WO2021108648A2 - Chimeric receptors to cea and methods of use thereof - Google Patents

Chimeric receptors to cea and methods of use thereof Download PDF

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WO2021108648A2
WO2021108648A2 PCT/US2020/062343 US2020062343W WO2021108648A2 WO 2021108648 A2 WO2021108648 A2 WO 2021108648A2 US 2020062343 W US2020062343 W US 2020062343W WO 2021108648 A2 WO2021108648 A2 WO 2021108648A2
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cell
cells
domain
seq
cea
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WO2021108648A3 (en
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Steven C. Katz
Prajna GUHA
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Prospect CharterCare RWMC, LLC d/b/a Roger Williams Medical Center
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46448—Cancer antigens from embryonic or fetal origin
    • A61K39/464482—Carcinoembryonic antigen [CEA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • C07K14/70564—Selectins, e.g. CD62
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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 A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 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 A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/50—Colon
    • 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
    • C07K16/3007—Carcino-embryonic Antigens
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells

Definitions

  • Carcinoembryonic antigen is a glycosyl phosphatidyl inositol (GPI) cell-surface-anchored glycoprotein that has been associated with a number of cancers including adenocarcinomas, such as colon, lung, breast, stomach, or pancreas cancers. It has been shown to affect many steps of liver metastasis from colorectal cells, and has thus been associated with liver cancers as well. Particularly, five amino acids (Pro-Glu-Leu-Pro-Lys, PELPK) existing between the N and A1 domain of CEA are known to be significant in liver metastasis. CEA is presently used as a diagnostic and prognostic tumor marker in cancer patients.
  • GPI glycosyl phosphatidyl inositol
  • CEA binds with heterogeneous RNA-binding protein M4 (“hnRNP M4” or “nlrCEA”) on the surface of Kupffer cells.
  • hnRNP M4 heterogeneous RNA-binding protein M4
  • nlrCEA heterogeneous RNA-binding protein M4
  • Anti-CEA therapeutic approaches have been attempted, including vaccines, dendritic cells and antibodies. Despite these attempts, their tumor- targe ting and tumor suppressing activities are still limited.
  • Engineered immune cells have been shown to possess desired qualities in therapeutic treatments, particularly in oncology.
  • Two main types of engineered immune cells are those that contain chimeric antigen receptors (termed “CARs” or “CAR-Ts”) and T-cell receptors (“TCRs”). These engineered cells are engineered to endow them with antigen specificity while retaining or enhancing their ability to recognize and kill a target cell.
  • CARs chimeric antigen receptors
  • TCRs T-cell receptors
  • Chimeric antigen receptors may comprise, for example, (i) an antigen-specific component (“antigen binding domain”), (ii) one or more costimulatory domains, and (iii) one or more activating domains. Each domain may be heterogeneous, that is, comprised of sequences derived from different protein chains. Chimeric antigen receptor-expressing immune cells (such as T cells) may be used in various therapies, including cancer therapies. It will be appreciated that costimulating polypeptides as defined herein may be used to enhance the activation of CAR-expressing cells against target antigens, and therefore increase the potency of adoptive immunotherapy. In addition to the CAR-T cells’ ability to recognize and destroy the targeted cells, successful T cell therapy benefits from the CAR-T cells’ ability to persist and maintain the ability to proliferate in response to antigen.
  • the invention relates to engineered immune cells (such as CARs), extracellular binding domains (derived from a naturally occurring CEA receptor) with specificity to CEA.
  • engineered immune cells such as CARs
  • extracellular binding domains derived from a naturally occurring CEA receptor
  • Chimeric antigen receptors of the invention typically comprise: (i) a CEA specific binding molecule, (ii) one or more costimulatory domain, and (iii) one or more activating domain. It will be appreciated that each domain may be heterogeneous, thus comprised of sequences derived from different protein chains.
  • the invention relates to a chimeric antigen receptor comprising a binding molecule that specifically binds to CEA (a “CEA-binding molecule”).
  • this CEA-binding molecule is derived from a receptor that is naturally expressed on a Kupffer cell.
  • the CEA-binding molecule is derived from hnRNP receptor.
  • the binding molecule is derived from hnRNP M4 protein.
  • the binding molecule comprises at least one RRM domain derived from an hnRNP, selected from the group consisting of RRM1, RRM2 and RRM3.
  • the chimeric antigen receptor further comprises at least one costimulatory domain. In further embodiments, the chimeric antigen receptor further comprises at least one activating domain.
  • the costimulatory domain comprises a signaling region of CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function- associated antigen-1 (LFA-1, CDl-la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR
  • the costimulatory domain is derived from 4-1BB. In other embodiments, the costimulatory domain is derived from 0X40. See also Hombach et al., ONCOIMMUNOLOGY, 2012, 1, 4, 458-466. In still other embodiments, the costimulatory domain comprises ICOS as described in Guedan et al. (BLOOD, 2014, 124, 7, 1070-1080) and Shen et al. (JOURNAL OF HEMATOLOGY & ONCOLOGY, 2013, 6, 33, 1-7). In still other embodiments, the costimulatory domain comprises CD27 as described in Song et al. (ONCOIMMUNOLOGY, 2012, 1, 4, 547-549).
  • the CD28 costimulatory domain comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.
  • the CD8 costimulatory domain comprises SEQ ID NO: 16.
  • the activating domain comprises CD3, CD3 zeta, or CD3 zeta having the sequence set forth in SEQ ID NO: 12.
  • the invention relates to a chimeric antigen receptor wherein the costimulatory domain comprises SEQ ID NO: 2 and the activating domain comprises SEQ ID NO: 12.
  • the invention further relates to polynucleotides encoding the chimeric antigen receptors, and vectors comprising the polynucleotides.
  • the vector can be, for example, a retroviral vector, a DNA vector, a plasmid, a RNA vector, an adenoviral vector, an adenovirus associated vector, a lentiviral vector, or any combination thereof.
  • the invention further relates to immune cells comprising the vectors.
  • the lentiviral vector is a pGAR vector.
  • Exemplary immune cells include, but are not limited to T cells, tumor infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, or NK-T cells.
  • TILs tumor infiltrating lymphocytes
  • NK cells TCR-expressing cells
  • dendritic cells dendritic cells
  • NK-T cells NK-T cells
  • the T cells can be autologous, allogeneic, or heterologous.
  • the invention relates to pharmaceutical compositions comprising the immune cells of described herein.
  • the invention relates to extracellular binding molecules (and chimeric antigen receptors comprising these molecules) comprising at a natural receptor to CEA.
  • that receptor is an hnRNP or a fragment thereof.
  • the hnRNP is the M4 isotype.
  • the M4 isotype may be any splice variant of M4, for example Isytope A (729 aa) or Isotype B (690 aa). See, e.g., Carpenter et al, 2006, BIOCHEMICA ET BIOPHYSICA ACTA, 1765, 85-100.
  • the extracellular binding molecule comprises, one or more RRM domains. In various embodiments the extracellular domain comprises at least SEQ ID NO. 1.
  • the invention relates to vectors encoding the polypeptides of the invention and to immune cells comprising these polypeptides.
  • Preferred immune cells include T cells, tumor infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, or NK-T cells.
  • TILs tumor infiltrating lymphocytes
  • NK cells TCR-expressing cells
  • dendritic cells dendritic cells
  • NK-T cells cytoplasmic cells
  • the invention includes a costimulatory domain, such as CD28, CD28T, 0X40, 4-1BB/CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD 33, CD37, CD40, CD 45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 (CD1 la/CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class I molecule, TNF, TNFr, integrin, signaling lymphocytic activation molecule, BTLA, Toll ligand receptor, ICAM-1,
  • the invention further relates to methods of treating a disease or disorder in a subject in need thereof comprising administering to the subject the antigen binding molecules, the CARs, TCRs, polynucleotides, vectors, cells, or compositions according to the invention.
  • Suitable diseases for treatment include, but are not limited to cancer metastasized from a primary tumor in the liver, breast, bladder, lymphoma, kidney, endometrial, cervical, ovarian, colon, rectum, esophagus, lung, pancreas and/or stomach.
  • the subject is diagnosed with unresectable metastatic liver tumors.
  • the subject is diagnosed with unresectable metastatic liver tumors from primary colorectal cancer.
  • the invention relates to a chimeric receptor comprising (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain, (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain.
  • the invention relates to a chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain, (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain.
  • the activating CD3 activating domain is CD3 zeta.
  • the activating CD3 zeta domain comprises the amino acid sequence of SEQ ID NO. 14.
  • the CEA binding domain is a Kupffer cell receptor or a fragment thereof.
  • the CEA binding domain is hnRNP M4 or a fragment thereof.
  • the CEA binding domain comprises the amino acid sequence of SEQ ID NO. 2.
  • the transmembrane domain comprises the amino acid sequence of SEQ ID NO. 4.
  • the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO. 6.
  • the chimeric receptor comprises the amino acid sequence of SEQ ID NO. 15.
  • the invention relates to a polynucleotide encoding a chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain, (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain.
  • the invention relates to a cell comprising a chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain, (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain.
  • the cell is a T cell, an NK cell, a stem cell or a red blood cell.
  • the invention relates to a polypeptide comprising the amino acid sequence of SEQ ID NO. 15. In various embodiments, the invention relates to the nucleotide sequence encoding the polypeptide of SEQ ID NO. 15. In various embodiments, the invention relates to a polypeptide comprising the CEA binding domain of SEQ ID NO. 2, a hinge domain, a transmembrane domain, a costimulatory domain, and a CD3 domain.
  • the costimulatory domain is a signaling region of CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD1 la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT,
  • the costimulatory domain comprises CD28.
  • the CD28 costimulatory domain comprises a sequence that differs at no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residues from the sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.
  • the invention relates to a method of enhancing T cell or an NK cell activity in a mammal comprising introducing into the mammal a T cell or NK cell, which T cell or NK cell comprises a chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain; (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain.
  • the invention relates to a method for treating a mammal suffering from cancer comprising introducing into the mammal a T cell or an NK cell, which T cell or NK cell comprises comprising a chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain; (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain.
  • the invention relates to a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal comprising administering to a mammal an effective amount of a cell genetically modified to express a comprising a chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain, (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain.
  • the mammal is suffering from a cancer metastasized from a primary tumor in the liver, breast, bladder, lymphoma, kidney, endometrial, cervical, ovarian, colon, rectum, esophagus, lung, pancreas and/or stomach.
  • the invention relates to a method of treating a patient suffering from cancer comprising: obtaining a plurality of cells from said patient; pre treating said patient with an agent capable of reducing the endogenous cell population in said patient; transducing said plurality of cells with comprising a chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain; (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain; expanding said transduced calls obtaining a population of said cells in the range of lxlO 6 to lxlO 9 , administering said cells to said patient.
  • a patient is administered an effective amount of one or more steroid compound.
  • FIG. 1 depicts the structure of an embodiment of the disclosed invention. Specifically, it discloses a chimeric T cell receptor encoding an extraceullar domain that binds to CEA, a CD28 extracellular, transmembrane and intracellular domain and a CD3 zeta intracellular costimulatory domain.
  • FIG. 2A illustrates flow cytometry analysis of nlrCEA expression on PG13 vims producing cells (VPC) of untransfected PG13 WT cells; and transfected PG13 nlrCEA VPCs.
  • PG13 nlrCEA unstained cells were used to set gates and voltages.
  • FIG. 2B illustrates Multiplicity of Infection (MOI) was evaluated for the retrovirus generated from PG13 nlrCEA VPCs overnight (o/n) or 8 hours.
  • FIG. 3B illustrates how extracellular staining was performed for Immunofluorescence (IF) to evaluate nlrCEA CAR expression. Untransduced cells were used as a control for nlrCEA CAR expression.
  • IF Immunofluorescence
  • FIG. 4A depicts Flow Cytometric analysis of nlrCEA expression on nlrCEA CAR-T cells (Td) were performed and untransduced (UnTd) cells were used a control.
  • FIG. 4B depicts total RNA isolated from 1 million nlrCEA CAR-T cells and their respective untransduced cells were used as a control. Quantitative PCR was performed using nlrCEA construct specific primer. Untransduced cells did not amplify using the nlrCEA specific primers. GAPDH was used to normalize mRNA expression.
  • FIG. 4C illustrates how western blot analysis was performed using protein lysate from UnTd, Td and Phoenix Eco (Phx Eco) cells were used for nlrCEA expression detection.
  • Phx Eco was used as a positive control as they endogenously express nlrCEA.
  • GAPDH protein was used as a loading control.
  • FIG. 4D depicts how In vitro cytotoxicity was performed at Target to Effector (T:E) ratios of 1:1, 1:5, 1:10 for 4 hours using CEA+ MC38 cells. Untransduced cells were used as a negative control.
  • FIG. 5A is a schematic representation of tumor generation, and treatment timeline. Mice were separated into two treatment groups (T1 and T2) and treated according to the schema depicted with UnTd (control) or nlrCEA CAR-T (Td). IP intraperitoneal.
  • FIG. 5B illustrates how nlrCEA CAR-T Cells were tested in vivo via intraperitoneal (IP) infusion, and peritoneal tumor killing was monitored by changes in bioluminescence. Each line on the plot is representative of the average of five mice.
  • FIG. 5C depicts how lavage was evaluated for nlrCEA CAR-T cells using flow cytometry.
  • FIG. 6 depicts additional embodiments of the present invention.
  • Each construct (Cl - C23 comprise various combinations of the RRM domain of the natural hnRNP (CEA) receptor, hinge domains, transmembrane and signaling domains. Even numbered constructs further comprise a myc domain.
  • FIG. 7 depicts a schematic representation of different CAR constructs.
  • CAR construct’s binding molecule comprises of at least one RNA recognition motif (RRM) domain from the group consisting of RRM1, RRM2 and RRM3 as derived from hnRNP M. Constructs either have CD8 hinge, CD28 (28) or 4- IBB (BB) or a combination of both (28-BB) as costimulatory domain followed by CD3z signaling domain.
  • the left panel denotes CAR constructs that have myc tag (m) for detection, while the right panel contains CAR constructs without the myc tag.
  • Nomenclature for CAR constructs Myc-m, RRM- RNA Recognition Motif, H-hinge, 28-CD28 costimulatory domain, BB- 4-1BB costimulatory domain, 28-BB- CD28 and 4- IBB costimulatory domains.
  • FIG. 8A illustrates how PG13 cells were transfected with mR3-H-28 or mR123-28 or mR123-H-28 to generate virus producing cells (VPC). Multiplicity of Infection (MOI) was evaluated for the retro vims generated from PG13 VPCs two days post transfection (P+2).
  • MOI Multiplicity of Infection
  • FIG. 8B illustrates flow cytometry analysis of myc tag expression performed on the PG13 VPCs of mR123-28, mR123-H-28 and mR3-H-28 to evaluate transfection efficiency.
  • Untransfected PG13 cells (WT) were used to set gates and voltages.
  • Zombie NIR viability dye was used to gate viable cells.
  • FIG. 9A illustrates how viruses harvested from mR3-H-28, mR123-28 and mR123-H-28 PG13 VPCs were used to transduce activated T cells from 2 separate donors (#1 and #2). Three rounds of transductions were performed over two days. Myc expression was evaluated to estimate transduction efficiency of mR3-H-28, mR123-28 and mR123-H-28 CAR- T cells for both donors, four days post transduction. CD3 was used as a marker for T cells. UnTd cells of corresponding donors were used as a control to set gates and voltages.
  • FIG. 9B illustrates how CD4 and CD 8 phenotyping was performed for mR123-28, mR123-H-18 and mR3-H-28 CAR-T cells for both donors, four days post transduction.
  • FIG. 10A depicts how the expansion profile and viability of mR3-H-28, mR123-28 and mR123-H-28 CAR-T were plotted for both donors (#1 and #2).
  • FIG. 10B depicts how the in vitro cytotoxic activity of mR123-28, mR123- H-28 and mR3-H-28 CAR-T cells were evaluated by incubating MC38CEA+ cells with individual CAR-T cells at a ratio of 1:10. MC38 cells were used as a negative control to evaluate CEA-specific cytotoxicity of CAR-T cells. Numbers on the graphs denote P values.
  • FIG. 11A illustrates a schematic of the CAR constructs used for the comparative study of mR123-H-28 with myc less R123-BB, R123-H-BB and R123-H-28-BB.
  • FIG. 11B depicts how viability and expansion graphs of mR123-H-28, R123-BB, R123-H-BB and R123-H-28-BB cells CAR-T cells were plotted for two donors (#5 and #6).
  • FIG. llC depicts how the in vitro cytotoxic activity of mR123-H-28, R123- BB, R123-H-BB and R123-H-28-BB cells were evaluated by incubating MC38CEA+ (target, T) cells with each of the CAR-T cells (E) at T:E ratio of 1:10. MC38 cells were used as a negative control to evaluate CEA-specific cytotoxicity of CAR-T cells. Data represented as mean + SEM. Assay performed in quadruplets. Numbers on the graphs denote P values.
  • FIG. 12 depicts how viruses harvested from mR123-H-28, R123-BB, R123- H-BB and R123-H-28-BB PG13 VPCs were used to transduce activated T cells from 2 separate donors (#5 and #6). Transduction efficiency was evaluated using PELPK reagent along with CD3 for mR123-H-28, R123-BB, R123-H-BB and R123-H-28-BB CAR-T cells, four days post transduction. UnTd cells of corresponding donors were used as a control to set gates and voltages.
  • FIG. 13B depicts how bioluminescence was expressed as fold over day 3.
  • the left panel shows data represented as mean + SEM.
  • the right panel shows the individual data points for each animal in respective groups.
  • FIG. 13C depicts how the T test (2-tailed) was performed for statistical analysis of the multiple groups. Outlined boxes indicate significance (p ⁇ 0.05).
  • FIG. 14A depicts how cells obtained from peritoneal lavage were analyzed for CAR-T persistence using human CD3 and PEFPK reagent. Zombie NIR was used to gate live cells as per the gating strategy. Unstained and isotype control for CD3 was used to set the gates and voltages.
  • FIG. 15 illustrates how immunofluorescence staining was performed on paraffin sections of tumors from UnTd, R123-28 and R123-H-28-BB treated groups. Top panel shows staining of sections with human CD3, murine cleaved caspase-3 and human CD-66 to assess tumor apoptosis. DAPI was used to stain nuclei. Bottom panel shows staining of sections with human CD3, murine Ki67 and human CD-66 to assess proliferation of tumors. Secondary antibody only control staining was performed to show specificity of the primary antibody staining. Images were taken at 20X magnification.
  • chimeric antigen receptors are genetically engineered receptors. These engineered receptors can be readily inserted into and expressed by immune cells, including T cells in accordance with techniques known in the art. With a CAR, a single receptor can be programmed to both recognize a specific antigen and, when bound to that antigen, activate the immune cell to attack and destroy the cell bearing that antigen. When these antigens exist on tumor cells, an immune cell that expresses the CAR can target and kill the tumor cell.
  • CARs can be engineered to bind to an antigen (such as a cell-surface antigen) by incorporating an antigen binding molecule that interacts with that targeted antigen.
  • the antigen binding molecule of the present invention binds to carcinoembryonic antigen (CEA).
  • CEA carcinoembryonic antigen
  • the CEA -binding domain is derived from a receptor found in nature that binds to CEA.
  • the antigen binding molecule is derived from a naturally occurring receptor.
  • the naturally occurring receptor is found on a Kupffer cell.
  • the naturally occurring receptor is hnRNP M or hnRNP M4.
  • the antigen binding protein comprises at least 1 at least 2, at least 3 or more RRM domains derived from hnRNP M or hnRNP M4.
  • the antigen binding domains comprises one or more of the following amino acid sequences: SEQ ID NOs. 67, 69, 71, 72 and 75.
  • the CEA-binding domain is typically contained within the extracellular portion of the CAR such that it is capable of recognizing and binding to CEA.
  • Bispecific and multispecific CARs i.e. immune cells genetically engineered to target more than one molecule are contemplated within the scope of the invention, with specificity to more than one target of interest.
  • a number of extracellular CEA-binding domains may be used:
  • nucleotide sequence of an nlrCEA extracellular domain is set forth in SEQ ID NO. 1:
  • amino acid sequence of the nlrCEA extracellular domain is set forth in SEQ ID NO. 2:
  • Chimeric antigen receptors may incorporate costimulatory (signaling) domains to increase their potency. See U.S. Pat. Nos. 7,741,465, and 6,319,494, as well as Krause et al and Finney et al (supra), Song et al, BLOOD 119:696-706 (2012); Kalos et al, Sci TRANSL. MED. 3:95 (2011); Porter et al, N. ENGL. J. MED. 365:725-33 (2011), and Gross etal, ANNU. REV. PHARMACOL. TOXICOL. 56:59-83 (2016).
  • CD28 is a costimulatory protein found naturally on T-cells.
  • CD28 The complete native amino acid sequence of CD28 is described in NCBI Reference Sequence: NP_006130.1.
  • the complete native CD28 nucleic acid sequence is described in NCBI Reference Sequence: NM_006139.1.
  • a number of CD28 domains have been used in chimeric antigen receptors.
  • the nucleotide sequence of the CD28 transmembrane domain is set forth in SEQ ID NO. 3:
  • nucleotide sequence of the CD28 intracellular signaling domain is set forth in SEQ ID NO. 5:
  • CD28 sequences suitable for use in the invention include the CD28 nucleotide sequence set forth in SEQ ID NO. 7:
  • CD28 nucleotide sequence set forth in SEQ ID NO. 9:
  • Suitable extracellular or transmembrane sequences can be derived from CD8.
  • the nucleotide sequence of a suitable CD8 extracellular and transmembrane domain is set forth in SEQ ID NO. 11:
  • Suitable costimulatory domains within the scope of the invention can be derived from, among other sources, CD28, CD28T, 0X40, 4-1BB/CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD 33, CD37, CD40, CD 45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 (CD1 la/CD18), CD247, CD276 (B7-H3), FIGHT (tumor necrosis factor superfamily
  • CD3 is an element of the T cell receptor on native T cells, and has been shown to be an important intracellular activating element in CARs and TCRs.
  • CD3 is CD3 zeta, the nucleotide sequence of which is set forth in SEQ ID NO: 13:
  • these domains correspond to locations relative to the immune cell.
  • these domains can be part of the (i) extracellular (EC) domain (EC), (ii) the transmembrane (TM) domain, and/or (iii) the intracellular (cytoplasmic) domain (IC).
  • the extracellular domain can further comprise a “hinge domain” (membrane bound or proximal) and a “spacer” domain to achieve optimal spatial orientation or distance to the desired antigen.
  • the intracellular component frequently comprises in part a member of the CD3 family, preferably CD3 zeta, which is capable of activating the T cell upon binding of the antigen binding molecule to its target.
  • the extracellular domain is typically comprised of at least one costimulatory domain as defined herein.
  • the extracellular domain may also contain some or all of a member of the immuno-globulin family such as IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragment thereof.
  • the engineered T cells of the invention comprise a CEA-binding domain, an extracellular domain (which may comprise a “hinge” domain), a transmembrane domain, and an intracellular domain.
