolarcarcinoma, epithelial adenocarcinoma, and liver metastases thereof, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma,
  • the neoplasia is selected from the group consisting of blood cancers (e.g. leukemias, lymphomas, and myelomas), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, and throat cancer.
  • blood cancers e.g. leukemias, lymphomas, and myelomas
  • ovarian cancer e.g. leukemias, lymphomas, and myelomas
  • the presently disclosed immunoresponsive cells and compositions comprising thereof can be used for treating and/or preventing blood cancers (e.g., leukemias, lymphomas, and myelomas) or ovarian cancer, which are not amenable to conventional therapeutic interventions.
  • the neoplasm is selected from the group consisting of melanoma, glioblastoma multiforme, anaplastic astrocytoma, ependymoma, meningioma, and oligodendroglioma.
  • the neoplasm is melanoma.
  • the method further comprises pre-conditioning the subject prior to administering the immunoresponsive cells or pharmaceutical composition.
  • Any suitable pre-conditioning treatments for immunotherapy can be applied.
  • pre-conditioning treatments include chemotherapy, radiotherapy, lymphodepleting treatment, total body irradiation, and a combination thereof.
  • the pre-conditioning treatment is myeloablative.
  • the pre-conditioning treatment is non-myeloablative.
  • the pre- conditioning treatment is lymphodepleting.
  • the pre-conditioning treatment can facilitate T cell expansion.
  • the pre-conditioning treatment is chemotherapy.
  • the method further comprises administering to the subject a chemotherapeutic agent.
  • the subject receives the
  • chemotherapeutic agent prior to the immunoresponsive cells or pharmaceutical composition comprising thereof.
  • chemotherapeutic agents include docetaxel, cyclophosphamide, capecitabine, doxorubic, fludarabin, and a combination thereof.
  • the chemotherapeutic agent is
  • the pre-conditioning treatment is performed about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days , about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 25 days, about 30 days, about 40 days, about 50 days or more, or any intermediate time period thereof, prior to the administration of the immunoresponsive cells or pharmaceutical composition comprising thereof.
  • the pre- conditioning treatment is performed between about 1 day to about 2 weeks between about 1 week to about 2 weeks, between about 2 weeks to about 3 weeks, between about 3 weeks to about 4 weeks, or between about 4 weeks to about 5 weeks, prior to the administration of the immunoresponsive cells or pharmaceutical composition comprising thereof.
  • the subjects can have an advanced form of disease, in which case the treatment objective can include mitigation or reversal of disease progression, and/or amelioration of side effects.
  • the subjects can have a history of the condition, for which they have already been treated, in which case the therapeutic objective will typically include a decrease or delay in the risk of recurrence.
  • Suitable human subjects for therapy typically comprise two treatment groups that can be distinguished by clinical criteria.
  • Subjects with“advanced disease” or“high tumor burden” are those who bear a clinically measurable tumor.
  • a clinically measurable tumor is one that can be detected on the basis of tumor mass (e.g., by palpation, CAT scan, sonogram, mammogram or X-ray; positive biochemical or histopathologic markers on their own are insufficient to identify this population).
  • a pharmaceutical composition is administered to these subjects to elicit an anti -tumor response, with the objective of palliating their condition.
  • reduction in tumor mass occurs as a result, but any clinical improvement constitutes a benefit.
  • Clinical improvement includes decreased risk or rate of progression or reduction in pathological consequences of the tumor.
  • a second group of suitable subjects is known in the art as the“adjuvant group.” These are individuals who have had a history of neoplasia, but have been responsive to another mode of therapy.
  • the prior therapy can have included, but is not restricted to, surgical resection, radiotherapy, and traditional chemotherapy.
  • these individuals have no clinically measurable tumor.
  • they are suspected of being at risk for progression of the disease, either near the original tumor site, or by metastases.
  • This group can be further subdivided into high-risk and low-risk individuals. The subdivision is made on the basis of features observed before or after the initial treatment. These features are known in the clinical arts, and are suitably defined for each different neoplasia.
  • Features typical of high-risk subgroups are those in which the tumor has invaded neighboring tissues, or who show involvement of lymph nodes.
  • Another group have a genetic predisposition to neoplasia but have not yet evidenced clinical signs of neoplasia. For instance, women testing positive for a genetic mutation associated with breast cancer, but still of childbearing age, can wish to receive one or more of the immunoresponsive cells described herein in treatment prophylactically to prevent the occurrence of neoplasia until it is suitable to perform preventive surgery.
  • adoptively transferred T or NK cells are endowed with augmented and selective cytolytic activity at the tumor site. Furthermore, subsequent to their localization to tumor or viral infection and their proliferation, the T cells turn the tumor or viral infection site into a highly conductive environment for a wide range of immune cells involved in the physiological anti-tumor or antiviral response (tumor infiltrating lymphocytes, NK-, NKT- cells, dendritic cells, and macrophages).
  • a pathogen infection e.g., viral infection, bacterial infection, fungal infection, parasite infection, or protozoal infection
  • a pathogen infection e.g., viral infection, bacterial infection, fungal infection, parasite infection, or protozoal infection
  • the method can comprise administering an effective amount of the presently disclosed immunoresponsive cells or a composition comprising thereof to a subject having a pathogen infection.
  • viral infections susceptible to treatment include, but are not limited to, Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Human Immunodeficiency Virus (HIV), and influenza virus infections.