  • the intracellular domain comprises at least in part an activating domain, preferably comprised of a CD3 family member such as CD3 zeta, CD3 epsilon, CD3 gamma, or portions thereof.
  • the CEA-binding domain is engineered such that it is located in the extracellular portion of the molecule/construct, such that it is capable of recognizing and binding to its target or targets.
  • Extracellular Domain comprises at minimum an antigen-binding domain capable of recognizing and binding to CEA as described above.
  • the extracellular domain may comprise additional elements that are beneficial for signaling and for an efficient response of lymphocytes to an antigen.
  • Extracellular domains of particular use in this invention may be derived from (i.e., comprise) CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CDl-la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1,
  • extracellular domains often comprise a hinge portion. This is a portion of the extracellular domain proximal to the cell membrane.
  • the extracellular domain may further comprise a spacer region.
  • a variety of hinges can be employed in accordance with the invention, including costimulatory molecules as discussed above, as well as immunoglobulin (Ig) sequences or other suitable molecules to achieve the desired special distance from the target cell.
  • the entire extracellular region comprises a hinge region.
  • the hinge region comprises the extracellular domain of CD28, or a portion thereof as described herein. .
  • the CAR can be designed to comprise a transmembrane domain that is fused or otherwise linked to the extracellular domain of the CAR. It can similarly be fused to the intracellular domain of the CAR.
  • the transmembrane domain that naturally is associated with one of the domains in a CAR is used.
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane- bound or transmembrane protein.
  • Transmembrane regions of particular use in this invention may be derived from (i.e. comprise) CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7,
  • CD27, CD30, CD40 programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CDl-la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a),
  • DAP- 10 Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL- 2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM,
  • short linkers may form linkages between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.
  • the transmembrane domain in the CAR of the invention is a CD8 transmembrane domain.
  • the CD8 transmembrane domain comprises the transmembrane portion of the nucleic acid sequence of SEQ ID NO: 15.
  • the CD8 transmembrane domain comprises the nucleic acid sequence that encodes the transmembrane amino acid sequence contained within SEQ ID NO: 16.
  • the transmembrane domain in the CAR of the invention is the CD28 transmembrane domain.
  • the CD28 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 5.
  • the CD28 transmembrane domain comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 6.
  • the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6.
  • Intracellular (Cytoplasmic) Domain The intracellular (IC, or cytoplasmic) domain of the engineered T cells of the invention can provide activation of at least one of the normal effector functions of the immune cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • suitable intracellular molecules include (i.e., comprise), but are not limited to CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death- 1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CDl-la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS,
  • PD-1 programmed death
  • the cytoplasmic domain of the CAR can be designed to comprise the CD3 zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention.
  • the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region.
  • cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention may be linked to each other in a random or specified order.
  • the cytoplasmic domain is designed to comprise the signaling domain of CD3 zeta and the signaling domain of CD28.
  • the cytoplasmic domain is designed to comprise the signaling domain of CD3 zeta and the signaling domain of 4- IBB.
  • the cytoplasmic domain in the CAR of the invention is designed to comprise a portion of CD28 and CD3 zeta, wherein the cytoplasmic CD28 comprises the nucleic acid sequence set forth in SEQ ID NO: 5 and the amino acid sequence set forth in SEQ ID NO: 6.
  • the CD3 zeta nucleic acid sequence is set forth in SEQ ID NO: 13, and the amino acid sequence is set forth in SEQ ID NO: 14.
  • one preferred orientation of the CARs in accordance with the invention comprises an antigen binding domain (such as a CEA-binding domain) in tandem with at least one costimulatory domain and an activating domain.
  • the costimulatory domain can comprise one or more of an extracellular portion, a transmembrane portion, and an intracellular portion. It will be further appreciated that multiple costimulatory domains can be utilized in tandem.
  • nucleic acids comprising a promoter operably linked to a first polynucleotide encoding an antigen binding molecule, at least one costimulatory molecule, and an activating domain.
  • the nucleic acid construct is contained within a viral vector.
  • the viral vector is selected from the group consisting of retro viral vectors, murine leukemia vims vectors, SFG vectors, adenoviral vectors, lentiviral vectors, adeno-associated vims (AAV) vectors, Herpes vims vectors, and vaccinia virus vectors.
  • the nucleic acid is contained within a plasmid.
  • the invention further relates to isolated polynucleotides encoding the chimeric antigen receptors, and vectors comprising the polynucleotides.
  • Any vector known in the art can be suitable for the present invention.
  • the vector is a viral vector.
  • the vector is a retroviral vector (such as pMSVGl), a DNA vector, a murine leukemia vims vector, an SFG vector, a plasmid, a RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated vector (AAV), a lentiviral vector (such as pGAR), or any combination thereof.
  • the vector sequence is as follows:
  • Suitable additional exemplary vectors include e.g., pBABE-puro, pBABE- neo largeTcDNA, pBABE-hygro-hTERT, pMKO.l GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES Luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pFncEXP, and pFXIN-Fuc.
  • the engineered immune cell is a T cell, tumor infiltrating lymphocyte (TIE), NK cell, TCR-expressing cell, dendritic cell, or NK-T cell.
  • TIE tumor infiltrating lymphocyte
  • NK cell TCR-expressing cell
  • dendritic cell dendritic cell
  • NK-T cell a T cell, tumor infiltrating lymphocyte (TIE), NK cell, TCR-expressing cell, dendritic cell, or NK-T cell.
  • the cell is obtained or prepared from peripheral blood.
  • the cell is obtained or prepared from peripheral blood mononuclear cells (PB- MCs).
  • PB- MCs peripheral blood mononuclear cells
  • the cell is obtained or prepared from bone marrow.
  • the cell is obtained or prepared from umbilical cord blood.
  • the cell is a human cell.
  • the cell is transfected or transduced by the nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., Gene Gun), lipid transfection, polymer transfection, nanoparticles, or polyplexes.
  • a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., Gene Gun), lipid transfection, polymer transfection, nanoparticles, or polyplexes.
  • chimeric antigen receptors are expressed in the engineered immune cells that comprise the nucleic acids of the present application.
  • These chimeric antigen receptors of the present application may comprise, in some embodiments, (i) an antigen binding molecule (such as an scFv), (ii) a transmembrane region, and (iii) a T cell activation molecule or region.
  • an antigen binding molecule such as an scFv
  • a transmembrane region such as an scFv
  • T cell activation molecule or region a T cell activation molecule or region.
  • Antigen binding molecules are within the scope of the invention.
  • CEA-binding domain means any protein that binds a CEA.
  • CEA-Binding Domains include, but are not limited to portions of naturally occurring proteins that bind to CEA, for example, the hnRNP M4 receptors that are expressed on the surface of Kupffer cells. .
  • the CEA-binding domain binds to an CEA on a tumor cell. In some embodiments, the CEA-binding domain binds to CEA on a cell involved in a hyperproliferative disease
  • compositions comprising nlrCEA-CAR T cell together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
  • pharmaceutical compositions will include more than one nlrCEA-CAR T cell.
  • pharmaceutical compositions will include more than one nlrCEA-CAR T cell with different CEA-binding domains wherein the CEA-binding domains bind more than one epitope on CEA.
  • the various CEA binding domains will not compete with one another for binding to CEA.
  • the pharmaceutical composition can be selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, such as orally.
  • a therapeutic composition can be in the form of a pyrogen- free, parenterally acceptable aqueous solution comprising a desired nlrCEA-CAR T Cell, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle.
  • a vehicle for parenteral injection is sterile distilled water in which an antigen binding molecule to CEA, with or without at least one additional therapeutic agent, is formulated as a sterile, isotonic solution, properly preserved.
  • antigen binding molecules will be further understood in view of the definitions and descriptions below.
  • a CEA-binding domain is said to “specifically bind” its target antigen (CEA) when the dissociation constant (K d ) is lxlO 7 M.
  • CEA target antigen
  • the CEA binding domain specifically binds antigen with “high affinity” when the K d is l-5xl0 9 M, and with “very high affinity” when the K d is l-5xlO 10 M.
  • the CEA-binding domain has a K d of 10 9 M.
  • the off-rate is ⁇ 1x10 5 .
  • the CEA-binding domain will bind to human CEA with a K d of between about 10 7 M and 10 13 M, and in yet another embodiment the CEA-binding domain will bind with a K d 1.0-5 x 10 _1° .
  • a CEA-binding domain is said to be “selective” when it binds to one target more tightly than it binds to a second target.
  • neutralizing refers to a CEA-binding domain that binds to a ligand and prevents or reduces the biological effect of that ligand. This can be done, for example, by directly blocking a binding site on the ligand or by binding to the ligand and altering the ligand’s ability to bind through indirect means (such as structural or energetic alterations in the ligand).
  • the term can also denote a CEA-binding domain that prevents the protein to which it is bound from performing a biological function.
  • target refers to a molecule or a portion of a molecule capable of being bound by an antigen binding molecule.
  • a target can have one or more epitopes.
  • Compet when used in the context of CEA-binding domains that compete for the same epitope means competition between CEA-binding domains as determined by an assay in which the CEA binding domain being tested prevents or inhibits (e.g., reduces) specific binding of a reference CEA binding domain to CEA
  • Numerous types of competitive binding assays can be used to determine if one CEA-binding domain competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (Stahli et al, METHODS IN ENZYMOLOGY, 1983, 9, 242253); solid phase direct biotin-avidin EIA (Kirkland et al, J.
  • epitope includes any determinant capable of being bound by the CEA-binding domain, such as a naturally occurring receptor, or a transduced immune cell of the invention.
  • An epitope is a region of CEA that is bound by a CEA-binding domain that targets that antigen, and when the antigen is a protein, includes specific amino acids that directly contact the antigen binding molecule.
  • label refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotin moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods).
  • marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods.
  • the label or marker can also be therapeutic.
  • Various methods of labeling polypeptides and glycoproteins are known in the art and can be used.
  • control switch techniques may be incorporated herein. These techniques may employ the use of dimerization domains and optional activators of such domain dimerization. These techniques include, e.g., those described by Wu et al., Science 2014350 (6258) utilizing FKBP/Rapalog dimerization systems in certain cells, the contents of which are incorporated by reference herein in their entirety. Additional dimerization technology is described in, e.g. , Fegan et al. CHEM. REV. 2010, 110, 3315-3336 as well as U.S. Pat. Nos.
  • dimerization pairs may include cyclosporine- A/cyclophilin, receptor, estrogen/estrogen receptor (optionally using tamoxifen), glucocorticoids/glucocorticoid receptor, tetracycline/tetracycline receptor, vitamin D/vitamin D receptor.
  • dimerization technology can be found in e.g., WO 2014/127261, WO 2015/090229, US 2014/0286987, US 2015/0266973, US 2016/0046700, U.S. Pat. No. 8,486,693, US 2014/0171649, and US 2012/0130076, the contents of which are further incorporated by reference herein in their entirety.
  • native T cells can be (i) removed from a patient, (ii) genetically engineered to express a chimeric antigen receptor (CAR) that binds to at least one tumor antigen (iii) expanded ex vivo into a larger population of engineered T cells, and (iv) reintroduced into the patient.
  • CAR chimeric antigen receptor
  • the engineered T cells After the engineered T cells are reintroduced into the patient, they mediate an immune response against cells expressing the tumor antigen. See, e.g., Krause et al. , J. EXP. MED., Volume 188, No. 4, 1998 (619-626). This immune response includes secretion of IL-2 and other cytokines by T cells, the clonal expansion of T cells recognizing the tumor antigen, and T cell-mediated specific killing of target-positive cells. See, e.g., Hombach et al. , JOURNAL OF IMMUN. 167 : 6123-6131 (2001).
  • the invention therefore comprises a method for treating or preventing a condition associated with undesired and/or elevated CEA levels in a patient, comprising administering to a patient in need thereof an effective amount of at least one nlrCEA-CAR disclosed herein.
  • the invention relates to creating a T cell-mediated immune response in a subject, comprising administering an effective amount of the engineered immune cells of the present application to the subject.
  • the T cell-mediated immune response is directed against a target cell or cells.
  • the engineered immune cell comprises a chimeric antigen receptor (CAR).
  • the target cell is a tumor cell.
  • the invention comprises a method for treating or preventing a malignancy, said method comprising administering to a subject in need thereof an effective amount of at least one isolated antigen binding molecule described herein.
  • the invention comprises a method for treating or preventing a malignancy, said method comprising administering to a subject in need thereof an effective amount of at least one immune cell, wherein the immune cell comprises at least one chimeric antigen receptor.
  • the invention comprises a pharmaceutical composition comprising at least one CEA-binding molecule as described herein and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition further comprises an additional active agent.
  • the subject is diagnosed with a metastatic disease localized to the liver.
  • the metastatic disease is a cancer.
  • the cancer metastasized from a primary tumor in the breast, colon, rectum, esophagus, lung, pancreas and/or stomach.
  • the subject is diagnosed with unresectable metastatic liver tumors.
  • the Subject is diagnosed with unresectable metastatic liver tumors from primary colorectal cancer.
  • the Subject is diagnosed with hepatocellular carcinoma.
  • target doses for CEA CAR-T cells can range from 1 xl0 6 -2xl0 10 cells/kg, preferably 2xl0 6 cells/kg, more preferably. It will be appreciated that doses above and below this range may be appropriate for certain subjects, and appropriate dose levels can be determined by the healthcare provider as needed. Additionally, multiple doses of cells can be provided in accordance with the invention.
  • Also provided are methods for reducing the size of a tumor in a subject comprising administering to the subject an engineered cell of the present invention to the subject, wherein the cell comprises a chimeric antigen receptor, a T cell receptor, or a T cell receptor based chimeric antigen receptor comprising CEA-binding domain binds to an antigen on the tumor.
  • the subject has a solid tumor, or a blood malignancy such as lymphoma or leukemia.
  • the engineered cell is delivered to a tumor bed.
  • the cancer is present in the bone marrow of the subject.
  • the engineered cells are autologous T cells. In some embodiments, the engineered cells are allogeneic T cells. In some embodiments, the engineered cells are heterologous T cells. In some embodiments, the engineered cells of the present application are transfected or transduced in vivo. In other embodiments, the engineered cells are transfected or transduced ex vivo.
  • the methods can further comprise administering one or more chemotherapeutic agent.
  • the chemotherapeutic agent is a lymphodepleting (preconditioning) chemotherapeutic.
  • beneficial preconditioning treatment regimens along with correlative beneficial biomarkers are described in U.S. Patent No. 10,322,146 which are hereby incorporated by reference in their entirety herein. These describe, e.g., methods of conditioning a patient in need of a T cell therapy comprising administering to the patient specified beneficial doses of cyclophosphamide (between 200 mg/m 2 / day and 2000 mg/m 2 /day) and specified doses of fludarabine (between 20 mg/m 2 /day and 900 mg/m 2 /day).
  • a preferred dose regimen involves treating a patient comprising administering daily to the patient about 500 mg/m 2 /day of cyclophosphamide and about 60 mg/m 2 /day of fludarabine for three days prior to administration of a therapeutically effective amount of engineered T cells to the patient.
  • the antigen binding molecule, transduced (or otherwise engineered) cells (such as CARs), and the chemotherapeutic agent are administered each in an amount effective to treat the disease or condition in the subject.
  • compositions comprising CAR-expressing immune effector cells disclosed herein may be administered in conjunction with any number of chemotherapeutic agents.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine resume; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nove
  • alkylating agents such
  • paclitaxel TAXOL®, Bristol-Myers Squibb
  • doxetaxel TAXO-TERE®, Rhone-Poulenc Rorer
  • chlorambucil gemcitabine
  • 6-thioguanine mercaptopurine
  • methotrexate platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as TargretinTM (bexarotene), PanretinTM, (alitretinoin); ONTAKTM (denileukin difti
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • Combinations of chemotherapeutic agents are also administered where appropriate, including, but not limited to CHOP, i.e., Cyclophosphamide (Cytoxan®) Doxorubicin (hydroxy doxorubicin), Vincristine (Oncovin®), and Prednisone.
  • CHOP Cyclophosphamide
  • Doxorubicin hydroxy doxorubicin
  • Vincristine Oncovin®
  • Prednisone Prednisone.
  • the chemotherapeutic agent is administered at the same time or within one week after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered from 1 to 4 weeks or from 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least 1 month before administering the cell or nucleic acid. In some embodiments, the methods further comprise administering two or more chemotherapeutic agents.
  • additional therapeutic agents may be used in conjunction with the compositions described herein.
  • additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab (Tecentriq ® ).
  • Additional therapeutic agents suitable for use in combination with the invention include, but are not limited to, ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (KADCYLA®), imatinib (Gleevec®), cetuximah (Erbitux®), panitumumab (Vectibix®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitini
  • the composition comprising CAR-containing immune can be administered with an anti-inflammatory agent.
  • Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.
  • steroids and glucocorticoids including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, tri
  • Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates.
  • Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride.
  • Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone.
  • Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists, (e.g., etanercept (ENBREL®), adaiimumab (HUMIRA®) and infliximab (REMICADE®)), chemokine inhibitors and adhesion molecule inhibitors.
  • TNF antagonists e.g., etanercept (ENBREL®), adaiimumab (HUMIRA®) and infliximab (REMICADE®
  • the biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules.
  • Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline.
  • compositions described herein are administered in conjunction with a cytokine.
  • cytokine as used herein is meant to refer to proteins released by one cell population that act on another cell as intercellular mediators. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones.
  • growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian- inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin- like growth factor-I and -II; erythropoietin (EPO); osteoinductive
  • the invention comprises an antigen binding molecule that binds to CEA with a K d that is smaller than 100 pM. In some embodiments, the antigen binding molecule binds with a Kd that is smaller than 10 pM. In other embodiments, the antigen binding molecule binds with a K d that is less than 5 pM.
  • the cells Prior to the in vitro manipulation or genetic modification of the immune cells described herein, the cells may be obtained from a subject.
  • the immune cells comprise T cells.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph nodes tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • PBMCs peripheral blood mononuclear cells
  • T cells can be obtained from a unit of blood collected from the subject using any number of techniques known to the skilled person, such as FICOLLTM separation.
  • Cells may preferably be obtained from the circulating blood of an individual by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction, and placed in an appropriate buffer or media for subsequent processing.
  • the cells may be washed with PBS.
  • a washing step may be used, such as by using a semi automated flowthrough centrifuge for example, the CobeTM 2991 cell processor, the Baxter Cyto-MateTM, or the like.
  • the cells may be resuspended in a variety of biocompatible buffers, or other saline solution with or without buffer.
  • the undesired components of the apheresis sample may be removed.
  • T cells are isolated from PBMCs by lysing the red blood cells and depleting the monocytes, for example, using centrifugation through a PERCOLLTM gradient.
  • a specific subpopulation of T cells, such as CD28 + , CD4 + , CD8 + , CD45RA + , and CD45R0 + T cells can be further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method for use herein is cell sorting and/or selection via negative magnetic immun oadh eren ce or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8.
  • Flow cytometry and cell sorting may also be used to isolate cell populations of interest for use in the present invention.
  • PBMCs may be used directly for genetic modification with the immune cells (such as CARs or TCRs) using methods as described herein.
  • T lymphocytes after isolating the PBMCs, T lymphocytes can be further isolated and both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and/or expansion.
  • CD8 + cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of these types of CD8 + cells.
  • the expression of phenotypic markers of central memory T cells include CD45RO, CD62L, CCR7, CD28, CD3, and CD 127 and are negative for granzyme B.
  • central memory T cells are CD45RO+, CD62L+, CD8 T cells.
  • effector T cells are negative for CD62L, CCR7, CD28, and CD 127, and positive for granzyme B and perforin.
  • T cells are further sorted into subpopulations.
  • CD4 + T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.
  • the immune cells can be genetically modified following isolation using known methods, or the immune cells can be activated and expanded (or differ entiated in the case of progenitors) in vitro prior to being genetically modified.
  • the immune cells such as T cells, are genetically modified with the chimeric antigen receptors described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then are activated and/or expanded in vitro.
  • Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Pat. Nos.
  • Such methods include contacting PBMC or isolated T cells with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2.
  • a stimulatory agent and costimulatory agent such as anti-CD3 and anti-CD28 antibodies
  • cytokines such as IL-2.
  • Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a “surrogate” antigen presenting cell (APC).
  • APC antigen presenting cell
  • One example is The Dynabeads® system, a CD3/CD28 activator/stimulator system for physiological activation of human T cells.
  • the T cells may be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Pat. Nos. 6,040,177; 5,827,642; and W02012129514, the contents of which are hereby incorporated by reference in their entirety.
  • PBMCs can further include other cytotoxic lymphocytes such as NK cells or NKT cells.
  • An expression vector carrying the coding sequence of a chimeric receptor as disclosed herein can be introduced into a population of human donor T cells, NK cells or NKT cells.
  • Successfully transduced T cells that carry the expression vector can be sorted using flow cytometry to isolate CD3 positive T cells and then further propagated to increase the number of these CAR expressing T cells in addition to cell activation using anti-CD3 antibodies and IL-2 or other methods known in the art as described elsewhere herein. Standard procedures are used for cryopreservation of T cells expressing the CAR for storage and/or preparation for use in a human subject.
  • the in vitro transduction, culture and/or expansion of T cells are performed in the absence of non human animal derived products such as fetal calf serum and fetal bovine serum.
  • the vector may be introduced into a host cell (an isolated host cell) to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein.
  • the cloning vectors may contain sequence components generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art.
  • the origin of replication may be selected to promote autonomous replication of the vector in the host cell.
  • the present disclosure provides isolated host cells containing the vector provided herein.
  • the host cells containing the vector may be useful in expression or cloning of the polynucleotide contained in the vector.
  • Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells.
  • Suitable prokaryotic cells for this purpose include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobactehaceae such as Escherichia, e.g., E.
  • the vector can be introduced to the host cell using any suitable methods known in the art, including, without limitation, DEAE-dextran mediated delivery, calcium phosphate precipitate method, cationic lipids mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of a vector of interest are well known in the art.
  • a mixture of different expression vectors can be used in genetically modifying a donor population of immune effector cells wherein each vector encodes a different CAR as disclosed herein.
  • the resulting transduced immune effector cells form a mixed population of engineered cells, with a proportion of the engineered cells expressing more than one different CARs.
  • the invention provides a method of storing genetically engineered cells expressing CARs, which target a CEA protein. This involves cryopreserving the immune cells such that the cells remain viable upon thawing. A fraction of the immune cells expressing the CARs can be cryopreserved by methods known in the art to provide a permanent source of such cells for the future treatment of patients afflicted with a malignancy. When needed, the cryopreserved transformed immune cells can be thawed, grown and expanded for more such cells.
  • cryopreserve refers to the preservation of cells by cooling to sub-zero temperatures, such as (typically) 77 Kelvin or — 196° C. (the boiling point of liquid nitrogen). Cryoprotective agents are often used at sub-zero temperatures to prevent the cells being preserved from damage due to freezing at low temperatures or warming to room temperature. Cryopreservative agents and optimal cooling rates can protect against cell injury.
  • Cryoprotective agents which can be used in accordance with the invention include but are not limited to: dimethyl sulfoxide (DMSO) (Lovelock & Bishop, NATURE, 1959, 183, 1394-1395; Ashwood-Smith, NATURE, 1961, 190, 1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, ANN. N.Y. ACAD. SCL, 1960, 85, 576), and polyethylene glycol (Sloviter & Ravdin, NATURE, 1962, 196, 48).
  • the preferred cooling rate is l°-3° C/minute.
  • the term, “substantially pure,” is used to indicate that a given component is present at a high level.
  • the component is desirably the predominant component present in a composition. Preferably it is present at a level of more than 30%, of more than 50%, of more than 75%, of more than 90%, or even of more than 95%, said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration. At very high levels (e.g.
  • the component can be regarded as being in “pure form.”
  • Biologically active substances of the present invention can be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated.
  • the contaminant will be at a low level (e.g., at a level of less than 10%, less than 5%, or less than 1% on the dry weight/dry weight basis set out above).
  • the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a “pharmaceutically acceptable” carrier) in a treatment- effective amount.
  • a medium and container system suitable for administration a “pharmaceutically acceptable” carrier
  • Suitable infusion media can be any isotonic medium formulation, typically normal saline, NormosolTM R (Abbott) or Plasma-LyteTM A (Baxter), but also 5% dextrose in water or Ringer’s lactate can be utilized.
  • the infusion medium can be supplemented with human serum albumin.
  • Desired treatment amounts of cells in the composition is generally at least 2 cells (for example, at least 1 CD8 + central memory T cell and at least 1 CD4 + helper T cell subset) or is more typically greater than 10 2 cells, and up to 10 6 , up to and including 10 8 or 10 9 cells and can be more than 10 10 cells.
  • the number of cells will depend upon the desired use for which the composition is intended, and the type of cells included therein.
  • the density of the desired cells is typically greater than 10 6 cells/ml and generally is greater than 10 7 cells/ml, generally 10 8 cells/ml or greater.
  • the clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 cells.
  • lower numbers of cells in the range of 10 6 /kilogram (10 6 -10 n per patient) may be administered.
  • CAR treatments may be administered multiple times at dosages within these ranges.
  • the cells may be autologous, allogeneic, or heterologous to the patient undergoing therapy.
  • the CAR expressing cell populations of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.
  • Pharmaceutical compositions of the present invention may comprise a CAR or TCR expressing cell population, such as T cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • the pharmaceutical compositions may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylene-diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • An injectable pharmaceutical composition is preferably sterile.
  • adverse events may be minimized by transducing the immune cells (containing one or more CARs) with a suicide gene. It may also be desired to incorporate an inducible “on” or “accelerator” switch into the immune cells. Suitable techniques include use of inducible caspase-9 (U.S. Appl. 2011/0286980) or a thymidine kinase, before, after or at the same time, as the cells are transduced with the CAR construct of the present invention. Additional methods for introducing suicide genes and/or “on” switches include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other techniques known in the art.
  • CEA activity includes any biological effect of CEA.
  • CEA activity includes the ability of CEA to interact or bind to a substrate or receptor.
  • polynucleotide includes both single- stranded and double- stranded nucleotide polymers.
  • the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2’,3’-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphoro-diselenoate, phosphoro-anilothioate, phoshoraniladate and phosphoroamidate.
  • base modifications such as bromouridine and inosine derivatives
  • ribose modifications such as 2’,3’-dideoxyribose
  • internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphoro-diselenoate, phosphoro-anilothioate, phoshoraniladate and phosphoroamidate.
  • oligonucleotide refers to a polynucleotide comprising 200 or fewer nucleotides. Oligonucleotides can be single stranded or double stranded, e.g., for use in the construction of a mutant gene. Oligonucleotides can be sense or antisense oligonucleotides. An oligonucleotide can include a label, including a radiolabel, a fluorescent label, a hapten or an antigenic label, for detection assays. Oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization probes.
  • control sequence refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is ligated. The nature of such control sequences can depend upon the host organism.
  • control sequences for prokaryotes can include a promoter, a ribosomal binding site, and a transcription termination sequence.
  • control sequences for eukaryotes can include promoters comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences, and transcription termination sequence.
  • Control sequences can include leader sequences (signal peptides) and/or fusion partner sequences.
  • operably linked means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions.
  • vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or vims) used to transfer protein coding information into a host cell.
  • expression vector or “expression construct” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control (in conjunction with the host cell) expression of one or more heterologous coding regions operatively linked thereto.
  • An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a coding region operably linked thereto.
  • host cell refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest.
  • the term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.
  • transformation refers to a change in a cell’s genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA.
  • a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques.
  • the transforming DNA can recombine with that of the cell by physically integrating into a chromosome of the cell, or can be maintained transiently as an episomal element without being replicated, or can replicate independently as a plasmid.
  • a cell is considered to have been “stably transformed” when the transforming DNA is replicated with the division of the cell.
  • transfection refers to the uptake of foreign or exogenous DNA by a cell.
  • a number of transfection techniques are well known in the art and are disclosed herein. See, e.g. , Graham et al, 1973, VIROLOGY, 52, 456; Sambrook et al, 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al, 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, Gene, 1981, 13, 197.
  • transduction refers to the process whereby foreign DNA is introduced into a cell via viral vector. See, e.g., Jones et al, Genetics: principles and analysis. (1998), Boston: Jones & Bartlett Publ.
  • polypeptide or “protein” refer to a macromolecule having the amino acid sequence of a protein, including deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence.
  • polypeptide and protein specifically encompass CEA antigen binding molecules, antibodies, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of antigen-binding protein.
  • polypeptide fragment refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion as compared with the full- length native protein. Such fragments can also contain modified amino acids as compared with the native protein.
  • Useful polypeptide fragments include immunologically functional fragments of antigen binding molecules.
  • isolated means (i) free of at least some other proteins with which it would normally be found, (ii) is essentially free of other proteins from the same source, e.g., from the same species, (iii) separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (iv) operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (v) does not occur in nature.
  • a “variant” of a polypeptide comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence.
  • Variants include fusion proteins.
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (/. ⁇ ? ., an “algorithm”).
  • the sequences being compared are typically aligned in a way that gives the largest match between the sequences.
  • One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al, 1984, NUCL. ACID RES. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.).
  • GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined.
  • the sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm).
  • a standard comparison matrix see, e.g., Dayhoff et al.
  • the twenty conventional (e.g., naturally occurring) amino acids and their abbreviations follow conventional usage. See, e.g., Immunology A Synthesis (2nd Edition, Golub and Gren, Eds., Sinauer Assoc., Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose.
  • Stereoisomers e.g., D-amino acids
  • unnatural amino acids such as alpha-, alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid
  • unconventional amino acids include: 4-hydroxyproline, . gamma.
  • -carboxy-glutamate epsilon- N,N,N-trimethyllysine, e-N-acetyllysine, 0-phosphoserine, N-acetylserine, N- formylmethionine, 3-methylhistidine, 5-hydroxylysine, . sigma.
  • -N-methylarginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
  • the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
  • Naturally occurring residues can be divided into classes based on common side chain properties: a) hydrophobic: norleucine, Met, Ala, Val, Leu, lie; b) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; c) acidic: Asp, Glu; d) basic: His, Lys, Arg; e) residues that influence chain orientation: Gly, Pro; and f) aromatic: Trp, Tyr, Phe.
  • non-conservative substitutions can involve the exchange of a member of one of these classes for a member from another class.
  • the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
  • amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. Exemplary amino acid substitutions are set forth in Table 2.
  • derivative refers to a molecule that includes a chemical modification other than an insertion, deletion, or substitution of amino acids (or nucleic acids).
  • derivatives comprise covalent modifications, including, but not limited to, chemical bonding with polymers, lipids, or other organic or inorganic moieties.
  • a chemically modified antigen binding molecule can have a greater circulating half-life than an antigen binding molecule that is not chemically modified.
  • a derivative antigen binding molecule is covalently modified to include one or more water soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.
  • Peptide analogs are commonly used in the pharmaceutical industry as non peptide drugs with properties analogous to those of the template peptide. These types of non peptide compound are termed “peptide mimetics” or “peptidomimetics.” Fauchere, J., ADV. DRUG RES., 15:29 (1986); Veber & Freidinger, TINS, p.392 (1985); and Evans et al, J. MED. CHEM., 30:1229 (1987), which are incorporated herein by reference for any purpose.
  • terapéuticaally effective amount refers to the amount of a nlrCEA -CAR T cells determined to produce a therapeutic response in a mammal. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art.
  • patient and “subject” are used interchangeably and include human and non-human animal subjects as well as those with formally diagnosed disorders, those without formally recognized disorders, those receiving medical attention, those at risk of developing the disorders, etc.
  • treat and “treatment” includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors.
  • prevent does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.
  • Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g. , electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer’ s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g. , Sambrook et al. , Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
  • CD28 AA Intracellular Domain CD28 AA Intracellular Domain
  • CD28 AA CD28 AA
  • CD8 AA extracellular & transmembrane Domain CD8 AA extracellular & transmembrane Domain
  • CRMMN GMKLS GREID VRIDRN ALDPKS AKPTTTPAPRPPTPAPTI AS QPLS LRPE

Abstract

Disclosed herein are chimeric antigen receptors that recognize and bind to CEA and methods of their use in the treatment of various cancers. Various embodiments the extracellular domain of the chimeric antigen receptors described herein, comprise various RRM domains found on naturally occurring is hnRNP M4 receptor. The chimeric antigen receptors can be used to treat various disorders including cancers involving CEA-expressing tumors.

Description

CHIMERIC RECEPTORS TO CEA AND METHODS OF USE THEREOF
BACKGROUND OF THE INVENTION
[001] Carcinoembryonic antigen (CEA) is a glycosyl phosphatidyl inositol (GPI) cell-surface-anchored glycoprotein that has been associated with a number of cancers including adenocarcinomas, such as colon, lung, breast, stomach, or pancreas cancers. It has been shown to affect many steps of liver metastasis from colorectal cells, and has thus been associated with liver cancers as well. Particularly, five amino acids (Pro-Glu-Leu-Pro-Lys, PELPK) existing between the N and A1 domain of CEA are known to be significant in liver metastasis. CEA is presently used as a diagnostic and prognostic tumor marker in cancer patients.
[002] In the liver, CEA binds with heterogeneous RNA-binding protein M4 (“hnRNP M4” or “nlrCEA”) on the surface of Kupffer cells. See, e.g. , Bajenova el al. , J BIOL CHEM, 2001 276(33):31067-31073. At least two known isoforms of M4 exist, both of which are capable of binding CEA. Bajenova et al, EXP. CELL RES., 2003, 292:282-291. Like most hnRNP proteins, the M4 isoforms primarily act as nuclear protein that bind to RNA, except when expressed in Kupffer cells. See, e.g. , Thomas et al. , CLIN. EXP. METASTASIS, 2011, 28:923-932. The role that this interaction between hnRNP M4 and CEA plays in liver metastasis is not well understood.
[003] Anti-CEA therapeutic approaches have been attempted, including vaccines, dendritic cells and antibodies. Despite these attempts, their tumor- targe ting and tumor suppressing activities are still limited.
[004] Engineered immune cells have been shown to possess desired qualities in therapeutic treatments, particularly in oncology. Two main types of engineered immune cells are those that contain chimeric antigen receptors (termed “CARs” or “CAR-Ts”) and T-cell receptors (“TCRs”). These engineered cells are engineered to endow them with antigen specificity while retaining or enhancing their ability to recognize and kill a target cell.
Chimeric antigen receptors may comprise, for example, (i) an antigen- specific component (“antigen binding domain”), (ii) one or more costimulatory domains, and (iii) one or more activating domains. Each domain may be heterogeneous, that is, comprised of sequences derived from different protein chains. Chimeric antigen receptor-expressing immune cells (such as T cells) may be used in various therapies, including cancer therapies. It will be appreciated that costimulating polypeptides as defined herein may be used to enhance the activation of CAR-expressing cells against target antigens, and therefore increase the potency of adoptive immunotherapy. In addition to the CAR-T cells’ ability to recognize and destroy the targeted cells, successful T cell therapy benefits from the CAR-T cells’ ability to persist and maintain the ability to proliferate in response to antigen.
[005] An effective method for CEA targeting CAR-T cells is desired.
SUMMARY OF THE INVENTION
[006] The invention relates to engineered immune cells (such as CARs), extracellular binding domains (derived from a naturally occurring CEA receptor) with specificity to CEA.
[007] Chimeric antigen receptors of the invention typically comprise: (i) a CEA specific binding molecule, (ii) one or more costimulatory domain, and (iii) one or more activating domain. It will be appreciated that each domain may be heterogeneous, thus comprised of sequences derived from different protein chains.
[008] In some embodiments, the invention relates to a chimeric antigen receptor comprising a binding molecule that specifically binds to CEA (a “CEA-binding molecule”). In various embodiments, this CEA-binding molecule is derived from a receptor that is naturally expressed on a Kupffer cell. In various embodiments, the CEA-binding molecule is derived from hnRNP receptor. In various embodiments, the binding molecule is derived from hnRNP M4 protein. In various embodiments, the binding molecule comprises at least one RRM domain derived from an hnRNP, selected from the group consisting of RRM1, RRM2 and RRM3.
[009] In other embodiments, the chimeric antigen receptor further comprises at least one costimulatory domain. In further embodiments, the chimeric antigen receptor further comprises at least one activating domain.
[0010] In certain embodiments the costimulatory domain comprises a signaling region of CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function- associated antigen-1 (LFA-1, CDl-la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof.
[0011] In some embodiments, the costimulatory domain is derived from 4-1BB. In other embodiments, the costimulatory domain is derived from 0X40. See also Hombach et al., ONCOIMMUNOLOGY, 2012, 1, 4, 458-466. In still other embodiments, the costimulatory domain comprises ICOS as described in Guedan et al. (BLOOD, 2014, 124, 7, 1070-1080) and Shen et al. (JOURNAL OF HEMATOLOGY & ONCOLOGY, 2013, 6, 33, 1-7). In still other embodiments, the costimulatory domain comprises CD27 as described in Song et al. (ONCOIMMUNOLOGY, 2012, 1, 4, 547-549).
[0012] In certain embodiments, the CD28 costimulatory domain comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In additional embodiments, the CD8 costimulatory domain comprises SEQ ID NO: 16. In further embodiments, the activating domain comprises CD3, CD3 zeta, or CD3 zeta having the sequence set forth in SEQ ID NO: 12.
[0013] In other embodiments, the invention relates to a chimeric antigen receptor wherein the costimulatory domain comprises SEQ ID NO: 2 and the activating domain comprises SEQ ID NO: 12.
[0014] The invention further relates to polynucleotides encoding the chimeric antigen receptors, and vectors comprising the polynucleotides. The vector can be, for example, a retroviral vector, a DNA vector, a plasmid, a RNA vector, an adenoviral vector, an adenovirus associated vector, a lentiviral vector, or any combination thereof. The invention further relates to immune cells comprising the vectors. In some embodiments, the lentiviral vector is a pGAR vector.
[0015] Exemplary immune cells include, but are not limited to T cells, tumor infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, or NK-T cells. The T cells can be autologous, allogeneic, or heterologous. In other embodiments, the invention relates to pharmaceutical compositions comprising the immune cells of described herein.
[0016] In certain embodiments, the invention relates to extracellular binding molecules (and chimeric antigen receptors comprising these molecules) comprising at a natural receptor to CEA. In various embodiments, that receptor is an hnRNP or a fragment thereof. In various embodiments the hnRNP is the M4 isotype. The M4 isotype may be any splice variant of M4, for example Isytope A (729 aa) or Isotype B (690 aa). See, e.g., Carpenter et al, 2006, BIOCHEMICA ET BIOPHYSICA ACTA, 1765, 85-100. In various embodiments, the extracellular binding molecule comprises, one or more RRM domains. In various embodiments the extracellular domain comprises at least SEQ ID NO. 1.
[0017] In other embodiments, the invention relates to vectors encoding the polypeptides of the invention and to immune cells comprising these polypeptides. Preferred immune cells include T cells, tumor infiltrating lymphocytes (TILs), NK cells, TCR-expressing cells, dendritic cells, or NK-T cells. The T cells may be autologous, allogeneic, or heterologous.
[0018] In other embodiments, the invention includes a costimulatory domain, such as CD28, CD28T, 0X40, 4-1BB/CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD 33, CD37, CD40, CD 45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 (CD1 la/CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class I molecule, TNF, TNFr, integrin, signaling lymphocytic activation molecule, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl-ld, ITGAE, CD103, ITGAL, CD1- 1 a, LFA- 1, ITGAM, CD1- 1 b, ITGAX, CDl-lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD83 ligand, or fragments or combinations thereof. Preferred costimulatory domains are recited herein below.
[0019] The invention further relates to methods of treating a disease or disorder in a subject in need thereof comprising administering to the subject the antigen binding molecules, the CARs, TCRs, polynucleotides, vectors, cells, or compositions according to the invention. Suitable diseases for treatment include, but are not limited to cancer metastasized from a primary tumor in the liver, breast, bladder, lymphoma, kidney, endometrial, cervical, ovarian, colon, rectum, esophagus, lung, pancreas and/or stomach. In other embodiments, the subject is diagnosed with unresectable metastatic liver tumors. In yet other embodiments, the subject is diagnosed with unresectable metastatic liver tumors from primary colorectal cancer.
[0020] In various embodiments, the invention relates to a chimeric receptor comprising (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain, (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain.
[0021] In various embodiments, the invention relates to a chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain, (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain. In various embodiments, the activating CD3 activating domain is CD3 zeta. In various embodiments, the activating CD3 zeta domain comprises the amino acid sequence of SEQ ID NO. 14. In various embodiments, the CEA binding domain is a Kupffer cell receptor or a fragment thereof. In various embodiments, the CEA binding domain is hnRNP M4 or a fragment thereof. In various embodiments, the CEA binding domain comprises the amino acid sequence of SEQ ID NO. 2. In various embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO. 4. In various embodiments, the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO. 6. In various embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO. 15.
[0022] In various embodiments, the invention relates to a polynucleotide encoding a chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain, (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain.
[0023] In various embodiments, the invention relates to a cell comprising a chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain, (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain. In various embodiments, the cell is a T cell, an NK cell, a stem cell or a red blood cell.
[0024] In various embodiments, the invention relates to a polypeptide comprising the amino acid sequence of SEQ ID NO. 15. In various embodiments, the invention relates to the nucleotide sequence encoding the polypeptide of SEQ ID NO. 15. In various embodiments, the invention relates to a polypeptide comprising the CEA binding domain of SEQ ID NO. 2, a hinge domain, a transmembrane domain, a costimulatory domain, and a CD3 domain. In various embodiments, the costimulatory domain is a signaling region of CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD1 la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof. In various embodiments, the costimulatory domain comprises CD28. In various embodiments, the CD28 costimulatory domain comprises a sequence that differs at no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residues from the sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.
[0025] In various embodiments, the invention relates to a method of enhancing T cell or an NK cell activity in a mammal comprising introducing into the mammal a T cell or NK cell, which T cell or NK cell comprises a chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain; (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain.
[0026] In various embodiments, the invention relates to a method for treating a mammal suffering from cancer comprising introducing into the mammal a T cell or an NK cell, which T cell or NK cell comprises comprising a chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain; (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain.
[0027] In various embodiments, the invention relates to a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal comprising administering to a mammal an effective amount of a cell genetically modified to express a comprising a chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain, (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain.
[0028] In various embodiments, the mammal is suffering from a cancer metastasized from a primary tumor in the liver, breast, bladder, lymphoma, kidney, endometrial, cervical, ovarian, colon, rectum, esophagus, lung, pancreas and/or stomach.
[0029] In various embodiments, the invention relates to a method of treating a patient suffering from cancer comprising: obtaining a plurality of cells from said patient; pre treating said patient with an agent capable of reducing the endogenous cell population in said patient; transducing said plurality of cells with comprising a chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain; (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain; expanding said transduced calls obtaining a population of said cells in the range of lxlO6 to lxlO9, administering said cells to said patient.
[0030] In various embodiments of the invention, a patient is administered an effective amount of one or more steroid compound.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1 depicts the structure of an embodiment of the disclosed invention. Specifically, it discloses a chimeric T cell receptor encoding an extraceullar domain that binds to CEA, a CD28 extracellular, transmembrane and intracellular domain and a CD3 zeta intracellular costimulatory domain.
[0032] FIG. 2A illustrates flow cytometry analysis of nlrCEA expression on PG13 vims producing cells (VPC) of untransfected PG13 WT cells; and transfected PG13 nlrCEA VPCs. PG13 nlrCEA unstained cells were used to set gates and voltages.
[0033] FIG. 2B illustrates Multiplicity of Infection (MOI) was evaluated for the retrovirus generated from PG13 nlrCEA VPCs overnight (o/n) or 8 hours.
[0034] FIG. 3 A illustrates how the retrovirus from PG13 nlrCEA VPCs harvested after 8 hrs was used to transduce activated T cells (n=3). Three rounds of transductions were performed and cells were counted every 48 hrs and expanded. Expansion curves and viabilities for n=3 nlrCEA CAR-T cells are shown.
[0035] FIG. 3B illustrates how extracellular staining was performed for Immunofluorescence (IF) to evaluate nlrCEA CAR expression. Untransduced cells were used as a control for nlrCEA CAR expression.
[0036] FIG. 4A depicts Flow Cytometric analysis of nlrCEA expression on nlrCEA CAR-T cells (Td) were performed and untransduced (UnTd) cells were used a control.
[0037] FIG. 4B depicts total RNA isolated from 1 million nlrCEA CAR-T cells and their respective untransduced cells were used as a control. Quantitative PCR was performed using nlrCEA construct specific primer. Untransduced cells did not amplify using the nlrCEA specific primers. GAPDH was used to normalize mRNA expression.
[0038] FIG. 4C illustrates how western blot analysis was performed using protein lysate from UnTd, Td and Phoenix Eco (Phx Eco) cells were used for nlrCEA expression detection. Phx Eco was used as a positive control as they endogenously express nlrCEA. GAPDH protein was used as a loading control.
[0039] FIG. 4D depicts how In vitro cytotoxicity was performed at Target to Effector (T:E) ratios of 1:1, 1:5, 1:10 for 4 hours using CEA+ MC38 cells. Untransduced cells were used as a negative control.
[0040] FIG. 5A is a schematic representation of tumor generation, and treatment timeline. Mice were separated into two treatment groups (T1 and T2) and treated according to the schema depicted with UnTd (control) or nlrCEA CAR-T (Td). IP intraperitoneal.
[0041] FIG. 5B illustrates how nlrCEA CAR-T Cells were tested in vivo via intraperitoneal (IP) infusion, and peritoneal tumor killing was monitored by changes in bioluminescence. Each line on the plot is representative of the average of five mice.
[0042] FIG. 5C depicts how lavage was evaluated for nlrCEA CAR-T cells using flow cytometry. [0043] FIG. 6 depicts additional embodiments of the present invention. Each construct (Cl - C23 comprise various combinations of the RRM domain of the natural hnRNP (CEA) receptor, hinge domains, transmembrane and signaling domains. Even numbered constructs further comprise a myc domain.