  • immunoresponsive cells e.g., T cells
  • T cells to avert or minimize the risks of immunological complications
  • immunological complications e.g, graft versus-host disease (GvHD)
  • GvHD graft versus-host disease
  • a potential solution to this problem is engineering a suicide gene into the presently disclosed immunoresponsive cells.
  • Suitable suicide genes include, but are not limited to, Herpes simplex virus thymidine kinase (hsv- tk), inducible Caspase 9 Suicide gene (iCasp-9), and a truncated human epidermal growth factor receptor (EGFRt) polypeptide.
  • the suicide gene is an EGFRt polypeptide.
  • the EGFRt polypeptide can enable T cell elimination by administering anti-EGFR monoclonal antibody (e.g ., cetuximab).
  • EGFRt can be covalently joined to the upstream of the antigen recognizing receptor of a presently disclosed CAR.
  • the suicide gene can be included within the vector comprising nucleic acids encoding a presently disclosed CAR.
  • a prodrug designed to activate the suicide gene e.g., a prodrug (e.g, AP1903 that can activate iCasp-9) during malignant T-cell transformation (e.g, GVHD) triggers apoptosis in the suicide gene-activated CAR-expressing T cells.
  • a prodrug e.g, AP1903 that can activate iCasp-9
  • GVHD malignant T-cell transformation
  • the above-described various methods can further comprise administering to the subject at least one checkpoint immune blockade agent.
  • checkpoint immune blockade agents include an anti-4-lBB antibody, an anti-OX40 antibody, an anti-GITR antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, an anti -PD- 1 antibody, an anti-LAG3 antibody, an anti-TNSF25 antibody, an anti-TIGT antibody, an anti-CD40 antibody, and combinations thereof.
  • kits for inducing and/or enhancing an immune response and/or treating and/or preventing a neoplasm or a pathogen infection in a subject comprises an effective amount of presently disclosed immunoresponsive cells or a pharmaceutical composition comprising thereof and an effective amount of a PS blocker.
  • the kit comprises a sterile container; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • the kit includes an isolated nucleic acid molecule encoding an antigen recognizing receptor (e.g., a CAR) directed toward an antigen of interest (e.g., an MDA or CD19) and a PS blocker (e.g., a PS-targeting antibody), which may optionally be comprised in the same or different vectors.
  • an antigen recognizing receptor e.g., a CAR
  • an antigen of interest e.g., an MDA or CD19
  • a PS blocker e.g., a PS-targeting antibody
  • the immunoresponsive cells and/or nucleic acid molecules are provided together with instructions for administering the cells or nucleic acid molecules to a subject having or at risk of developing a neoplasia or pathogen or immune disorder.
  • the instructions generally include information about the use of the composition for the treatment and/or prevention of neoplasia or a pathogen infection.
  • the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of a neoplasm, pathogen infection, or immune disorder or symptoms thereof; precautions; warnings; indications; counter-indications; over-dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • Example 1 Phosphatidylserine Tarsetins Antibody Enhances Anti-Tumor Activity of CAR T cell Therapy in a Mouse Melanoma Model
  • a viable strategy to treat advanced cancers includes transferring of tumor-specific T cells.
  • T cells that recognize tumor antigens can be expanded and reinvigorated ex- vivo.
  • autologous T cells can be genetically modified to express anti -turn or chimeric antigen receptors (CARs).
  • CARs chimeric antigen receptors
  • An important immune checkpoint regulator within tumors is phosphatidylserine (PS), a phospholipid that is exposed on apoptotic cells, tumor cells and tumor endothelium.
  • PS phosphatidylserine
  • M2 anti-inflammatory
  • scFv that specifically binds to Trpl was generated from a murine monoclonal antibody TA99 disclosed in International Patent Publication No. WO96/40249. This scFv was cloned into an eGFP cassette. The binding specificity of this scFv to the Trpl polypeptide was evaluated using a soluble protein comprising this scFv and a Fc domain fused to the scFv.
  • the binding specificity of the scFv-Fc fusion protein to B16 melanoma cells (expressing Trpl), a B16 variant B78H1 (not expressing Trpl), and B78Hl-Trpl (B78H1 engineered to express TRP1 on the surface) was evaluated by flow cytometry.
  • a second generation CAR comprising this Trpl -specific scFv, a transmembrane domain comprising a CD8 polypeptide, an intracellular domain comprising a CD28 polypeptide, an intracellular domain comprising a CD3zeta polypeptide, and a co-stimulatory signaling region that comprises a CD28 polypeptide, was generated, which CAR is referred to as“TA99 CAR”,“TA99-CAR” or“CAR TA99”.
  • TA99 CAR is also disclosed in International Patent Application No. PCT/US17/057098, which is incorporated by reference herein.
  • a retroviral cell packaging line was transfected with the TA99 CAR construct, and an empty vector (MIG) that served as a negative control.
  • the virus supernatant produced by these cells was then used to transduce CD4 and CD8 T cells isolated from the spleen and lymph nodes of naive mice.
  • the transduction rate of the TA99 CAR and empty vector were both about 30%, as shown in Figure 1, upper panels.
  • phosphatidyl serine were sorted and cultured in vitro with B16 melanoma at a 10: 1 effector to target ratio with and without mchlNl 1.
  • plates that were treated with mchlNl 1 showed enhanced killing compared to those treated with the control, indicating that mchlNl 1 directly enhanced the cytolytic activity of antigen specific T cells.
  • Example 2 mchlNl 1 enhanced survival of mice treated with CD1928z CAR T cells in a mouse model of EL4-CD19 thymoma
  • T cell modified to express a CAR that binds to CD 19 (a CAR named“l928z”) on the surface of EL4-mCDl9 tumor cells, in combination with mchlNl 1 improved anti-tumor activity and overall survival in EL4-mCDl9 tumor cells bearing mice.
  • mice were inoculated with lxlO 6 EL4-mCDl9 tumor cells intravenously and then treated with 500,000 CAR T cells intravenously the following day. Mice were treated with either 0.2mg mchlNl 1 or a control IgG
  • the CDl9Delta CAR has a truncated signaling domain and serves as a control.