[0044] FIG. 7 depicts a schematic representation of different CAR constructs. CAR construct’s binding molecule comprises of at least one RNA recognition motif (RRM) domain from the group consisting of RRM1, RRM2 and RRM3 as derived from hnRNP M. Constructs either have CD8 hinge, CD28 (28) or 4- IBB (BB) or a combination of both (28-BB) as costimulatory domain followed by CD3z signaling domain. The left panel denotes CAR constructs that have myc tag (m) for detection, while the right panel contains CAR constructs without the myc tag. Nomenclature for CAR constructs: Myc-m, RRM- RNA Recognition Motif, H-hinge, 28-CD28 costimulatory domain, BB- 4-1BB costimulatory domain, 28-BB- CD28 and 4- IBB costimulatory domains.
[0045] FIG. 8A illustrates how PG13 cells were transfected with mR3-H-28 or mR123-28 or mR123-H-28 to generate virus producing cells (VPC). Multiplicity of Infection (MOI) was evaluated for the retro vims generated from PG13 VPCs two days post transfection (P+2).
[0046] FIG. 8B illustrates flow cytometry analysis of myc tag expression performed on the PG13 VPCs of mR123-28, mR123-H-28 and mR3-H-28 to evaluate transfection efficiency. Untransfected PG13 cells (WT) were used to set gates and voltages. Zombie NIR viability dye was used to gate viable cells.
[0047] FIG. 9A illustrates how viruses harvested from mR3-H-28, mR123-28 and mR123-H-28 PG13 VPCs were used to transduce activated T cells from 2 separate donors (#1 and #2). Three rounds of transductions were performed over two days. Myc expression was evaluated to estimate transduction efficiency of mR3-H-28, mR123-28 and mR123-H-28 CAR- T cells for both donors, four days post transduction. CD3 was used as a marker for T cells. UnTd cells of corresponding donors were used as a control to set gates and voltages.
[0048] FIG. 9B illustrates how CD4 and CD 8 phenotyping was performed for mR123-28, mR123-H-18 and mR3-H-28 CAR-T cells for both donors, four days post transduction.
[0049] FIG. 10A depicts how the expansion profile and viability of mR3-H-28, mR123-28 and mR123-H-28 CAR-T were plotted for both donors (#1 and #2).
[0050] FIG. 10B depicts how the in vitro cytotoxic activity of mR123-28, mR123- H-28 and mR3-H-28 CAR-T cells were evaluated by incubating MC38CEA+ cells with individual CAR-T cells at a ratio of 1:10. MC38 cells were used as a negative control to evaluate CEA-specific cytotoxicity of CAR-T cells. Numbers on the graphs denote P values.
[0051] FIG. 11A illustrates a schematic of the CAR constructs used for the comparative study of mR123-H-28 with myc less R123-BB, R123-H-BB and R123-H-28-BB.
[0052] FIG. 11B depicts how viability and expansion graphs of mR123-H-28, R123-BB, R123-H-BB and R123-H-28-BB cells CAR-T cells were plotted for two donors (#5 and #6).
[0053] FIG. llC depicts how the in vitro cytotoxic activity of mR123-H-28, R123- BB, R123-H-BB and R123-H-28-BB cells were evaluated by incubating MC38CEA+ (target, T) cells with each of the CAR-T cells (E) at T:E ratio of 1:10. MC38 cells were used as a negative control to evaluate CEA-specific cytotoxicity of CAR-T cells. Data represented as mean + SEM. Assay performed in quadruplets. Numbers on the graphs denote P values.
[0054] FIG. 12 depicts how viruses harvested from mR123-H-28, R123-BB, R123- H-BB and R123-H-28-BB PG13 VPCs were used to transduce activated T cells from 2 separate donors (#5 and #6). Transduction efficiency was evaluated using PELPK reagent along with CD3 for mR123-H-28, R123-BB, R123-H-BB and R123-H-28-BB CAR-T cells, four days post transduction. UnTd cells of corresponding donors were used as a control to set gates and voltages.
[0055] FIG. 13A illustrates a schematic representation of the timeline of intraperitoneal (IP) tumor generation in SCID mice, IVIS imaging (arrows) and CAR-T/UnTd/ PBS treatments. Number of mice used for each group are as follows: PBS (n=5), UnTd (n=8), R123-28 (n=12) and R123-H-28-BB (n=5).
[0056] FIG. 13B depicts how bioluminescence was expressed as fold over day 3. The left panel shows data represented as mean + SEM. The right panel shows the individual data points for each animal in respective groups.
[0057] FIG. 13C depicts how the T test (2-tailed) was performed for statistical analysis of the multiple groups. Outlined boxes indicate significance (p<0.05).
[0058] FIG. 14A depicts how cells obtained from peritoneal lavage were analyzed for CAR-T persistence using human CD3 and PEFPK reagent. Zombie NIR was used to gate live cells as per the gating strategy. Unstained and isotype control for CD3 was used to set the gates and voltages.
[0059] FIG. 14B depicts how CAR-T cell detection was performed on peritoneal lavage cells isolated from UnTd (n=3), R123-28 (n=2), R123-H-28-BB (n=5). Data are plotted as mean ± SEM. [0060] FIG. 15 illustrates how immunofluorescence staining was performed on paraffin sections of tumors from UnTd, R123-28 and R123-H-28-BB treated groups. Top panel shows staining of sections with human CD3, murine cleaved caspase-3 and human CD-66 to assess tumor apoptosis. DAPI was used to stain nuclei. Bottom panel shows staining of sections with human CD3, murine Ki67 and human CD-66 to assess proliferation of tumors. Secondary antibody only control staining was performed to show specificity of the primary antibody staining. Images were taken at 20X magnification.
DETAILED DESCRIPTION OF THE INVENTION
[0061] It will be appreciated that chimeric antigen receptors (CARs) are genetically engineered receptors. These engineered receptors can be readily inserted into and expressed by immune cells, including T cells in accordance with techniques known in the art. With a CAR, a single receptor can be programmed to both recognize a specific antigen and, when bound to that antigen, activate the immune cell to attack and destroy the cell bearing that antigen. When these antigens exist on tumor cells, an immune cell that expresses the CAR can target and kill the tumor cell.
[0062] Antigen Binding Domain. CARs can be engineered to bind to an antigen (such as a cell-surface antigen) by incorporating an antigen binding molecule that interacts with that targeted antigen. Preferably, the antigen binding molecule of the present invention binds to carcinoembryonic antigen (CEA). Preferably, the CEA -binding domain is derived from a receptor found in nature that binds to CEA. In certain aspects, the antigen binding molecule is derived from a naturally occurring receptor. In certain embodiments, the naturally occurring receptor is found on a Kupffer cell. In certain embodiments, the naturally occurring receptor is hnRNP M or hnRNP M4. In various embodiments, the antigen binding protein comprises at least 1 at least 2, at least 3 or more RRM domains derived from hnRNP M or hnRNP M4. In various embodiments, the antigen binding domains comprises one or more of the following amino acid sequences: SEQ ID NOs. 67, 69, 71, 72 and 75. It will be appreciated that the CEA-binding domain is typically contained within the extracellular portion of the CAR such that it is capable of recognizing and binding to CEA. Bispecific and multispecific CARs (i.e. immune cells genetically engineered to target more than one molecule) are contemplated within the scope of the invention, with specificity to more than one target of interest.
[0063] A number of extracellular CEA-binding domains may be used:
[0064] The nucleotide sequence of an nlrCEA extracellular domain is set forth in SEQ ID NO. 1:
ATGGCCAATCCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTT
GATGTGAAATGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAG
GTAACATACGTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGT
GCTGTTGTTGAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGT
CCTAAACAAGCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATC
CTGATGGTGAACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGA
AGCACAGTATTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTG
AAGGAAGTATTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAA
GATAAAGATGGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCC
ATTGAAGCTGTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTTGATA
GACCAATGCACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGG
GTGGCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTC
ACATGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTAC
GCCGACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAA
GTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCAT
GAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGA
TCCCAAA
[0065] The amino acid sequence of the nlrCEA extracellular domain is set forth in SEQ ID NO. 2:
M ANPTKRYRAFITNIPFD VKW QS LKDLVKEKV GE VT Y VELLMD AEGKSRGC A
VVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHARRAMQKAGRLGST
VFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKSRGIGTVTFEQSIEA
VQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQIFVRNLPFDFTWKM
LKD KFNECGH VLY ADIKMEN GKS KGCG V VKFESPE V AER ACRMMN GMKLS G
REIDVRIDRNALDPK
[0066] Costimulatory Domains. Chimeric antigen receptors may incorporate costimulatory (signaling) domains to increase their potency. See U.S. Pat. Nos. 7,741,465, and 6,319,494, as well as Krause et al and Finney et al (supra), Song et al, BLOOD 119:696-706 (2012); Kalos et al, Sci TRANSL. MED. 3:95 (2011); Porter et al, N. ENGL. J. MED. 365:725-33 (2011), and Gross etal, ANNU. REV. PHARMACOL. TOXICOL. 56:59-83 (2016). For example, CD28 is a costimulatory protein found naturally on T-cells. The complete native amino acid sequence of CD28 is described in NCBI Reference Sequence: NP_006130.1. The complete native CD28 nucleic acid sequence is described in NCBI Reference Sequence: NM_006139.1. [0067] A number of CD28 domains have been used in chimeric antigen receptors. [0068] The nucleotide sequence of the CD28 transmembrane domain is set forth in SEQ ID NO. 3:
TTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCG
TCACCGTGGCTTTTATAATCTTCTGGGTT
[0069] The amino acid sequence of the CD28 transmembrane domain is set forth in
SEQ ID NO. 4:
FWVLVVV GGVLACY SLLVTV AFIIFWV
[0070] The nucleotide sequence of the CD28 intracellular signaling domain is set forth in SEQ ID NO. 5:
AGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCA
CGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCAC
CACCTAGAGATTTCGCTGCCTATCGGAGC
[0071] The amino acid sequence of the CD28 intracellular signaling domain is set forth in SEQ ID NO. 6:
RSKRSRLLHSD YMNMTPRRPGPTRKHY QPY APPRDFAA YRS [0072] Additional CD28 sequences suitable for use in the invention include the CD28 nucleotide sequence set forth in SEQ ID NO. 7:
ATTGAGGTGATGTATCCACCGCCTTACCTGGATAACGAAAAGAGTAACGGT
ACCATCATTCACGTGAAAGGTAAACACCTGTGTCCTTCTCCCC
TCTTCCCCGGGCCATCAAAGCCC
[0073] The corresponding amino acid sequence is set forth in SEQ ID NO. 8: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP [0074] Additional CD28 sequences suitable for use in the invention include the CD28 nucleotide sequence set forth in SEQ ID NO. 9:
GGACCGGTAGGCTGCGAAGTCGCGTGGTGGGGCATAGGGCTGGTAATGCTT
GCGGGTGGGCCCGGGGCGGCGGGGAGTCATGTTCATGTAGTCACTGTGCAG
GAGCCGGCTCCTCTTACTCCTCACCCAGAAAATAATAAAGGCCACTGTTAC
TAGCAAGCTATAGCAAGCCAGGACTCCACCAACCACCACCAGCA
[0075] The corresponding amino acid sequence is set forth in SEQ ID NO. 10:
PKVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKH
YQPY APPRDFAA YRS
[0076] Other suitable extracellular or transmembrane sequences can be derived from CD8. The nucleotide sequence of a suitable CD8 extracellular and transmembrane domain is set forth in SEQ ID NO. 11:
GCTGCAGCATTGAGCAACTCAATAATGTATTTTAGTCACTTTGTACCAGTGT
TCTTGCCGGCT A AGCCT ACT ACC AC ACCCGCTCC ACGGCC ACCT ACCCC AGC
TCCTACCATCGCTTCACAGCCTCTGTCCCTGCGCCCAGAGGCTTGCCGACCG
GCCGCAGGGGGCGCTGTTCATACCAGAGGACTGGATTTCGCCTGCGATATC
TATATCTGGGCACCCCTGGCCGGAACCTCCGGCGTACTCCTGCTGTCCCTGG
TCATCACGCTCTATTGTAATCACAGGAAC
[0077] The corresponding amino acid sequence is set forth in SEQ ID NO. 12: AAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASOPLSLRPEACRPAAGG AVHTRGFDFACDIYIWAPFAGTCGVFFFSFVITFYCNHRN [0078] Suitable costimulatory domains within the scope of the invention can be derived from, among other sources, CD28, CD28T, 0X40, 4-1BB/CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD 33, CD37, CD40, CD 45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 (CD1 la/CD18), CD247, CD276 (B7-H3), FIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class I molecule, TNF, TNFr, integrin, signaling lymphocytic activation molecule, BTFA, Toll ligand receptor, ICAM-1, B7-H3, CD5, ICAM-1, GITR, BAFFR, FIGHT, HVEM (FIGHTR), KIRDS2, SFAMF7, NKp80 (KFRF1), NKp44, NKp30, NKp46, CD 19, CD4, CD8alpha, CD8beta, IF-2R beta, IF-2R gamma, IF-7R alpha, ITGA4, VFA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VFA-6, CD49f, ITGAD, CD1 -Id, ITGAE, CD103, ITGAE, CD1- 1 a, FFA-1, ITGAM, CD1- 1 b, ITGAX, CD1- 1 c, ITGB1, CD29, ITGB2, CD 18, FFA-1, ITGB7, NKG2D, TNFR2, TRANCE/ RANKE, DNAM1 (CD226), SFAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Fy9 (CD229), CD160 (BY55), PSGF1, CD100 (SEMA4D), CD69, SFAMF6 (NTB-A, Fyl08), SEAM (SEAMF1, CD150, IPO-3), BEAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD 19a, CD 83 ligand, or fragments or combinations thereof.
[0079] Activating Domains. CD3 is an element of the T cell receptor on native T cells, and has been shown to be an important intracellular activating element in CARs and TCRs. In a preferred embodiment, the CD3 is CD3 zeta, the nucleotide sequence of which is set forth in SEQ ID NO: 13:
AGAGTGAAGTTCAGCCGGTCCGCAGACGCCCCCGCGTACCAGCAGGGCCA
GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATG
TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGA AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT
GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA
AGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCT
ACGACGCCCTTCACATGCAAGCTTTGCCCCCTCGC
[0080] The corresponding amino acid of intracellular CD3 zeta is set forth in SEQ ID NO: 14:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDTYD ALHMQALPPR
Domain Orientation
[0081] Structurally, it will appreciated that these domains correspond to locations relative to the immune cell. Thus, these domains can be part of the (i) extracellular (EC) domain (EC), (ii) the transmembrane (TM) domain, and/or (iii) the intracellular (cytoplasmic) domain (IC). Those skilled in the art will appreciate that the extracellular domain can further comprise a “hinge domain” (membrane bound or proximal) and a “spacer” domain to achieve optimal spatial orientation or distance to the desired antigen. The intracellular component frequently comprises in part a member of the CD3 family, preferably CD3 zeta, which is capable of activating the T cell upon binding of the antigen binding molecule to its target. In one embodiment, the extracellular domain is typically comprised of at least one costimulatory domain as defined herein.
[0082] It will also be appreciated that the extracellular domain may also contain some or all of a member of the immuno-globulin family such as IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragment thereof.
[0083] Exemplary CAR constructs in accordance with the invention are set forth in
Table 1.
TABLE 1
Construct Costimulatory Activating
Extracellular Domain Hinge Sequence
Name(s) Domain Domain
C1/C2 RRM1-RRM2-RRM3 CD28 CD3 zeta 21-24
C3/C4 RRM1-RRM2-RRM3 CD 8 CD28 CD3 zeta 25-28
C5/C6 RRM3 CD 8 CD28 CD3 zeta 29-32
C7/C8 RRM2-RRM3 CD 8 CD28 CD3 zeta 33-36
C9/C10 RRM1-RRM3 CD 8 CD28 CD3 zeta 37-40
C11/C12 RRM1-RRM2-RRM3- CD8 CD28 CD3 zeta 41-44
RRM1-RRM2-RRM3
C13/C14 RRM1-RRM2-RRM3 4- IBB CD3 zeta 45-48
C15/C16 RRM1-RRM2-RRM3 CD 8 4- IBB CD3 zeta 49-52
C17/C18 RRM3 CD 8 4- IBB CD3 zeta 53-56
C19/C20 RRM1-RRM2-RRM3 CD 8 CD28+4-1BB CD3 zeta 57-60
C21/C22 RM3 CD 8 CD28+4-1BB CD3 zeta 61-64
Domains Relative to the Cell
[0084] It will be appreciated that relative to the cell bearing the receptor, the engineered T cells of the invention comprise a CEA-binding domain, an extracellular domain (which may comprise a “hinge” domain), a transmembrane domain, and an intracellular domain. The intracellular domain comprises at least in part an activating domain, preferably comprised of a CD3 family member such as CD3 zeta, CD3 epsilon, CD3 gamma, or portions thereof. It will further be appreciated that the CEA-binding domain is engineered such that it is located in the extracellular portion of the molecule/construct, such that it is capable of recognizing and binding to its target or targets.
[0085] Extracellular Domain. The extraceullar domain comprises at minimum an antigen-binding domain capable of recognizing and binding to CEA as described above. The extracellular domain may comprise additional elements that are beneficial for signaling and for an efficient response of lymphocytes to an antigen. Extracellular domains of particular use in this invention may be derived from (i.e., comprise) CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CDl-la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL- 2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof. The extracellular domain may be derived either from a natural or from a synthetic source.
[0086] As described herein, extracellular domains often comprise a hinge portion. This is a portion of the extracellular domain proximal to the cell membrane. The extracellular domain may further comprise a spacer region. A variety of hinges can be employed in accordance with the invention, including costimulatory molecules as discussed above, as well as immunoglobulin (Ig) sequences or other suitable molecules to achieve the desired special distance from the target cell. In some embodiments, the entire extracellular region comprises a hinge region. In some embodiments, the hinge region comprises the extracellular domain of CD28, or a portion thereof as described herein. .
[0087] Transmembrane Domain. The CAR can be designed to comprise a transmembrane domain that is fused or otherwise linked to the extracellular domain of the CAR. It can similarly be fused to the intracellular domain of the CAR. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in a CAR is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane- bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i.e. comprise) CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7,
CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CDl-la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a),
DAP- 10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL- 2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof.
[0088] Optionally, short linkers may form linkages between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.
[0089] In one embodiment, the transmembrane domain in the CAR of the invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the transmembrane portion of the nucleic acid sequence of SEQ ID NO: 15. In another embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence that encodes the transmembrane amino acid sequence contained within SEQ ID NO: 16.
[0090] In certain embodiments, the transmembrane domain in the CAR of the invention is the CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 5. In one embodiment, the CD28 transmembrane domain comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 6. In another embodiment, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6.
[0091] Intracellular (Cytoplasmic) Domain. The intracellular (IC, or cytoplasmic) domain of the engineered T cells of the invention can provide activation of at least one of the normal effector functions of the immune cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
[0092] It will be appreciated that suitable intracellular molecules include (i.e., comprise), but are not limited to CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death- 1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CDl-la/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/ RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof
[0093] In a preferred embodiment, the cytoplasmic domain of the CAR can be designed to comprise the CD3 zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region.
[0094] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention may be linked to each other in a random or specified order.
[0095] In one preferred embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3 zeta and the signaling domain of CD28. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3 zeta and the signaling domain of 4- IBB. In another embodiment, the cytoplasmic domain in the CAR of the invention is designed to comprise a portion of CD28 and CD3 zeta, wherein the cytoplasmic CD28 comprises the nucleic acid sequence set forth in SEQ ID NO: 5 and the amino acid sequence set forth in SEQ ID NO: 6. The CD3 zeta nucleic acid sequence is set forth in SEQ ID NO: 13, and the amino acid sequence is set forth in SEQ ID NO: 14.
[0096] It will be appreciated that one preferred orientation of the CARs in accordance with the invention comprises an antigen binding domain (such as a CEA-binding domain) in tandem with at least one costimulatory domain and an activating domain. The costimulatory domain can comprise one or more of an extracellular portion, a transmembrane portion, and an intracellular portion. It will be further appreciated that multiple costimulatory domains can be utilized in tandem.
[0097] In some embodiments, nucleic acids are provided comprising a promoter operably linked to a first polynucleotide encoding an antigen binding molecule, at least one costimulatory molecule, and an activating domain. [0098] In some embodiments, the nucleic acid construct is contained within a viral vector. In some embodiments, the viral vector is selected from the group consisting of retro viral vectors, murine leukemia vims vectors, SFG vectors, adenoviral vectors, lentiviral vectors, adeno-associated vims (AAV) vectors, Herpes vims vectors, and vaccinia virus vectors. In some embodiments, the nucleic acid is contained within a plasmid.
[0099] The invention further relates to isolated polynucleotides encoding the chimeric antigen receptors, and vectors comprising the polynucleotides. Any vector known in the art can be suitable for the present invention. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector (such as pMSVGl), a DNA vector, a murine leukemia vims vector, an SFG vector, a plasmid, a RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated vector (AAV), a lentiviral vector (such as pGAR), or any combination thereof. In various embodiments, the vector sequence is as follows:
(SEQ ID NO: 15)
TAGGCGCCCCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACT
TCCCTGACCCTGACATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCA
CTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGC
AGCCTACCAAGAACAACTGGACCGGCCGGTGGTACCTCACCCTTACCGAGT
CGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCG
CTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGT
AGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCC
CGGGGGTGGACCATCCTCTAGACTGGCCACCATGGCCAATCCAACTAAAAG
ATACAGAGCCTTCATTACAAACATACCTTTTGATGTGAAATGGCAGTCACTT
AAAGACCTGGTTAAAGAAAAAGTTGGTGAGGTAACATACGTGGAGCTCTTA
ATGGACGCTGAAGGAAAGTCAAGGGGATGTGCTGTTGTTGAATTCAAGATG
GAAGAGAGCATGAAAAAAGCTGCGGAAGTCCTAAACAAGCATAGTCTGAG
CGGAAGACCACTGAAAGTCAAAGAAGATCCTGATGGTGAACATGCCAGGA
GAGCAATGCAAAAGGCTGGAAGACTTGGAAGCACAGTATTTGTAGCAAAT
CTGGATTATAAAGTTGGCTGGAAGAAACTGAAGGAAGTATTTAGTATGGCT
GGTGTGGTGGTCCGAGCAGACATTCTTGAAGATAAAGATGGAAAAAGTCGT
GGAATAGGCACTGTTACTTTTGAACAGTCCATTGAAGCTGTGCAAGCTATA
TCTATGTTCAATGGCCAGCTGCTATTTGATAGACCAATGCACGTCAAGATG
GATGAGAGGGCCTTACCAAAAGGATCCTGGGGTGGCCAGGAAGGCCTGCC
AGATATTTGTGAGAAATCTGCCATTCGATTTCACATGGAAGATGCTAAAGG
ACAAATTCAACGAGTGCGGCCACGTGCTGTACGCCGACATCAAGATGGAGA
ATGGGAAGTCCAAGGGGTGTGGTGTGGTTAAGTTCGAGTCGCCAGAGGTGG
CCGAGAGAGCCTGCCGGATGATGAATGGCATGAAGCTGAGTGGCCGAGAG
ATTGACGTTCGAATTGATAGAAACGCTCTAGATCCCAAAGTGCTGGTGGTG
GTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTA
TTTTCTGGGTGAGGAGTAAGAGGAGCCGGCTCCTGCACAGTGACTACATGA
ACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATG
CCCCACCACGCGACTTCGCAGCCTACCGGTCCAGAGTGAAGTTCAGCCGGT CCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGC
TCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGC
CGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG
CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGA
TTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTAC
CAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAA
GCTTTGCCCCCTCGCTAAGCGGCCGCTCCGGATTAGTCCAATTTGTTAAAGA
CAGGATATCAGTGGTCCAGGCTCTAGTTTTGACTCAACAATATCACCAGCT
G A AGCCT ATAG AGT ACGAGCC AT AG AT A A A ATA A A AG ATTTT ATTT AGTCT
CCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAG
CTTAAGTAACGCCATTTTGCAAGGCATGGAAAAATACATAACTGAGAATAG
AGAAGTTCAGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCA
AACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACA
GATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCC
TGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCT
CAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTG
AAATGACCCTGT
[00100] Suitable additional exemplary vectors include e.g., pBABE-puro, pBABE- neo largeTcDNA, pBABE-hygro-hTERT, pMKO.l GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES Luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pFncEXP, and pFXIN-Fuc.
[00101] In some embodiments, the engineered immune cell is a T cell, tumor infiltrating lymphocyte (TIE), NK cell, TCR-expressing cell, dendritic cell, or NK-T cell. In some embodiments, the cell is obtained or prepared from peripheral blood. In some embodiments, the cell is obtained or prepared from peripheral blood mononuclear cells (PB- MCs). In some embodiments, the cell is obtained or prepared from bone marrow. In some embodiments, the cell is obtained or prepared from umbilical cord blood. In some embodiments, the cell is a human cell. In some embodiments, the cell is transfected or transduced by the nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., Gene Gun), lipid transfection, polymer transfection, nanoparticles, or polyplexes.
[00102] In some embodiments, chimeric antigen receptors are expressed in the engineered immune cells that comprise the nucleic acids of the present application. These chimeric antigen receptors of the present application may comprise, in some embodiments, (i) an antigen binding molecule (such as an scFv), (ii) a transmembrane region, and (iii) a T cell activation molecule or region. CEA-Binding Domains
[00103] Antigen binding molecules are within the scope of the invention.
[00104] An “CEA-binding domain” as used herein means any protein that binds a CEA. CEA-Binding Domains include, but are not limited to portions of naturally occurring proteins that bind to CEA, for example, the hnRNP M4 receptors that are expressed on the surface of Kupffer cells. .
[00105] In some embodiments, the CEA-binding domain binds to an CEA on a tumor cell. In some embodiments, the CEA-binding domain binds to CEA on a cell involved in a hyperproliferative disease
[00106] The invention further provides for pharmaceutical compositions comprising nlrCEA-CAR T cell together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. In certain embodiments, pharmaceutical compositions will include more than one nlrCEA-CAR T cell. In certain embodiments, pharmaceutical compositions will include more than one nlrCEA-CAR T cell with different CEA-binding domains wherein the CEA-binding domains bind more than one epitope on CEA. In some embodiments, the various CEA binding domains will not compete with one another for binding to CEA.
[00107] In other embodiments, the pharmaceutical composition can be selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, such as orally.
The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. In certain embodiments, when parenteral administration is contemplated, a therapeutic composition can be in the form of a pyrogen- free, parenterally acceptable aqueous solution comprising a desired nlrCEA-CAR T Cell, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. In certain embodiments, a vehicle for parenteral injection is sterile distilled water in which an antigen binding molecule to CEA, with or without at least one additional therapeutic agent, is formulated as a sterile, isotonic solution, properly preserved.
[00108] The antigen binding molecules will be further understood in view of the definitions and descriptions below.
[00109] A CEA-binding domain is said to “specifically bind” its target antigen (CEA) when the dissociation constant (Kd) is lxlO 7 M. The CEA binding domain specifically binds antigen with “high affinity” when the Kd is l-5xl09 M, and with “very high affinity” when the Kd is l-5xlO 10 M. In one embodiment, the CEA-binding domain has a Kd of 109 M. In one embodiment, the off-rate is <1x105. In other embodiments, the CEA-binding domain will bind to human CEA with a Kd of between about 107 M and 1013 M, and in yet another embodiment the CEA-binding domain will bind with a Kd 1.0-5 x 10_1°.
[00110] A CEA-binding domain is said to be “selective” when it binds to one target more tightly than it binds to a second target.
[00111] The term “neutralizing” refers to a CEA-binding domain that binds to a ligand and prevents or reduces the biological effect of that ligand. This can be done, for example, by directly blocking a binding site on the ligand or by binding to the ligand and altering the ligand’s ability to bind through indirect means (such as structural or energetic alterations in the ligand). In some embodiments, the term can also denote a CEA-binding domain that prevents the protein to which it is bound from performing a biological function.
[00112] The term “target” or “antigen” refers to a molecule or a portion of a molecule capable of being bound by an antigen binding molecule. In certain embodiments, a target can have one or more epitopes.
[00113] The term “compete” when used in the context of CEA-binding domains that compete for the same epitope means competition between CEA-binding domains as determined by an assay in which the CEA binding domain being tested prevents or inhibits (e.g., reduces) specific binding of a reference CEA binding domain to CEA Numerous types of competitive binding assays can be used to determine if one CEA-binding domain competes with another, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (Stahli et al, METHODS IN ENZYMOLOGY, 1983, 9, 242253); solid phase direct biotin-avidin EIA (Kirkland et al, J. IMMUNOL., 1986, 137, 3614-3619), solid phase direct labeled assay, solid phase direct labeled sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (Morel et al, MOLEC. IMMUNOL., 1988, 25:7-15); solid phase direct biotin-avidin EIA (Cheung, et al. , VIROLOGY, 1990, 176, 546-552); and direct labeled RIA (Moldenhauer et al, J. IMMUNOL., 1990, 32, 77- 82). The term “epitope” includes any determinant capable of being bound by the CEA-binding domain, such as a naturally occurring receptor, or a transduced immune cell of the invention. An epitope is a region of CEA that is bound by a CEA-binding domain that targets that antigen, and when the antigen is a protein, includes specific amino acids that directly contact the antigen binding molecule.
[00114] As used herein, the terms “label” or “labeled” refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotin moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In certain embodiments, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used.
[00115] In accordance with the invention, on-off or other types of control switch techniques may be incorporated herein. These techniques may employ the use of dimerization domains and optional activators of such domain dimerization. These techniques include, e.g., those described by Wu et al., Science 2014350 (6258) utilizing FKBP/Rapalog dimerization systems in certain cells, the contents of which are incorporated by reference herein in their entirety. Additional dimerization technology is described in, e.g. , Fegan et al. CHEM. REV. 2010, 110, 3315-3336 as well as U.S. Pat. Nos. 5,830,462; 5,834,266; 5,869,337; and 6,165,787, the contents of which are also incorporated by reference herein in their entirety. Additional dimerization pairs may include cyclosporine- A/cyclophilin, receptor, estrogen/estrogen receptor (optionally using tamoxifen), glucocorticoids/glucocorticoid receptor, tetracycline/tetracycline receptor, vitamin D/vitamin D receptor. Further examples of dimerization technology can be found in e.g., WO 2014/127261, WO 2015/090229, US 2014/0286987, US 2015/0266973, US 2016/0046700, U.S. Pat. No. 8,486,693, US 2014/0171649, and US 2012/0130076, the contents of which are further incorporated by reference herein in their entirety.
Methods of Treatment
[00116] Using adoptive immunotherapy, native T cells can be (i) removed from a patient, (ii) genetically engineered to express a chimeric antigen receptor (CAR) that binds to at least one tumor antigen (iii) expanded ex vivo into a larger population of engineered T cells, and (iv) reintroduced into the patient. See, e.g., U.S. Pat. Nos. 7,741,465, and 6,319,494, Eshhar et al. (CANCER IMMUNOL, supra); Krause et al. (supra); Finney et al. (supra). After the engineered T cells are reintroduced into the patient, they mediate an immune response against cells expressing the tumor antigen. See, e.g., Krause et al. , J. EXP. MED., Volume 188, No. 4, 1998 (619-626). This immune response includes secretion of IL-2 and other cytokines by T cells, the clonal expansion of T cells recognizing the tumor antigen, and T cell-mediated specific killing of target-positive cells. See, e.g., Hombach et al. , JOURNAL OF IMMUN. 167 : 6123-6131 (2001). [00117] In some aspects, the invention therefore comprises a method for treating or preventing a condition associated with undesired and/or elevated CEA levels in a patient, comprising administering to a patient in need thereof an effective amount of at least one nlrCEA-CAR disclosed herein.
[00118] Methods are provided for treating diseases or disorders, including cancer. In some embodiments, the invention relates to creating a T cell-mediated immune response in a subject, comprising administering an effective amount of the engineered immune cells of the present application to the subject. In some embodiments, the T cell-mediated immune response is directed against a target cell or cells. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR). In some embodiments, the target cell is a tumor cell. In some aspects, the invention comprises a method for treating or preventing a malignancy, said method comprising administering to a subject in need thereof an effective amount of at least one isolated antigen binding molecule described herein. In some aspects, the invention comprises a method for treating or preventing a malignancy, said method comprising administering to a subject in need thereof an effective amount of at least one immune cell, wherein the immune cell comprises at least one chimeric antigen receptor.
[00119] In some aspects, the invention comprises a pharmaceutical composition comprising at least one CEA-binding molecule as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.
[00120] In some embodiments, the subject is diagnosed with a metastatic disease localized to the liver. In other embodiments, the metastatic disease is a cancer. In still other embodiments, the cancer metastasized from a primary tumor in the breast, colon, rectum, esophagus, lung, pancreas and/or stomach. In still other embodiments, the subject is diagnosed with unresectable metastatic liver tumors. In yet other embodiments, the Subject is diagnosed with unresectable metastatic liver tumors from primary colorectal cancer. In some embodiments, the Subject is diagnosed with hepatocellular carcinoma.
[00121] It will be appreciated that target doses for CEA CAR-T cells can range from 1 xl06-2xl010 cells/kg, preferably 2xl06 cells/kg, more preferably. It will be appreciated that doses above and below this range may be appropriate for certain subjects, and appropriate dose levels can be determined by the healthcare provider as needed. Additionally, multiple doses of cells can be provided in accordance with the invention.
[00122] Also provided are methods for reducing the size of a tumor in a subject, comprising administering to the subject an engineered cell of the present invention to the subject, wherein the cell comprises a chimeric antigen receptor, a T cell receptor, or a T cell receptor based chimeric antigen receptor comprising CEA-binding domain binds to an antigen on the tumor. In some embodiments, the subject has a solid tumor, or a blood malignancy such as lymphoma or leukemia. In some embodiments, the engineered cell is delivered to a tumor bed. In some embodiments, the cancer is present in the bone marrow of the subject.
[00123] In some embodiments, the engineered cells are autologous T cells. In some embodiments, the engineered cells are allogeneic T cells. In some embodiments, the engineered cells are heterologous T cells. In some embodiments, the engineered cells of the present application are transfected or transduced in vivo. In other embodiments, the engineered cells are transfected or transduced ex vivo.
[00124] The methods can further comprise administering one or more chemotherapeutic agent. In certain embodiments, the chemotherapeutic agent is a lymphodepleting (preconditioning) chemotherapeutic. Beneficial preconditioning treatment regimens, along with correlative beneficial biomarkers are described in U.S. Patent No. 10,322,146 which are hereby incorporated by reference in their entirety herein. These describe, e.g., methods of conditioning a patient in need of a T cell therapy comprising administering to the patient specified beneficial doses of cyclophosphamide (between 200 mg/m2/ day and 2000 mg/m2/day) and specified doses of fludarabine (between 20 mg/m2/day and 900 mg/m2/day). A preferred dose regimen involves treating a patient comprising administering daily to the patient about 500 mg/m2/day of cyclophosphamide and about 60 mg/m2/day of fludarabine for three days prior to administration of a therapeutically effective amount of engineered T cells to the patient.
[00125] In other embodiments, the antigen binding molecule, transduced (or otherwise engineered) cells (such as CARs), and the chemotherapeutic agent are administered each in an amount effective to treat the disease or condition in the subject.
[00126] In certain embodiments, compositions comprising CAR-expressing immune effector cells disclosed herein may be administered in conjunction with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine resume; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esombicin, idambicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, que-lamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2’,2”-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb) and doxetaxel (TAXO-TERE®, Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™, (alitretinoin); ONTAK™ (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Combinations of chemotherapeutic agents are also administered where appropriate, including, but not limited to CHOP, i.e., Cyclophosphamide (Cytoxan®) Doxorubicin (hydroxy doxorubicin), Vincristine (Oncovin®), and Prednisone.
[00127] In some embodiments, the chemotherapeutic agent is administered at the same time or within one week after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered from 1 to 4 weeks or from 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least 1 month before administering the cell or nucleic acid. In some embodiments, the methods further comprise administering two or more chemotherapeutic agents.
[00128] A variety of additional therapeutic agents may be used in conjunction with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab (Tecentriq®).
[00129] Additional therapeutic agents suitable for use in combination with the invention include, but are not limited to, ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (KADCYLA®), imatinib (Gleevec®), cetuximah (Erbitux®), panitumumab (Vectibix®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, mTOR inhibitors such as Everolimus and Temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, CDK inhibitors such as CDK inhibitor (palbociclib).
[00130] In additional embodiments, the composition comprising CAR-containing immune can be administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists, (e.g., etanercept (ENBREL®), adaiimumab (HUMIRA®) and infliximab (REMICADE®)), chemokine inhibitors and adhesion molecule inhibitors. The biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline.
[00131] In certain embodiments, the compositions described herein are administered in conjunction with a cytokine. “Cytokine” as used herein is meant to refer to proteins released by one cell population that act on another cell as intercellular mediators. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian- inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin- like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-lalpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines.
[00132] In some aspects, the invention comprises an antigen binding molecule that binds to CEA with a Kd that is smaller than 100 pM. In some embodiments, the antigen binding molecule binds with a Kd that is smaller than 10 pM. In other embodiments, the antigen binding molecule binds with a Kd that is less than 5 pM.
Methods of Making
[00133] A variety of known techniques can be utilized in making the polynucleotides, polypeptides, vectors, antigen binding molecules, immune cells, compositions, and the like according to the invention.
[00134] Prior to the in vitro manipulation or genetic modification of the immune cells described herein, the cells may be obtained from a subject. In some embodiments, the immune cells comprise T cells. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph nodes tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from the subject using any number of techniques known to the skilled person, such as FICOLL™ separation. Cells may preferably be obtained from the circulating blood of an individual by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, the cells collected by apheresis may be washed to remove the plasma fraction, and placed in an appropriate buffer or media for subsequent processing. The cells may be washed with PBS. As will be appreciated, a washing step may be used, such as by using a semi automated flowthrough centrifuge for example, the Cobe™ 2991 cell processor, the Baxter Cyto-Mate™, or the like. After washing, the cells may be resuspended in a variety of biocompatible buffers, or other saline solution with or without buffer. In certain embodiments, the undesired components of the apheresis sample may be removed.
[00135] In certain embodiments, T cells are isolated from PBMCs by lysing the red blood cells and depleting the monocytes, for example, using centrifugation through a PERCOLL™ gradient. A specific subpopulation of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45R0+ T cells can be further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method for use herein is cell sorting and/or selection via negative magnetic immun oadh eren ce or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting may also be used to isolate cell populations of interest for use in the present invention.
[00136] PBMCs may be used directly for genetic modification with the immune cells (such as CARs or TCRs) using methods as described herein. In certain embodiments, after isolating the PBMCs, T lymphocytes can be further isolated and both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and/or expansion.
[00137] In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers of central memory T cells include CD45RO, CD62L, CCR7, CD28, CD3, and CD 127 and are negative for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8 T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD 127, and positive for granzyme B and perforin. In certain embodiments, CD4+
T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.
[00138] The immune cells, such as T cells, can be genetically modified following isolation using known methods, or the immune cells can be activated and expanded (or differ entiated in the case of progenitors) in vitro prior to being genetically modified. In another embodiment, the immune cells, such as T cells, are genetically modified with the chimeric antigen receptors described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then are activated and/or expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Pat. Nos. 6,905,874; 6,867,041; 6,797,514; and PCT W02012/079000, the contents of which are hereby incorporated by reference in their entirety. Generally, such methods include contacting PBMC or isolated T cells with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a “surrogate” antigen presenting cell (APC). One example is The Dynabeads® system, a CD3/CD28 activator/stimulator system for physiological activation of human T cells.
[00139] In other embodiments, the T cells may be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Pat. Nos. 6,040,177; 5,827,642; and W02012129514, the contents of which are hereby incorporated by reference in their entirety.
[00140] Certain methods for making the constructs and engineered immune cells of the invention are described in PCT application PCT/US15/14520, the contents of which are hereby incorporated by reference in their entirety. Additional methods of making the constructs and cells can be found in U.S. provisional patent application No. 62/244,036 the contents of which are hereby incorporated by reference in their entirety.
[00141] It will be appreciated that PBMCs can further include other cytotoxic lymphocytes such as NK cells or NKT cells. An expression vector carrying the coding sequence of a chimeric receptor as disclosed herein can be introduced into a population of human donor T cells, NK cells or NKT cells. Successfully transduced T cells that carry the expression vector can be sorted using flow cytometry to isolate CD3 positive T cells and then further propagated to increase the number of these CAR expressing T cells in addition to cell activation using anti-CD3 antibodies and IL-2 or other methods known in the art as described elsewhere herein. Standard procedures are used for cryopreservation of T cells expressing the CAR for storage and/or preparation for use in a human subject. In one embodiment, the in vitro transduction, culture and/or expansion of T cells are performed in the absence of non human animal derived products such as fetal calf serum and fetal bovine serum.
[00142] For cloning of polynucleotides, the vector may be introduced into a host cell (an isolated host cell) to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein. The cloning vectors may contain sequence components generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art. For example, the origin of replication may be selected to promote autonomous replication of the vector in the host cell.
[00143] In certain embodiments, the present disclosure provides isolated host cells containing the vector provided herein. The host cells containing the vector may be useful in expression or cloning of the polynucleotide contained in the vector. Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. Suitable prokaryotic cells for this purpose include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobactehaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marc-escans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.
[00144] The vector can be introduced to the host cell using any suitable methods known in the art, including, without limitation, DEAE-dextran mediated delivery, calcium phosphate precipitate method, cationic lipids mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of a vector of interest are well known in the art. In a further embodiment, a mixture of different expression vectors can be used in genetically modifying a donor population of immune effector cells wherein each vector encodes a different CAR as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, with a proportion of the engineered cells expressing more than one different CARs.
[00145] In one embodiment, the invention provides a method of storing genetically engineered cells expressing CARs, which target a CEA protein. This involves cryopreserving the immune cells such that the cells remain viable upon thawing. A fraction of the immune cells expressing the CARs can be cryopreserved by methods known in the art to provide a permanent source of such cells for the future treatment of patients afflicted with a malignancy. When needed, the cryopreserved transformed immune cells can be thawed, grown and expanded for more such cells.
[00146] As used herein, “cryopreserve” refers to the preservation of cells by cooling to sub-zero temperatures, such as (typically) 77 Kelvin or — 196° C. (the boiling point of liquid nitrogen). Cryoprotective agents are often used at sub-zero temperatures to prevent the cells being preserved from damage due to freezing at low temperatures or warming to room temperature. Cryopreservative agents and optimal cooling rates can protect against cell injury. Cryoprotective agents which can be used in accordance with the invention include but are not limited to: dimethyl sulfoxide (DMSO) (Lovelock & Bishop, NATURE, 1959, 183, 1394-1395; Ashwood-Smith, NATURE, 1961, 190, 1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, ANN. N.Y. ACAD. SCL, 1960, 85, 576), and polyethylene glycol (Sloviter & Ravdin, NATURE, 1962, 196, 48). The preferred cooling rate is l°-3° C/minute.
[00147] The term, “substantially pure,” is used to indicate that a given component is present at a high level. The component is desirably the predominant component present in a composition. Preferably it is present at a level of more than 30%, of more than 50%, of more than 75%, of more than 90%, or even of more than 95%, said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration. At very high levels (e.g. at levels of more than 90%, of more than 95% or of more than 99%) the component can be regarded as being in “pure form.” Biologically active substances of the present invention (including polypeptides, nucleic acid molecules, antigen binding molecules, moieties) can be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated. When a composition is substantially free of a given contaminant, the contaminant will be at a low level (e.g., at a level of less than 10%, less than 5%, or less than 1% on the dry weight/dry weight basis set out above).
[00148] In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a “pharmaceutically acceptable” carrier) in a treatment- effective amount. Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol™ R (Abbott) or Plasma-Lyte™ A (Baxter), but also 5% dextrose in water or Ringer’s lactate can be utilized. The infusion medium can be supplemented with human serum albumin.
[00149] Desired treatment amounts of cells in the composition is generally at least 2 cells (for example, at least 1 CD8+ central memory T cell and at least 1 CD4+ helper T cell subset) or is more typically greater than 102 cells, and up to 106, up to and including 108 or 109 cells and can be more than 1010 cells. The number of cells will depend upon the desired use for which the composition is intended, and the type of cells included therein. The density of the desired cells is typically greater than 106 cells/ml and generally is greater than 107 cells/ml, generally 108 cells/ml or greater. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 105, 106, 107, 108, 109, 1010, 1011, or 1012 cells. In some aspects of the present invention, particularly since all the infused cells will be redirected to a particular target antigen (CEA), lower numbers of cells, in the range of 106/kilogram (106-10n per patient) may be administered. CAR treatments may be administered multiple times at dosages within these ranges. The cells may be autologous, allogeneic, or heterologous to the patient undergoing therapy.
[00150] The CAR expressing cell populations of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations. Pharmaceutical compositions of the present invention may comprise a CAR or TCR expressing cell population, such as T cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are preferably formulated for intravenous administration.
[00151] The pharmaceutical compositions (solutions, suspensions or the like), may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylene-diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.
[00152] It will be appreciated that adverse events may be minimized by transducing the immune cells (containing one or more CARs) with a suicide gene. It may also be desired to incorporate an inducible “on” or “accelerator” switch into the immune cells. Suitable techniques include use of inducible caspase-9 (U.S. Appl. 2011/0286980) or a thymidine kinase, before, after or at the same time, as the cells are transduced with the CAR construct of the present invention. Additional methods for introducing suicide genes and/or “on” switches include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other techniques known in the art.
[00153] It will be understood that descriptions herein are exemplary and explanatory only and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise.
[00154] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
[00155] In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.
[00156] The term “CEA activity” includes any biological effect of CEA. In certain embodiments, CEA activity includes the ability of CEA to interact or bind to a substrate or receptor.
[00157] The term “polynucleotide”, “nucleotide”, or “nucleic acid” includes both single- stranded and double- stranded nucleotide polymers. The nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2’,3’-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphoro-diselenoate, phosphoro-anilothioate, phoshoraniladate and phosphoroamidate.