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Abstract

La présente invention concerne des méthodes et des compositions visant à amplifier la réponse immunitaire aux cancers et aux agents pathogènes. L'invention concerne des agents bloquant la phosphatidylsérine (PS) et des cellules immunoréactives comprenant des récepteurs reconnaissant l'antigène. Plus particulièrement, les méthodes comprennent l'administration à un patient : (a) d'une quantité efficace de cellules immunoréactives ou d'une composition pharmaceutique comprenant celles-ci, les cellules immunoréactives comportant un récepteur de reconnaissance de l'antigène qui se lie à un antigène, et (b) une quantité efficace d'un agent bloquant la PS ; le récepteur de reconnaissance de l'antigène étant, de plus, un récepteur antigénique chimérique (CAR) ou un récepteur de cellule T (TCR).
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US20160362472A1 (en) * 2015-04-08 2016-12-15 Hans Bitter Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car)- expressing cell

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160362472A1 (en) * 2015-04-08 2016-12-15 Hans Bitter Cd20 therapies, cd22 therapies, and combination therapies with a cd19 chimeric antigen receptor (car)- expressing cell

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Title
BELZILE ET AL.: "Antibody targeting of phosphatidylserine for the detection and immunotherapy of cancer", IMMUNOTARGETS THER., vol. 7, 23 January 2018 (2018-01-23), pages 1 - 14, XP055510905, doi:10.2147/ITT.S134834 *
BIRGE ET AL.: "Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer", CELL DEATH DIFFER, vol. 23, no. 6, 2016, pages 962 - 978, XP055636827 *
KELLEHER ET AL.: "Extracellular Vesicles Present in Human Ovarian Tumor Microenvironments Induce a Phosphatidylserine-Dependent Arrest in the T- cell Signaling Cascade", CANCER IMMUNOL RES., vol. 3, no. 11, 2015, pages 1269 - 1278, XP055636830 *

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