[00158] The term “oligonucleotide” refers to a polynucleotide comprising 200 or fewer nucleotides. Oligonucleotides can be single stranded or double stranded, e.g., for use in the construction of a mutant gene. Oligonucleotides can be sense or antisense oligonucleotides. An oligonucleotide can include a label, including a radiolabel, a fluorescent label, a hapten or an antigenic label, for detection assays. Oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization probes.
[00159] The term “control sequence” refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is ligated. The nature of such control sequences can depend upon the host organism. In particular embodiments, control sequences for prokaryotes can include a promoter, a ribosomal binding site, and a transcription termination sequence. For example, control sequences for eukaryotes can include promoters comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences, and transcription termination sequence. “Control sequences” can include leader sequences (signal peptides) and/or fusion partner sequences.
[00160] As used herein, “operably linked” means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions.
[00161] The term “vector” means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or vims) used to transfer protein coding information into a host cell. The term “expression vector” or “expression construct” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control (in conjunction with the host cell) expression of one or more heterologous coding regions operatively linked thereto. An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, if introns are present, affect RNA splicing of a coding region operably linked thereto.
[00162] The term “host cell” refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest.
The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.
[00163] The term “transformation” refers to a change in a cell’s genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques. Following transfection or transduction, the transforming DNA can recombine with that of the cell by physically integrating into a chromosome of the cell, or can be maintained transiently as an episomal element without being replicated, or can replicate independently as a plasmid. A cell is considered to have been “stably transformed” when the transforming DNA is replicated with the division of the cell.
[00164] The term “transfection” refers to the uptake of foreign or exogenous DNA by a cell. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g. , Graham et al, 1973, VIROLOGY, 52, 456; Sambrook et al, 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al, 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, Gene, 1981, 13, 197.
[00165] The term “transduction” refers to the process whereby foreign DNA is introduced into a cell via viral vector. See, e.g., Jones et al, Genetics: principles and analysis. (1998), Boston: Jones & Bartlett Publ.
[00166] The terms “polypeptide” or “protein” refer to a macromolecule having the amino acid sequence of a protein, including deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The terms “polypeptide” and “protein” specifically encompass CEA antigen binding molecules, antibodies, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of antigen-binding protein. The term “polypeptide fragment” refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion as compared with the full- length native protein. Such fragments can also contain modified amino acids as compared with the native protein. Useful polypeptide fragments include immunologically functional fragments of antigen binding molecules.
[00167] The term “isolated” means (i) free of at least some other proteins with which it would normally be found, (ii) is essentially free of other proteins from the same source, e.g., from the same species, (iii) separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (iv) operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (v) does not occur in nature.
[00168] A “variant” of a polypeptide (e.g., an antigen binding molecule) comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins.
[00169] The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (/.<?., an “algorithm”).
[00170] To calculate percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al, 1984, NUCL. ACID RES. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm). In certain embodiments, a standard comparison matrix (see, e.g., Dayhoff et al. , 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al, 1992, PROC. NATL. ACAD. SCI. U.S.A. 89:10915-10919 for the BLO-SUM 62 comparison matrix) is also used by the algorithm.
[00171] As used herein, the twenty conventional (e.g., naturally occurring) amino acids and their abbreviations follow conventional usage. See, e.g., Immunology A Synthesis (2nd Edition, Golub and Gren, Eds., Sinauer Assoc., Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as alpha-, alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids can also be suitable components for polypep-tides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, . gamma. -carboxy-glutamate, epsilon- N,N,N-trimethyllysine, e-N-acetyllysine, 0-phosphoserine, N-acetylserine, N- formylmethionine, 3-methylhistidine, 5-hydroxylysine, . sigma. -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
[00172] Conservative amino acid substitutions can encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties. Naturally occurring residues can be divided into classes based on common side chain properties: a) hydrophobic: norleucine, Met, Ala, Val, Leu, lie; b) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; c) acidic: Asp, Glu; d) basic: His, Lys, Arg; e) residues that influence chain orientation: Gly, Pro; and f) aromatic: Trp, Tyr, Phe.
[00173] For example, non-conservative substitutions can involve the exchange of a member of one of these classes for a member from another class.
[00174] In making changes to the antigen binding molecule, the costimulatory or activating domains of the engineered T cell, according to certain embodiments, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). See, e.g., Kyte et al, J. MOL. BIOL., 157:105-131 (1982). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. Exemplary amino acid substitutions are set forth in Table 2.
TABLE 2
Original Residues Exemplary Substitutions Preferred Substitutions
Ala Val, Leu, lie Val
Arg Lys, Gin, Asn Lys
Asn Gin Gin
Asp Glu Glu
Cys Ser, Ala Ser
Gin Asn Asn
Glu Asp Asp
Gly Pro, Ala Ala
His Asn, Gin, Lys, Arg Arg lie Leu, Val, Met, Ala, Phe, Leu
Norleucine
Leu Norleucine, lie, Va, Met, Ala, He
Phe
Lys Arg, 1, 4 Diamino-butyric Arg
Acid, Gin, Asn
Met Leu, Phe, He Leu
Phe Leu, Val, He, Ala, Tyr Leu
Pro Ala Gly
Ser Thr, Ala, Cys Thr
Thr Ser Ser
Trp Tyr, Phe Tyr
Tyr Trp, Phe, Thr, Ser Phe
Val He, Met, Leu, Phe, Leu
Ala, Norleucine
[00175] The term “derivative” refers to a molecule that includes a chemical modification other than an insertion, deletion, or substitution of amino acids (or nucleic acids). In certain embodiments, derivatives comprise covalent modifications, including, but not limited to, chemical bonding with polymers, lipids, or other organic or inorganic moieties. In certain embodiments, a chemically modified antigen binding molecule can have a greater circulating half-life than an antigen binding molecule that is not chemically modified. In some embodiments, a derivative antigen binding molecule is covalently modified to include one or more water soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. [00176] Peptide analogs are commonly used in the pharmaceutical industry as non peptide drugs with properties analogous to those of the template peptide. These types of non peptide compound are termed “peptide mimetics” or “peptidomimetics.” Fauchere, J., ADV. DRUG RES., 15:29 (1986); Veber & Freidinger, TINS, p.392 (1985); and Evans et al, J. MED. CHEM., 30:1229 (1987), which are incorporated herein by reference for any purpose.
[00177] The term “therapeutically effective amount” refers to the amount of a nlrCEA -CAR T cells determined to produce a therapeutic response in a mammal. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art.
[00178] The terms “patient” and “subject” are used interchangeably and include human and non-human animal subjects as well as those with formally diagnosed disorders, those without formally recognized disorders, those receiving medical attention, those at risk of developing the disorders, etc.
[00179] The term “treat” and “treatment” includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors. The term “prevent” does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.
[00180] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g. , electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer’ s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g. , Sambrook et al. , Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
[00181] The following sequences will further exemplify the invention. nlrCEA DNA Extracellular Domain
(SEQ ID NO: 1)
ATGGCCAATCCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTT
GATGTGAAATGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAG
GTAACATACGTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGT
GCTGTTGTTGAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGT CCTAAACAAGCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATC
CTGATGGTGAACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGA
AGCACAGTATTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTG
AAGGAAGTATTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAA
GATAAAGATGGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCC
ATTGAAGCTGTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTTGATA
GACCAATGCACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGG
GTGGCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTC
ACATGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTAC
GCCGACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAA
GTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCAT
GAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGA
TCCCAAA nlrCEA AA Extracellular Domain
(SEQ ID NO: 2)
M ANPTKRYRAFITNIPFD VKW QS LKDLVKEKV GE VT Y VELLMD AEGKSRGC A
VVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHARRAMQKAGRLGST
VFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKSRGIGTVTFEQSIEA
VQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQIFVRNLPFDFTWKM
LKD KFNECGH VLY ADIKMEN GKS KGCG V VKFESPE V AER ACRMMN GMKLS G
REIDVRIDRNALDPK
CD28 DNA Transmembrane Domain
(SEQ ID NO: 3)
TTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCG
TCACCGTGGCTTTTATAATCTTCTGGGTT
CD28 AA Transmembrane Domain:
(SEQ ID NO: 4)
FWVLVVV GGVLACY SLLVTV AFIIFWV
CD28 DNA Intracellular Domain:
(SEQ ID NO: 5)
AGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCA
CGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGA
GATTTCGCTGCCTATCGGAGC
CD28 AA Intracellular Domain:
(SEQ ID NO: 6)
RSKRSRLLHSD YMNMTPRRPGPTRKHY QPY APPRDFAA YRS
CD28 DNA:
(SEQ ID NO: 7) ATTGAGGTGATGTATCCACCGCCTTACCTGGATAACGAAAAGAGTAACGGT ACCATCATTCACGTGAAAGGTAAACACCTGTGTCCTTCTCCCC TCTTCCCCGGGCCATCAAAGCCC CD28 AA:
(SEQ ID NO: 8)
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP
CD28 DNA:
(SEQ ID NO: 9)
GGACCGGTAGGCTGCGAAGTCGCGTGGTGGGGCATAGGGCTGGTAATGCTT
GCGGGTGGGCCCGGGGCGGCGGGGAGTCATGTTCATGTAGTCACTGTGCAG
GAGCCGGCTCCTCTTACTCCTCACCCAGAAAATAATAAAGGCCACTGTTAC
TAGCAAGCTATAGCAAGCCAGGACTCCACCAACCACCACCAGCA
CD28 AA:
(SEQ ID NO: 10)
PKVL ///GGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKH Y QPY APPRDFAA YRS
CD8 DNA extracellular & transmembrane domain:
(SEQ ID NO: 11)
GCTGCAGCATTGAGCAACTCAATAATGTATTTTAGTCACTTTGTACCAGTGT
TCTTGCCGGCT A AGCCT ACT ACC AC ACCCGCTCC ACGGCC ACCT ACCCC AGC
TCCTACCATCGCTTCACAGCCTCTGTCCCTGCGCCCAGAGGCTTGCCGACCG
GCCGCAGGGGGCGCTGTTCATACCAGAGGACTGGATTTCGCCTGCGATATC
TATATCTGGGCACCCCTGGCCGGAACCTCCGGCGTACTCCTGCTGTCCCTGG
TCATCACGCTCTATTGTAATCACAGGAAC
CD8 AA extracellular & transmembrane Domain:
(SEQ ID NO: 12)
AAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASOPLSLRPEACRPAAGG
AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN
CD3 zeta DNA:
(SEQ ID NO: 13)
AGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGG
CCAGAACCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGT
ATGACGTTTTGGACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGC
AAACCAAGACGAAAAAACCCCCAGGAGGGTCTCTATAATGAGCTGCA
GAAGGATAAGATGGCTGAAGCCTATTCTGAAATAGGCATGAAAGGAG
AGCGGAGAAGGGGAAAAGGGCACGACGGTTTGTACCAGGGACTCAGC
ACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAGCCCTGCC
ACCTAGG CD3 zeta AA
(SEQ ID NO: 14)
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEA Y SEIGMKGERRRGKGHDGLY OGLS TATKDTYDALHMQALPPR nlrCEA.CD28.CD3zeta Vector DNA
(SEQ ID NO: 15)
TAGGCGCCCCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACT
TCCCTGACCCTGACATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCA
CTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGC
AGCCTACCAAGAACAACTGGACCGGCCGGTGGTACCTCACCCTTACCGAGT
CGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCG
CTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGT
AGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCC
CGGGGGTGGACCATCCTCTAGACTGGCCACCATGGCCAATCCAACTAAAAG
ATACAGAGCCTTCATTACAAACATACCTTTTGATGTGAAATGGCAGTCACTT
AAAGACCTGGTTAAAGAAAAAGTTGGTGAGGTAACATACGTGGAGCTCTTA
ATGGACGCTGAAGGAAAGTCAAGGGGATGTGCTGTTGTTGAATTCAAGATG
GAAGAGAGCATGAAAAAAGCTGCGGAAGTCCTAAACAAGCATAGTCTGAG
CGGAAGACCACTGAAAGTCAAAGAAGATCCTGATGGTGAACATGCCAGGA
GAGCAATGCAAAAGGCTGGAAGACTTGGAAGCACAGTATTTGTAGCAAAT
CTGGATTATAAAGTTGGCTGGAAGAAACTGAAGGAAGTATTTAGTATGGCT
GGTGTGGTGGTCCGAGCAGACATTCTTGAAGATAAAGATGGAAAAAGTCGT
GGAATAGGCACTGTTACTTTTGAACAGTCCATTGAAGCTGTGCAAGCTATA
TCTATGTTCAATGGCCAGCTGCTATTTGATAGACCAATGCACGTCAAGATG
GATGAGAGGGCCTTACCAAAAGGATCCTGGGGTGGCCAGGAAGGCCTGCC
AGATATTTGTGAGAAATCTGCCATTCGATTTCACATGGAAGATGCTAAAGG
ACAAATTCAACGAGTGCGGCCACGTGCTGTACGCCGACATCAAGATGGAGA
ATGGGAAGTCCAAGGGGTGTGGTGTGGTTAAGTTCGAGTCGCCAGAGGTGG
CCGAGAGAGCCTGCCGGATGATGAATGGCATGAAGCTGAGTGGCCGAGAG
ATTGACGTTCGAATTGATAGAAACGCTCTAGATCCCAAAGTGCTGGTGGTG
GTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTA
TTTTCTGGGTGAGGAGTAAGAGGAGCCGGCTCCTGCACAGTGACTACATGA
ACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATG
CCCCACCACGCGACTTCGCAGCCTACCGGTCCAGAGTGAAGTTCAGCCGGT
CCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGC
TCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGC
CGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGG
CCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGA
TTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTAC
CAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAA
GCTTTGCCCCCTCGCTAAGCGGCCGCTCCGGATTAGTCCAATTTGTTAAAGA
CAGGATATCAGTGGTCCAGGCTCTAGTTTTGACTCAACAATATCACCAGCT
G A AGCCT ATAG AGT ACGAGCC AT AG AT A A A ATA A A AG ATTTT ATTT AGTCT
CCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAG
CTTAAGTAACGCCATTTTGCAAGGCATGGAAAAATACATAACTGAGAATAG AGAAGTTCAGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCA
AACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACA
GATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCC
TGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCT
CAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTG
AAATGACCCTGT nlrCEA.CD28.CD3zeta DNA
(SEQ ID NO: 16)
TTAGCGAGGGGGCAAAGCTTGCATGTGAAGGGCGTCGTAGGTGTCCTTGGT
GGCTGTACTGAGACCCTGGTAAAGGCCATCGTGCCCCTTGCCCCTCCGGCG
CTCGCCTTTCATCCCAATCTCACTGTAGGCCTCCGCCATCTTATCTTTCTGCA
GTTCATTGTACAGGCCTTCCTGAGGGTTCTTCCTTCTCGGCTTTCCCCCCATC
TCAGGGTCCCGGCCACGTCTCTTGTCCAAAACATCGTACTCCTCTCTTCGTC
CTAGATTGAGCTCGTTATAGAGCTGGTTCTGGCCCTGCTGGTACGCGGGGG
CGTCTGCGGACCGGCTGAACTTCACTCTGGACCGGTAGGCTGCGAAGTCGC
GTGGTGGGGCATAGGGCTGGTAATGCTTGCGGGTGGGCCCGGGGCGGCGG
GGAGTCATGTTCATGTAGTCACTGTGCAGGAGCCGGCTCCTCTTACTCCTCA
CCCAGAAAATAATAAAGGCCACTGTTACTAGCAAGCTATAGCAAGCCAGG
ACTCCACCAACCACCACCAGCACTTTGGGATCTAGAGCGTTTCTATCAATTC
GAACGTCAATCTCTCGGCCACTCAGCTTCATGCCATTCATCATCCGGCAGGC
TCTCTCGGCCACCTCTGGCGACTCGAACTTAACCACACCACACCCCTTGGAC
TTCCCATTCTCCATCTTGATGTCGGCGTACAGCACGTGGCCGCACTCGTTGA
ATTTGTCCTTTAGCATCTTCCATGTGAAATCGAATGGCAGATTTCTCACAAA
TATCTGGCAGGCCTTCCTGGCCACCCCGGATCCTTTTGGTAAGGCCCTCTCA
TCCATCTTGACGTGCATTGGTCTATCAAATAGCAGCTGGCCATTGAACATAG
ATATAGCTTGCACAGCTTCAATGGACTGTTCAAAAGTAACAGTGCCTATTCC
ACGACTTTTTCCATCTTTATCTTCAAGAATGTCTGCTCGGACCACCACACCA
GCCATACTAAATACTTCCTTCAGTTTCTTCCAGCCAACTTTATAATCCAGAT
TTGCTACAAATACTGTGCTTCCAAGTCTTCCAGCCTTTTGCATTGCTCTCCTG
GCATGTTCACCATCAGGATCTTCTTTGACTTTCAGTGGTCTTCCGCTCAGAC
TATGCTTGTTTAGGACTTCCGCAGCTTTTTTCATGCTCTCTTCCATCTTGAAT
TCAACAACAGCACATCCCCTTGACTTTCCTTCAGCGTCCATTAAGAGCTCCA
CGTATGTTACCTCACCAACTTTTTCTTTAACCAGGTCTTTAAGTGACTGCCA
TTTCACATCAAAAGGTATGTTTGTAATGAAGGCTCTGTATCTTTTAGTTGGA
TTGGCCAT nlrCEA.CD28.CD3zeta AA
(SEQ ID NO: 17)
M ANPTKRYRAFITNIPFD VKW QS LKDLVKEKV GE VT Y VELLMD AEGKSRGC A
VVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHARRAMQKAGRLGST
VFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKSRGIGTVTFEQSIEA
VQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQIFVRNLPFDFTWKM
LKD KFNECGH VLY ADIKMEN GKS KGCG V VKFESPE V AER ACRMMN GMKLS G
REIDVRIDRNALDPKVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM
NMTPRRPGPTRKHY QPY APPRDFAA YRSRVKFSRS ADAPA Y QQGQNQLYNEL
NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG
MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR - IBB Nucleic Acid Sequence (intracellular domain)
(SEQ ID NO: 18)
AAGCGCGGCAGGAAGAAGCTCCTCTACATTTTTAAGCAGCCTTTTAT
GAGGCCCGTACAGACAACACAGGAGGAAGATGGCTGTAGCTGCAGAT
TTCCCGAGGAGGAGGAAGGTGGGTGCGAGCTG - IBB A A (intracellular domain)
(SEQ ID NO: 19)
KRGRKKLLYIFKQPFMRPVOTTQEEDGCSCRFPEEEEGGCEL X40 AA
(SEQ ID NO: 20)
RRDORLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI l Construct DNA
(SEQ ID NO: 21)
ATGGCCAATCCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTT
GATGTGAAATGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAG
GTAACATACGTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGT
GCTGTTGTTGAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGT
CCTAAACAAGCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATC
CTGATGGTGAACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGA
AGCACAGTATTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTG
AAGGAAGTATTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAA
GATAAAGATGGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCC
ATTGAAGCTGTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTTGATA
GACCAATGCACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGG
GTGGCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTC
ACATGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTAC
GCCGACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAA
GTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCAT
GAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGA
TCCCAAAGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTA
GTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCCGGCTC
CTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGC
AAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTACCGGTCC
AGAGTGAAGTTCAGCCGGTCCGCAGACGCCCCCGCGTACCAGCAGGGCCA
GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATG
TTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGA
AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT
GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA
AGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCT
ACGACGCCCTTCACATGCAAGCTTTGCCCCCTCGCTAA struct AA
(SEQ ID NO: 22)
M ANPTKRYRAFITNIPFD VKW QS LKDLVKEKV GE VT Y VELLMD AEGKSRGC A
VVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHARRAMQKAGRLGST
VFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKSRGIGTVTFEQSIEA
VQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQIFVRNLPFDFTWKM
LKD KFNECGH VLY ADIKMEN GKS KGCG V VKFESPE V AER ACRMMN GMKLS G
REIDVRIDRNALDPKVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM
NMTPRRPGPTRKHY QPY APPRDFAA YRSRVKFSRS ADAPA Y QQGQNQLYNEL
NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG
MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR struct DNA
(SEQ ID NO: 23)
ATGGAACAAAAACTCATCTCAGAAGAGGATCTGGGCGGCTCCGGCGCCAAT
CCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTTGATGTGAAA
TGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAGGTAACATAC
GTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGTGCTGTTGTT
GAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGTCCTAAACAA
GCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATCCTGATGGTG
AACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGAAGCACAGTA
TTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTGAAGGAAGTA
TTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAAGATAAAGAT
GGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCCATTGAAGCT
GTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTTGATAGACCAATGC
ACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGGGTGGCCAGG
AAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTCACATGGAAG
ATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTACGCCGACATC
AAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAAGTTCGAGTC
GCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCATGAAGCTGA
GTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGATCCCAAAG
TGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGT
GGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCCGGCTCCTGCACAG
TGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTA
CCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTACCGGTCCAGAGTGAA
GTTCAGCCGGTCCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCT
CTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAA
GAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC
CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCC
TACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGA
TGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCT
TCACATGCAAGCTTTGCCCCCTCGCTAA struct AA
(SEQ ID NO: 24)
MEQKLISEEDLGGS G ANPTKRYRAFITNIPFD VKW QS LKDLVKEKV GE VT Y VE LLMDAEGKSRGCAVVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHA RRAMQKAGRLGSTVFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKS
RGIGTVTFEQSIEAVQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQI
FVRNLPFDFTWKMLKDKFNECGHVLYADIKMENGKSKGCGVVKFESPEVAER
ACRMMN GMKLS GREID VRIDRN ALDPKVL V V V GG VL AC Y S LL VT V AFIIFW V
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA
YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ
KDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDTYD ALHMQALPPRstruct DNA
(SEQ ID NO: 25)
ATGGCCAATCCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTT
GATGTGAAATGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAG
GTAACATACGTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGT
GCTGTTGTTGAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGT
CCTAAACAAGCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATC
CTGATGGTGAACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGA
AGCACAGTATTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTG
AAGGAAGTATTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAA
GATAAAGATGGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCC
ATTGAAGCTGTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTTGATA
GACCAATGCACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGG
GTGGCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTC
ACATGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTAC
GCCGACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAA
GTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCAT
GAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGA
TCCCAAAAGTGCTAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACC
GGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGGCTCG
GCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCCCTA
GGAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCA
ATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTAT
TTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCT
GGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGG
AGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC
CGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGAC
TTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGAGCCCCCC
GCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGA
AGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGAT
GGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAAC
TGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGC
GAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTAC
AGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCG
CTAA struct AA
(SEQ ID NO: 26)
M ANPTKRYRAFITNIPFD VKW QS LKDLVKEKV GE VT Y VELLMD AEGKSRGC A VVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHARRAMQKAGRLGST VFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKSRGIGTVTFEQSIEA
VQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQIFVRNLPFDFTWKM
LKD KFNECGH VLY ADIKMEN GKS KGCG V VKFESPE V AER ACRMMN GMKLS G
REIDVRIDRNALDPKSAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVH
TRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLV
VVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA
PPRDFAAYRSRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP
EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS
TATKDTYDALHMQALPPR struct DNA
(SEQ ID NO: 27)
ATGGAACAAAAACTCATCTCAGAAGAGGATCTGGGCGGCTCCGGCGCCAAT
CCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTTGATGTGAAA
TGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAGGTAACATAC
GTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGTGCTGTTGTT
GAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGTCCTAAACAA
GCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATCCTGATGGTG
AACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGAAGCACAGTA
TTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTGAAGGAAGTA
TTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAAGATAAAGAT
GGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCCATTGAAGCT
GTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTTGATAGACCAATGC
ACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGGGTGGCCAGG
AAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTCACATGGAAG
ATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTACGCCGACATC
AAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAAGTTCGAGTC
GCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCATGAAGCTGA
GTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGATCCCAAAA
GTGCTAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCA
CCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGGCTCGGCCAGCGG
CGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCCCTAGGAAAATT
GAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACC
ATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGAC
CTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTA
TAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGG
AGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGG
CCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCC
TATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCGTACCAG
CAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGA
GTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA
AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAA
GATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCG
GAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCA
AGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA struct AA
(SEQ ID NO: 28)
MEQKLISEEDLGGS G ANPTKRYRAFITNIPFD VKW QS LKDL VKEKV GE VT Y VE
LLMDAEGKSRGCAVVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHA
RRAMQKAGRLGSTVFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKS
RGIGTVTFEQSIEAVQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQI
FVRNLPFDFTWKMLKDKFNECGHVLYADIKMENGKSKGCGVVKFESPEVAER
ACRMMNGMKLSGREIDVRIDRNALDPKSAKPTTTPAPRPPTPAPTIASQPLSLRP
EAARPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPL
FPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR
PGPTRKHYQPYAPPRDFAAYRSRVKFSRSAEPPAYQQGQNQLYNELNLGRREE
YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR
GKGHDGLY QGLSTATKDTYD ALHMQALPPR struct DNA
(SEQ ID NO: 29)
ATGGAGGAATTCGCAGGTTCCTTTGGTGGAGCTGGAGGCCATGCTCCTGGG
GTGGCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTC
ACATGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTAC
GCCGACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGCGTGGTTAA
GTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCAT
GAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCAGGTA
CCGGATCCAAGCCTACTACAACTCCAGCGCCGCGACCACCAACACCGGCGC
CCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAA
CGGCTGGAGGTGCAGTGCACACGAGGGGGCTGGACTTCGCAGATCCCAAA
GTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAG
TGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCCGGCTCCTGCACA
GTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATT
ACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTACCGGTCCAGAGTGA
AGTTCAGCCGGTCCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGC
TCTATAACGAGCTCAATCTACGAAAGAGGAGTACGATGTTTTGGACAGAGA
CGTGGCCGGGACCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAG
GAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAG
TGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCC
TTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACA
TGCAAGCTTTGCCCCCTCGCTAA struct AA
(SEQ ID NO: 30)
MEEFAGSFGGAGGHAPGVARKACQIFVRNLPFDFTWKMLKDKFNECGHVLYA DIKMENGKSKGCGVVKFESPEVAERACRMMNGMKLSGREIDVRIDRNAGTGS KPTTTPAPRPPTPAPTI AS QPLS LRPEACRPT AGG A VHTRGLDFADPKVL V V V G GVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLRKRSTMFWTETWPGPEMG GKPRRKNPQEGLYNELQKDKMAEA Y SEIGMKGERRRGKGHDGLY QGLSTAT KDTYD ALHMQALPPR struct DNA
(SEQ ID NO: 31)
ATGGAACAAAAACTCATCTCAGAAGAGGATCTCGAGGAATTCGCAGGTTCC
TTTGGTGGAGCTGGAGGCCATGCTCCTGGGGTGGCCAGGAAGGCCTGCCAG
ATATTTGTGAGAAATCTGCCATTCGATTTCACATGGAAGATGCTAAAGGAC
AAATTCAACGAGTGCGGCCACGTGCTGTACGCCGACATCAAGATGGAGAAT
GGGAAGTCCAAGGGGTGTGGCGTGGTTAAGTTCGAGTCGCCAGAGGTGGCC
GAGAGAGCCTGCCGGATGATGAATGGCATGAAGCTGAGTGGCCGAGAGAT
TGACGTTCGAATTGATAGAAACGCAGGTACCGGATCCAAGCCTACTACAAC
TCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCT
GTCCCTGCGCCCAGAGGCGTGCCGGCCAACGGCTGGAGGTGCAGTGCACAC
GAGGGGGCTGGACTTCGCAGATCCCAAAGTGCTGGTGGTGGTTGGTGGAGT
CCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG
AGGAGTAAGAGGAGCCGGCTCCTGCACAGTGACTACATGAACATGACTCCC
CGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGC
GACTTCGCAGCCTACCGGTCCAGAGTGAAGTTCAGCCGGTCCGCAGACGCC
CCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTACGA
AAGAGGAGTACGATGTTTTGGACAGAGACGTGGCCGGGACCTGAGATGGG
GGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGC
AGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAG
CGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCC
ACCAAGGACACCTACGACGCCCTTCACATGCAAGCTTTGCCCCCTCGCTAA struct AA
(SEQ ID NO: 32)
MEQKLISEEDLEEFAGSFGGAGGHAPGVARKACQIFVRNLPFDFTWKMLKDKF NECGHVLY ADIKMEN GKS KGCG V VKFESPE V AER ACRMMN GMKLS GREID V RIDRNAGTGSKPTTTPAPRPPTPAPTIASQPLSLRPEACRPTAGGAVHTRGLDFA DPKVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRK HY QPYAPPRDFAAYRSRVKFSRS ADAPAY QQGQNQLYNELNLRKRSTMFWTE TWPGPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL Y QGLSTATKDTYD ALHMQALPPR struct DNA
(SEQ ID NO: 33)
ATGGCTGGAAGACTTGGAAGCACAGTATTTGTAGCAAATCTGGATTATAAA
GTTGGCTGGAAGAAACTGAAGGAAGTATTTAGTATGGCTGGTGTGGTGGTC
CGAGCAGACATTCTTGAAGATAAAGATGGAAAAAGTCGTGGAATAGGCAC
TGTTACTTTTGAACAGTCCATTGAAGCTGTGCAAGCTATATCTATGTTCAAT
GGCCAGCTGCTATTTGATAGACCAATGCACGTCAAGATGGATGAGAGGGCC
TTACCAAAAGGATCCGGGGTGGCCAGGAAGGCCTGCCAGATATTTGTGAGA
AATCTGCCATTCGATTTCACATGGAAGATGCTAAAGGACAAATTCAACGAG
TGCGGCCACGTGCTGTACGCCGACATCAAGATGGAGAATGGGAAGTCCAA
GGGGTGTGGTGTGGTTAAGTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTG
CCGGATGATGAATGGCATGAAGCTGAGTGGCCGAGAGATTGACGTTCGAAT
TGATAGAAACGCCGCTCTAGATCCCAAAAGTGCTAAGCCCACCACGACGCC
AGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTC CCTGCGCCCAGAGGCGGCTCGGCCAGCGGCGGGGGGCGCAGTGCACACGA
GGGGGCTGGACTTCGCCCCTAGGAAAATTGAAGTTATGTATCCTCCTCCTTA
CCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAAC
ACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCT
GGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC
CTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGA
CTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCA
GCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTT
CAGCAGGAGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCT
ATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAG
AGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCC
TCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCT
ACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGAT
GGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTT
CACATGCAGGCCCTGCCCCCTCGCTAA struct AA
(SEQ ID NO: 34)
MAGRLGSTVFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKSRGIGT
VTFEQSIEAVQAISMFNGQEEFDRPMHVKMDERAEPKGSGVARKACQIFVRNE
PFDFTWKMEKDKFNECGHVEYADIKMENGKSKGCGVVKFESPEVAERACRM
MNGMKESGREIDVRIDRNAAEDPKSAKPTTTPAPRPPTPAPTIASQPESERPEAA
RPAAGGAVHTRGEDFAPRKIEVMYPPPYEDNEKSNGTIIHVKGKHECPSPEFPG
PSKPFWVEVVVGGVEACYSEEVTVAFIIFWVRSKRSREEHSDYMNMTPRRPGP
TRKH Y QP Y APPRDFA A YRSR VKFSRS AEPP A Y QQGQN QE YNEENEGRREE YD
VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK
GHDGLY QGLSTATKDTYDALHMQALPPR struct DNA
(SEQ ID NO: 35)
ATGGAACAAAAACTCATCTCAGAAGAGGATCTGGGCGGCTCCGGCGCTGG
AAGACTTGGAAGCACAGTATTTGTAGCAAATCTGGATTATAAAGTTGGCTG
GAAGAAACTGAAGGAAGTATTTAGTATGGCTGGTGTGGTGGTCCGAGCAGA
CATTCTTGAAGATAAAGATGGAAAAAGTCGTGGAATAGGCACTGTTACTTT
TGAACAGTCCATTGAAGCTGTGCAAGCTATATCTATGTTCAATGGCCAGCT
GCTATTTGATAGACCAATGCACGTCAAGATGGATGAGAGGGCCTTACCAAA
AGGATCCGGGGTGGCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCC
ATTCGATTTCACATGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCA
CGTGCTGTACGCCGACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTG
GTGTGGTTAAGTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGA
TGAATGGCATGAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAA
ACGCCGCTCTAGATCCCAAAAGTGCTAAGCCCACCACGACGCCAGCGCCGC
GACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCC
CAGAGGCGGCTCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTG
GACTTCGCCCCTAGGAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACA
ATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTC
CAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGT
TGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATT TTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAAC
ATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCC
CCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGC
GCAGAGCCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC
AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCG
GGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCC
TGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATT
GGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCA
GGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGC
CCTGCCCCCTCGCTAA struct AA
(SEQ ID NO: 36)
MEQKLISEEDLGGSGAGRLGSTVFVANLDYKVGWKKLKEVFSMAGVVVRADI LEDKDGKSRGIGTVTFEQSIEAVQAISMFNGQLLFDRPMHVKMDERALPKGSG V ARKACQIF VRNLPFDFTWKMLKD KFNECGH VLY ADIKMEN GKS KGCG V VKF ESPEVAERACRMMNGMKLSGREIDVRIDRNAALDPKSAKPTTTPAPRPPTPAPT IAS QPLS LRPE A ARPA AGG A VHTRGLDFAPRKIE VM YPPPYLDNEKSNGTIIH V KGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHS D YMNMTPRRPGPTRKH Y QPY APPRDFAA YRSRVKFSRS AEPPA Y QQGQNQLY NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR struct DNA
(SEQ ID NO: 37)
ATGGCCAATCCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTT
GATGTGAAATGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAG
GTAACATACGTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGT
GCTGTTGTTGAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGT
CCTAAACAAGCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATC
CTGATGGTGAACATGCCAGGAGAGCAATGCAAAAGTCCGGGGTGGCCAGG
AAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTCACATGGAAG
ATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTACGCCGACATC
AAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAAGTTCGAGTC
GCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCATGAAGCTGA
GTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCCGCTCTAGATCCCA
AAAGTGCTAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCG
CCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGGCTCGGCCA
GCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCCCTAGGAA
AATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGG
AACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCC
GGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTT
GCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAA
GAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCC
CGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGC
AGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCGTA
CCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAG
AGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA
GAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGC
GCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCC
ACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA
C9 Construct AA
(SEQ ID NO: 38)
M ANPTKRYRAFITNIPFD VKW QS LKDLVKEKV GE VT Y VELLMD AEGKSRGC A
VVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHARRAMQKSGVARKA
CQIFVRNLPFDFTWKMLKDKFNECGHVLYADIKMENGKSKGCGVVKFESPEV
AERACRMMNGMKLSGREIDVRIDRNAALDPKSAKPTTTPAPRPPTPAPTIASQP
LSLRPEAARPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHL
CPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMN
MTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSAEPPAYQQGQNQLYNELNL
GRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK
GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
CIO Construct DNA
(SEQ ID NO: 39)
ATGGAACAAAAACTCATCTCAGAAGAGGATCTGGGCGGCTCCGGCGCCAAT
CCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTTGATGTGAAA
TGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAGGTAACATAC
GTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGTGCTGTTGTT
GAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGTCCTAAACAA
GCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATCCTGATGGTG
AACATGCCAGGAGAGCAATGCAAAAGTCCGGGGTGGCCAGGAAGGCCTGC
CAGATATTTGTGAGAAATCTGCCATTCGATTTCACATGGAAGATGCTAAAG
GACAAATTCAACGAGTGCGGCCACGTGCTGTACGCCGACATCAAGATGGAG
AATGGGAAGTCCAAGGGGTGTGGTGTGGTTAAGTTCGAGTCGCCAGAGGTG
GCCGAGAGAGCCTGCCGGATGATGAATGGCATGAAGCTGAGTGGCCGAGA
GATTGACGTTCGAATTGATAGAAACGCCGCTCTAGATCCCAAAAGTGCTAA
GCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGC
GTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGGCTCGGCCAGCGGCGGGGGG
CGCAGTGCACACGAGGGGGCTGGACTTCGCCCCTAGGAAAATTGAAGTTAT
GTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCA
TGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAG
CCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGC
TAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGC
TCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCC
GCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCT
CCAGAGTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCGTACCAGCAGGGC
CAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGA
TGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGA
GAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAG
ATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGG
CAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACAC
CTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA CIO Construct DNA
(SEQ ID NO: 40)
MEQKLISEEDLGGS G ANPTKRYRAFITNIPFD VKW QS LKDL VKEKV GE VT Y VE LLMDAEGKSRGCAVVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHA RRAMQKSGVARKACQIFVRNLPFDFTWKMLKDKFNECGHVLYADIKMENGK SKGCGVVKFESPEVAERACRMMNGMKLSGREIDVRIDRNAALDPKSAKPTTTP APRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNE KSN GTIIH VKGKHLCPSPLFPGPS KPFWVLV V V GG VL AC Y S LLVT V AFIIFW VRS KRSRLLHSD YMNMTPRRPGPTRKHY QPY APPRDFAA YRSRVKFSRS AEPPA Y Q QGQN QL YNELNLGRREEYD VLD KRRGRDPEMGGKPRRKNPQEGL YNELQKD KMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR
Cll Construct DNA
(SEQ ID NO: 41)
ATGGCCAATCCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTT
GATGTGAAATGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAG
GTAACATACGTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGT
GCTGTTGTTGAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGT
CCTAAACAAGCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATC
CTGATGGTGAACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGA
AGCACAGTATTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTG
AAGGAAGTATTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAA
GATAAAGATGGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCC
ATTGAAGCTGTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTTGATA
GACCAATGCACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGG
GTGGCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTC
ACATGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTAC
GCCGACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAA
GTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCAT
GAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGA
TCCCAAAGCCAATCCAACTAAAAGATACAGAGCCTTCATTACAAACATACC
TTTTGATGTGAAATGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGG
TGAGGTAACATACGTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGG
GATGTGCTGTTGTTGAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGG
AAGTCCTAAACAAGCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAA
GATCCTGATGGTGAACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACT
TGGAAGCACAGTATTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAA
ACTGAAGGAAGTATTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCT
TGAAGATAAAGATGGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACA
GTCCATTGAAGCTGTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTT
GATAGACCAATGCACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATC
CGGGGTGGCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGA
TTTCACATGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCT
GTACGCCGACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGG
TTAAGTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATG
GCATGAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTC
TAGATCCCAAAAGTGCTAAGCCCACCACGACGCCAGCGCCGCGACCACCAA
CACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGG CTCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCC
CCTAGGAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAG
AGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCC
CTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAG
TCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGT
GAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTC
CCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCAC
GCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGAGC
CCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAG
GACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCT
GAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAA
TGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGA
AAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTC
AGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCC
CCTCGCTAA
Cll Construct AA
(SEQ ID NO: 42)
M ANPTKRYRAFITNIPFD VKW QS LKDLVKEKV GE VT Y VELLMD AEGKSRGC A
VVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHARRAMQKAGRLGST
VFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKSRGIGTVTFEQSIEA
VQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQIFVRNLPFDFTWKM
LKD KFNECGH VLY ADIKMEN GKS KGCG V VKFESPE V AER ACRMMN GMKLS G
REIDVRIDRNALDPKANPTKRYRAFITNIPFDVKWQSLKDLVKEKVGEVTYVEL
LMDAEGKSRGCAVVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHAR
RAMQKAGRLGSTVFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKSR
GIGTVTFEQSIEA V QAISMFNGQLLFDRPMHVKMDERALPKGS GV ARKACQIF
VRNLPFDFTWKMLKDKFNECGHVLYADIKMENGKSKGCGVVKFESPEVAERA
CRMMN GMKLS GREID VRIDRN ALDPKS AKPTTTPAPRPPTPAPTI AS QPLS LRPE
AARPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLF
PGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRP
GPTRKHY QPY APPRDFAAYRSRVKFSRS AEPPA Y QQGQNQLYNELNLGRREEY
DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG
KGHDGLY QGLSTATKDTYD ALHMQ ALPPR
C12 Construct DNA
(SEQ ID NO: 43)
ATGGAACAAAAACTCATCTCAGAAGAGGATCTGGGCGGCTCCGGCGCCAAT
CCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTTGATGTGAAA
TGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAGGTAACATAC
GTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGTGCTGTTGTT
GAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGTCCTAAACAA
GCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATCCTGATGGTG
AACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGAAGCACAGTA
TTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTGAAGGAAGTA
TTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAAGATAAAGAT
GGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCCATTGAAGCT
GTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTTGATAGACCAATGC ACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGGGTGGCCAGG
AAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTCACATGGAAG
ATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTACGCCGACATC
AAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAAGTTCGAGTC
GCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCATGAAGCTGA
GTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGATCCCAAAG
CCAATCCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTTGATG
TGAAATGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAGGTAA
CATACGTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGTGCTG
TTGTTGAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGTCCTA
AACAAGCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATCCTGA
TGGTGAACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGAAGCA
CAGTATTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTGAAGG
AAGTATTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAAGATA
AAGATGGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCCATTG
A AGCTGTGC A AGCT AT ATCT ATGTTC A AT GGCC AGCTGCT ATTTG AT AGACC
AATGCACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGGGTGG
CCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTCACAT
GGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTACGCC
GACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAAGTTC
GAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCATGAA
GCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGATCC
CAAAAGTGCTAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGG
CGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGGCTCGGC
CAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCCCTAGG
AAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAAT
GGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTC
CCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGC
TTGCT AT AGCTTGCT AGT A AC AGTGGCCTTT ATT ATTTTCTGGGT GAGG AGT
AAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGC
CCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTC
GCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCG
TACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAG
AGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGG
GGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG
CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGA
GCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAG
CCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCT
AA
C12 Construct AA
(SEQ ID NO: 44)
MEQKLISEEDLGGS G ANPTKRYRAFITNIPFD VKW QS LKDL VKEKV GE VT Y VE
LLMDAEGKSRGCAVVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHA
RRAMQKAGRLGSTVFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKS
RGIGTVTFEQSIEAVQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQI
FVRNLPFDFTWKMLKDKFNECGHVLYADIKMENGKSKGCGVVKFESPEVAER
ACRMMNGMKLSGREIDVRIDRNALDPKANPTKRYRAFITNIPFDVKWQSLKDL
VKEKVGEVTYVELLMDAEGKSRGCAVVEFKMEESMKKAAEVLNKHSLSGRP LKVKEDPDGEHARRAMQKAGRLGSTVFVANLDYKVGWKKLKEVFSMAGVV
VRADILEDKDGKSRGIGTVTFEQSIEAVQAISMFNGQLLFDRPMHVKMDERAL
PKGSGVARKACQIFVRNLPFDFTWKMLKDKFNECGHVLYADIKMENGKSKGC
GVVKFESPEVAERACRMMNGMKLSGREIDVRIDRNALDPKSAKPTTTPAPRPP
TPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNG
TIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSR
LLHSD YMNMTPRRPGPTRKHY QPY APPRDFAAYRSRVKFSRS AEPPA Y QQGQ
NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA
EAY SEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR
C13 Construct DNA
(SEQ ID NO: 45)
ATGGCCAATCCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTT
GATGTGAAATGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAG
GTAACATACGTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGT
GCTGTTGTTGAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGT
CCTAAACAAGCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATC
CTGATGGTGAACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGA
AGCACAGTATTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTG
AAGGAAGTATTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAA
GATAAAGATGGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCC
ATTGAAGCTGTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTTGATA
GACCAATGCACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGG
GTGGCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTC
ACATGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTAC
GCCGACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAA
GTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCAT
GAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGA
TCCCAAAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATT
TATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATT
TCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGCGTTAAAAGAGTGAAGT
TCAGCAGGAGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAGAACCAGCTC
TATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAG
AGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCC
TCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCT
ACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGAT
GGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTT
CACATGCAGGCCCTGCCCCCTCGCTAA
C13 Construct AA
(SEQ ID NO: 46)
M ANPTKRYRAFITNIPFD VKW QS LKDLVKEKV GE VT Y VELLMD AEGKSRGC A
VVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHARRAMQKAGRLGST
VFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKSRGIGTVTFEQSIEA
VQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQIFVRNLPFDFTWKM
LKD KFNECGH VLY ADIKMEN GKS KGCG V VKFESPE V AER ACRMMN GMKLS G
REIDVRIDRNALDPKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG
CELRVKRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEA Y SEIGMKGERRRGKGHDGLY QGLSTAT KDTYDALHMQALPPR
C14 Construct DNA
(SEQ ID NO: 47)
ATGGAACAAAAACTCATCTCAGAAGAGGATCTGGGCGGCTCCGGCGCCAAT
CCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTTGATGTGAAA
TGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAGGTAACATAC
GTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGTGCTGTTGTT
GAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGTCCTAAACAA
GCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATCCTGATGGTG
AACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGAAGCACAGTA
TTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTGAAGGAAGTA
TTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAAGATAAAGAT
GGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCCATTGAAGCT
GTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTTGATAGACCAATGC
ACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGGGTGGCCAGG
AAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTCACATGGAAG
ATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTACGCCGACATC
AAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAAGTTCGAGTC
GCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCATGAAGCTGA
GTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGATCCCAAAA
A ACGGGGC AGA A AG A AACTCCTGT AT AT ATTC A AAC A ACC ATTT ATG AG AC
CAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAG
AAGAAGAAGGAGGATGTGAACTGCGCGTTAAAAGAGTGAAGTTCAGCAGG
AGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGA
GCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTG
GCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA
GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA
GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTT
ACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC
AGGCCCTGCCCCCTCGCT A A
C14 Construct AA
(SEQ ID NO: 48)
MEQKLISEEDLGGS G ANPTKRYRAFITNIPFD VKW QS LKDL VKEKV GE VT Y VE
LLMDAEGKSRGCAVVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHA
RRAMQKAGRLGSTVFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKS
RGIGTVTFEQSIEAVQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQI
FVRNLPFDFTWKMLKDKFNECGHVLYADIKMENGKSKGCGVVKFESPEVAER
ACRMMNGMKLSGREIDVRIDRNALDPKKRGRKKLLYIFKQPFMRPVQTTQEE
DGCSCRFPEEEEGGCELRVKRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYD
VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK
GHDGLY QGLSTATKDTYDALHMQALPPR C15 Construct DNA
(SEQ ID NO: 49)
ATGGCCAATCCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTT
GATGTGAAATGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAG
GTAACATACGTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGT
GCTGTTGTTGAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGT
CCTAAACAAGCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATC
CTGATGGTGAACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGA
AGCACAGTATTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTG
AAGGAAGTATTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAA
GATAAAGATGGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCC
ATTGAAGCTGTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTTGATA
GACCAATGCACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGG
GTGGCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTC
ACATGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTAC
GCCGACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAA
GTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCAT
GAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGA
TCCCAAAAGTGCTAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACC
GGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGGCTCG
GCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCCCTA
GGAGAAACAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCA
TTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGA
TTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGCGTTAAAAGAGTGAA
GTTCAGCAGGAGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAGAACCAGC
TCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACA
AGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAA
CCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGG
CCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC
GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCC
CTTCACATGCAGGCCCTGCCCCCTCGCTAA
C15 Construct AA
(SEQ ID NO: 50)
M ANPTKRYRAFITNIPFD VKW QS LKDLVKEKV GE VT Y VELLMD AEGKSRGC A
VVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHARRAMQKAGRLGST
VFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKSRGIGTVTFEQSIEA
VQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQIFVRNLPFDFTWKM
LKD KFNECGH VLY ADIKMEN GKS KGCG V VKFESPE V AER ACRMMN GMKLS G
REIDVRIDRNALDPKSAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVH
TRGLDFAPRRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR
VKRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDTY
DALHMQALPPR C16 Construct DNA
(SEQ ID NO: 51)
ATGGAACAAAAACTCATCTCAGAAGAGGATCTGGGCGGCTCCGGCGCCAAT
CCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTTGATGTGAAA
TGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAGGTAACATAC
GTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGTGCTGTTGTT
GAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGTCCTAAACAA
GCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATCCTGATGGTG
AACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGAAGCACAGTA
TTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTGAAGGAAGTA
TTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAAGATAAAGAT
GGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCCATTGAAGCT
GTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTTGATAGACCAATGC
ACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGGGTGGCCAGG
AAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTCACATGGAAG
ATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTACGCCGACATC
AAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAAGTTCGAGTC
GCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCATGAAGCTGA
GTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGATCCCAAAA
GTGCTAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCA
CCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGGCTCGGCCAGCGG
CGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCCCTAGGAGAAAC
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGA
CCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAA
GAAGAAGAAGGAGGATGTGAACTGCGCGTTAAAAGAGTGAAGTTCAGCAG
GAGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACG
AGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGT
GGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGA
AGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTG
AGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTT
TACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATG
CAGGCCCTGCCCCCTCGCTAA
C16 Construct AA
(SEQ ID NO: 52)
MEQKLISEEDLGGS G ANPTKRYRAFITNIPFD VKW QS LKDL VKEKV GE VT Y VE
LLMDAEGKSRGCAVVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHA
RRAMQKAGRLGSTVFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKS
RGIGTVTFEQSIEAVQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQI
FVRNLPFDFTWKMLKDKFNECGHVLYADIKMENGKSKGCGVVKFESPEVAER
ACRMMNGMKLSGREIDVRIDRNALDPKSAKPTTTPAPRPPTPAPTIASQPLSLRP
EAARPAAGGAVHTRGLDFAPRRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC
RFPEEEEGGCELRVKRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKR
RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL
Y QGLSTATKDTYD ALHMQALPPR C17 Construct DNA
(SEQ ID NO: 53)
ATGGAGGAATTCGCAGGTTCCTTTGGTGGAGCTGGAGGCCATGCTCCTGGG
GTGGCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTC
ACATGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTAC
GCCGACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGCGTGGTTAA
GTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCAT
GAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGA
TCCCAAAAGTGCTAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACC
GGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGGCTCG
GCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCCCTA
GGAGAAACAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCA
TTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGA
TTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGCGTTAAAAGAGTGAA
GTTCAGCAGGAGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAGAACCAGC
TCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACA
AGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAA
CCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGG
CCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC
GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCC
CTTCACATGCAGGCCCTGCCCCCTCGCTAA
C17 Construct AA
(SEQ ID NO: 54)
MEEFAGSFGGAGGHAPGVARKACQIFVRNLPFDFTWKMLKDKFNECGHVLYA
DIKMENGKSKGCGVVKFESPEVAERACRMMNGMKFSGREIDVRIDRNAFDPK
SAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAPRRNKRG
RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKRVKFSRSAEPPA
YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ
KDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDTYD ALHMQALPPR
C18 Construct DNA
(SEQ ID NO: 55)
ATGGAACAAAAACTCATCTCAGAAGAGGATCTGGGCGGCTCCGGCGAGGA
ATTCGCAGGTTCCTTTGGTGGAGCTGGAGGCCATGCTCCTGGGGTGGCCAG
GAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTCACATGGAA
GATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTACGCCGACAT
CAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGCGTGGTTAAGTTCGAGT
CGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCATGAAGCTG
AGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGATCCCAAA
AGTGCTAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCC
ACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGGCTCGGCCAGCG
GCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCCCTAGGAGAAA
CAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAG
ACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGA
AGAAGAAGAAGGAGGATGTGAACTGCGCGTTAAAAGAGTGAAGTTCAGCA
GGAGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAAC GAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACG
TGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGG
AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT
GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCT
TTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACAT
GCAGGCCCTGCCCCCTCGCTAA
C18 Construct AA
(SEQ ID NO: 56)
MEQKLISEEDLGGSGEEFAGSFGGAGGHAPGVARKACQIFVRNLPFDFTWKML
KDKFNECGHVLYADIKMENGKSKGCGVVKFESPEVAERACRMMNGMKLSGR
EIDVRIDRNALDPKSAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHT
RGLDFAPRRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV
KRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR
KNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDTYD
ALHMQALPPR
C19 Construct DNA
(SEQ ID NO: 57)
ATGGCCAATCCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTT
GATGTGAAATGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAG
GTAACATACGTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGT
GCTGTTGTTGAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGT
CCTAAACAAGCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATC
CTGATGGTGAACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGA
AGCACAGTATTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTG
AAGGAAGTATTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAA
GATAAAGATGGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCC
ATTGAAGCTGTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTTGATA
GACCAATGCACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGG
GTGGCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTC
ACATGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTAC
GCCGACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAA
GTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCAT
GAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGA
TCCCAAAAGTGCTAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACC
GGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGGCTCG
GCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCCCTA
GGAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCA
ATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTAT
TTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCT
GGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGG
AGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC
CGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGAC
TTCGCAGCCTATCGCTCCAGAAACAAACGGGGCAGAAAGAAACTCCTGTAT
ATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT
GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCG
CGTTAAAAGAGTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCGTACCAGC AGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAG
TACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAA
GCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAG
ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG
AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAA
GGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA
C19 Construct AA
(SEQ ID NO: 58)
M ANPTKRYRAFITNIPFD VKW QS LKDLVKEKV GE VT Y VELLMD AEGKSRGC A
VVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHARRAMQKAGRLGST
VFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKSRGIGTVTFEQSIEA
VQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQIFVRNLPFDFTWKM
LKD KFNECGH VLY ADIKMEN GKS KGCG V VKFESPE V AER ACRMMN GMKLS G
REIDVRIDRNALDPKSAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVH
TRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLV
VVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA
PPRDFAAYRSRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
RVKRVKFSRS AEPPA Y QQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKP
RRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDT
YDALHMQALPPR
C20 Construct DNA
(SEQ ID NO: 59)
ATGGAACAAAAACTCATCTCAGAAGAGGATCTGGGCGGCTCCGGCGCCAAT
CCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTTGATGTGAAA
TGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAGGTAACATAC
GTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGTGCTGTTGTT
GAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGTCCTAAACAA
GCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATCCTGATGGTG
AACATGCCAGGAGAGCAATGCAAAAGGCTGGAAGACTTGGAAGCACAGTA
TTTGTAGCAAATCTGGATTATAAAGTTGGCTGGAAGAAACTGAAGGAAGTA
TTTAGTATGGCTGGTGTGGTGGTCCGAGCAGACATTCTTGAAGATAAAGAT
GGAAAAAGTCGTGGAATAGGCACTGTTACTTTTGAACAGTCCATTGAAGCT
GTGCAAGCTATATCTATGTTCAATGGCCAGCTGCTATTTGATAGACCAATGC
ACGTCAAGATGGATGAGAGGGCCTTACCAAAAGGATCCGGGGTGGCCAGG
AAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTCACATGGAAG
ATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTACGCCGACATC
AAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGTGGTTAAGTTCGAGTC
GCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCATGAAGCTGA
GTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGATCCCAAAA
GTGCTAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCA
CCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGGCTCGGCCAGCGG
CGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCCCTAGGAAAATT
GAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACC
ATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGAC
CTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTA
TAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGG AGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGG
CCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCC
TATCGCTCCAGAAACAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAA
CAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGC
TGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGCGTTAAAAG
AGTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAGA
ACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT
TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGG
AAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGC
GGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGG
GGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACG
ACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA
C20 Construct DNA
(SEQ ID NO: 60)
MEQKLISEEDLGGS G ANPTKRYRAFITNIPFD VKW QS LKDL VKEKV GE VT Y VE
LLMDAEGKSRGCAVVEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHA
RRAMQKAGRLGSTVFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKS
RGIGTVTFEQSIEAVQAISMFNGQLLFDRPMHVKMDERALPKGSGVARKACQI
FVRNLPFDFTWKMLKDKFNECGHVLYADIKMENGKSKGCGVVKFESPEVAER
ACRMMNGMKLSGREIDVRIDRNALDPKSAKPTTTPAPRPPTPAPTIASQPLSLRP
EAARPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPL
FPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR
PGPTRKHYQPYAPPRDFAAYRSRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCS
CRFPEEEEGGCELRVKRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDK
RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG
LY QGLSTATKDTYD ALHMQALPPR
C21 Construct DNA
(SEQ ID NO: 61)
ATGGAGGAATTCGCAGGTTCCTTTGGTGGAGCTGGAGGCCATGCTCCTGGG
GTGGCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTC
ACATGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTAC
GCCGACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGCGTGGTTAA
GTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCAT
GAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGA
TCCCAAAAGTGCTAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACC
GGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGGCTCG
GCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCCCTA
GGAAAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCA
ATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTAT
TTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCT
GGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGG
AGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC
CGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGAC
TTCGCAGCCTATCGCTCCAGAAACAAACGGGGCAGAAAGAAACTCCTGTAT
ATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT
GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCG CGTTAAAAGAGTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCGTACCAGC
AGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAG
TACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAA
GCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAG
ATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG
AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAA
GGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA
C21 Construct AA
(SEQ ID NO: 62)
MEEFAGSFGGAGGHAPGVARKACQIFVRNLPFDFTWKMLKDKFNECGHVLYA
DIKMENGKSKGCGVVKFESPEVAERACRMMNGMKFSGREIDVRIDRNAFDPK
SAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAPRKIEVMY
PPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTV
AFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRNKRG
RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKRVKFSRSAEPPA
YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ
KDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDTYD ALHMQALPPR
C22 Construct DNA
(SEQ ID NO: 63)
ATGGAACAAAAACTCATCTCAGAAGAGGATCTGGGCGGCTCCGGCGAGGA
ATTCGCAGGTTCCTTTGGTGGAGCTGGAGGCCATGCTCCTGGGGTGGCCAG
GAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTCACATGGAA
GATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTACGCCGACAT
CAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGCGTGGTTAAGTTCGAGT
CGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCATGAAGCTG
AGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGCTCTAGATCCCAAA
AGTGCTAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCC
ACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGGCTCGGCCAGCG
GCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCCCTAGGAAAAT
TGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAAC
CATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGA
CCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCT
ATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAG
GAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGG
GCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGC
CTATCGCTCCAGAAACAAACGGGGCAGAAAGAAACTCCTGTATATATTCAA
ACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAG
CTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGCGTTAAAA
GAGTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAG
AACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTT
TTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAG
GAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGG
CGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAG
GGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTAC
GACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA C22 Construct DNA
(SEQ ID NO: 63)
MEQKLISEEDLGGSGEEFAGSFGGAGGHAPGVARKACQIFVRNLPFDFTWKML
KDKFNECGHVLYADIKMENGKSKGCGVVKFESPEVAERACRMMNGMKLSGR
EIDVRIDRNALDPKSAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHT
RGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVV
VGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY QPY AP
PRDFAAYRSRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR
VKRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDTY
DALHMQALPPR
MYC DNA
(SEQ ID NO: 64)
GAACAAAAACTCATCTCAGAAGAGGATCTGGGCGGCTCCGGC
MYC AA
(SEQ ID NO: 65)
EQKLISEEDLGGSG RRM1 DNA
(SEQ ID NO: 66)
GCCAATCCAACTAAAAGATACAGAGCCTTCATTACAAACATACCTTTTGAT
GTGAAATGGCAGTCACTTAAAGACCTGGTTAAAGAAAAAGTTGGTGAGGTA
ACATACGTGGAGCTCTTAATGGACGCTGAAGGAAAGTCAAGGGGATGTGCT
GTTGTTGAATTCAAGATGGAAGAGAGCATGAAAAAAGCTGCGGAAGTCCT
AAACAAGCATAGTCTGAGCGGAAGACCACTGAAAGTCAAAGAAGATCCTG
ATGGTGAACATGCCAGGAGAGCAATGCAAAAG
RRM1 A A
(SEQ ID NO: 67)
ANPTKRYRAFITNIPFDVKWQSLKDLVKEKVGEVTYVELLMDAEGKSRGCAV
VEFKMEESMKKAAEVLNKHSLSGRPLKVKEDPDGEHARRAMQK
RRM2 DNA
(SEQ ID NO: 68)
GCTGGAAGACTTGGAAGCACAGTATTTGTAGCAAATCTGGATTATAAAGTT
GGCTGGAAGAAACTGAAGGAAGTATTTAGTATGGCTGGTGTGGTGGTCCGA
GCAGACATTCTTGAAGATAAAGATGGAAAAAGTCGTGGAATAGGCACTGTT
ACTTTTGAACAGTCCATTGAAGCTGTGCAAGCTATATCTATGTTCAATGGCC
AGCTGCTATTTGATAGACCAATGCACGTCAAGATGGATGAGAGGGCCTTAC
CAAAAGGA RRM2 AA
(SEQ ID NO: 69)
AGRLGSTVFVANLDYKVGWKKLKEVFSMAGVVVRADILEDKDGKSRGIGTVT
FEQSIEAVQAISMFNGQLLFDRPMHVKMDERALPKG
RRM3 DNA
(SEQ ID NO: 70)
TCCGGGGTGGCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTC
GATTTCACATGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTG
CTGTACGCCGACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGTGT
GGTTAAGTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGA
ATGGCATGAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACG
CTCT AG ATCCC A AA
RRM3 AA
(SEQ ID NO: 71)
S GV ARKACQIFVRNLPFDFTWKMLKDKFNECGH VLY ADIKMEN GKS KGCG V V KFESPEVAERACRMMNGMKLSGREID VRIDRN ALDPK
RRM3 DNA
(SEQ ID NO: 72)
GTGGTTAAGTTCGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATG
AATGGCATGAAGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAAC
GC
RRM3 AA
(SEQ ID NO: 73)
V VKFES PE V AER ACRMMN GMKLS GREID VRIDRN RRM3 DNA
(SEQ ID NO: 74)
GAGGAATTCGCAGGTTCCTTTGGTGGAGCTGGAGGCCATGCTCCTGGGGTG
GCCAGGAAGGCCTGCCAGATATTTGTGAGAAATCTGCCATTCGATTTCACA
TGGAAGATGCTAAAGGACAAATTCAACGAGTGCGGCCACGTGCTGTACGCC
GACATCAAGATGGAGAATGGGAAGTCCAAGGGGTGTGGCGTGGTTAAGTT
CGAGTCGCCAGAGGTGGCCGAGAGAGCCTGCCGGATGATGAATGGCATGA
AGCTGAGTGGCCGAGAGATTGACGTTCGAATTGATAGAAACGC
RRM3 AA
(SEQ ID NO: 75)
EEFAGSFGGAGGHAPGVARKACQIFVRNLPFDFTWKMLKDKFNECGHVLYAD IKMENGKSKGCGVVKFESPEVAERACRMMNGMKFSGREID VRIDRN INCORPORATION BY REFERENCE
[00182] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention. To the extent that any of the definitions or terms provided in the references incorporated by reference differ from the terms and discussion provided herein, the present terms and definitions control.
EXAMPLES
[00183] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The foregoing description and examples detail certain preferred embodiments of the invention and describe the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof.
[00184] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.
Example 1 nlrCEA CAR molecular construction and expression.
[00185] An exemplary nlrCEA CAR of the present invention is depicted in FIG. 1 and comprises a nucleic acid sequence of SEQ ID NO. 1. To clone DNA encoding the extracellular domain of v, the RRM1, RRM2 and RRM3 domains were isolated from the plasmid pT7-V5-SBP-Cl-HshnRNPM (Addgene, Cat# 64924) using the following primer pairs:
CEA5pr : 5 ’ GACTCCATGGCCAATCCAACTAAAAGATAC 3 ’ ;
CEA3pr : 5 ’ TGGAAGATCTAGAGCTTTCTATC AATTCG 3 ’ ;
CEARRM3 : 5’ CTATGGATCCGGGGTGGCCAGGAAG 3’;
CEARRM2 : 5’ CATCGGATCCTTTTGGTAAGGCCCTCTC 3’.
The final construct had 3 RRM domains. See, e.g., FIG. 1. The nlrCEA fragment generated using the primers was then joined with a second generation CAR construct (comprising a CD8 hinge domain, portions of the extracellular, transmembrane and intracellular CD28 domain; and the Oϋ3z intracellular domains) using known techniques in the art. The resulting construct was then inserted between the Ncol and BamHl sites of an MFG retroviral backbone.
Example 2 Expression of nlrCEA in Virus Producing Cells
[00186] Next, viral supernatant for transducing T cells with the nlrCEA-CAR was manufactured by creating stable Vims Producing Cell (“VPC”) lines. First, the PG13 cell line was transfected with rMR0-hM3EA^ϋ28^03z vector as shown in FIG 1 to generate PG13 nlrCEA VPC line. RNA was isolated from PG13 nlrCEA VPC and sequencing was performed for confirmation. The viral supernatant produced from the packaging cell line is then used to transfect the PG13 cells.
[00187] After infection, the PG13 cells were assayed for expression of the CAR transgene by flow cytometry. Multiple groups were stained to determine, which cell lines would be optimal for moving forward with transduction of T cells. Cells were prepared by staining with an anti-nlrCEA (hnRNPM) antibody (Abeam® Cat# abl26103) and with an anti- CD66-FITC antibody and then sorted using FAC sorting. The isolated cells were then tested for viability and nlrCEA expression. PG13 nlrCEA VPCs were cultured for 48 hours and replaced by T cell expansion media (AIM V + 5% Human Serum + 3000 IU/ml IL2) and the viral supernatant was harvested after 8 hours.
Example 3 Expression of nlrCEA in human T cells
[00188] Human T cells were transduced with C nlrCEA EAr CAR viral supernatant from the VPCs described above. Human peripheral blood mononuclear cells (PBMCs) were isolated from leukapheresis product using Ficoll (Sigma®, St. Louis, MO). The PBMCs were activated for 48 hours in tissue culture flasks (BD Falcon®, Franklin Lakes, N.J.) containing AIM V media (Life Technologies®, Grand Island, NY) supplemented with 5% sterile human AB serum (Valley Biomedical®, Winchester, VA), 50 ng/mL of anti-CD3 monoclonal antibody (OKT3; Ortho Biotech®, Horsham, PA) and 30000 U/mL of IL-2 (Prometheus®, San Diego, CA).
[00189] Using the spinoculation method (Quintas-Cardama et al. , HUM GENE THER, 2007, 18, 1253-1260), T cells obtained from patients were transduced in retronectin (Takara Bio Inc., Japan) coated 6-well plates in AIM V media with 5% human AB serum, 3000 U/mL of IL-2, and protamine sulfate (MP Biomedicals®) at low speed centrifugation for 1 hour at room temperature. The transduction step was carried out a total of three times over 36-hrs. Transduction efficiency (“TE”) was evaluated by flow cytometry with fluorescently labeled antibodies specific for CD3 (Biolegend®, Cat# 300440), unconjugated hnRNPM (Abeam®, Cat# abl26103) followed by secondary antibody (Thermofisher®, APC goat anti-rabbit Cat# A10931). See FIG. 3 A. Untransduced T cells were used as a control for TE.
[00190] Expression of nlrCEA CAR on transduced T cells was measured with immunofluorescence (FIG. 3B), quantitative PCR (“qPCR”) (FIG. 3C) and western blot analysis (FIG. 3D). Immunofluorescence was performed by cytospinning 50,000 transduced and untransduced cells on slides and staining using CD3 and incubating with soluble CEACAM5 recombinant protein followed by staining with CD66 antibodies mentioned above. Images were taken using confocal microscopy at 40x magnification. FIG. 3A.
[00191] For the qPCR analysis, mRNA was isolated (Rneasy Kit Cat# 74104, Qiagen) from transduced and untransduced cells and quantified using Spectrostar Nano Plate® reader (BMG Fabtech). qPCR was performed on CFX 96 Touch Real Time® PCR Detection System (Biorad). Primers to CD3z and CD28 were designed to quantify the relative amount of RNA encoding the nlrCEA-CAR found in the transduced and untransduced T cells: 28-S: 5’- GC AAGCATTACCAGCCCTAT-3 ’ and Z-A: 5 ’ -GTTCTGGCCCTGCTGGTA-3 ’ . Primers designed to quantify GAPDH were used as a control. Relative to untranduced controls, nlrCEA-CAR T Cells had significantly greater mRNA expression.
[00192] Western blot analysis was also performed using 50 pg of transduced and untransduced cell lysates and probing for hnRNPM (1:500) using the above-mentioned antibody followed by incubation with HRP conjugated anti-rabbit secondary antibody (1:1000, Cell Signaling®; Cat# 7074). Phoenix Eco® cell lysate was used a positive control for nlrCEA detection. GAPDH (1:500, Cell Signaling; Cat# 5174) was used as a loading control for the cell lysates followed by incubation with HRP conjugated anti-rabbit secondary antibody (1:1000, Cell Signaling®; Cat# 7074).
Example 4 Cytotoxicity TE Ratios of transduced T cells
[00193] Killing by the transduced CAR-T cells was tested in vitro using as target cells MC38 cells which were stably transfected with CEA and firefly luciferase. MC38CEA+ cells were first generated by stably transfecting MC38 cells with the human CEA gene. MC38-luc was generated by transfecting the MC38CEA+ cells with pLenti-III-UbC-Lucif erase (Applied Biological Materials®, Inc., Richmond, BC Canada). Effector cells were either CEA CAR-T cells generated as described above or untransduced splenic T cells which were used as a negative control. Briefly, 20,000 MC38-CEA+ tumor cells (Target) were plated overnight in a 37°C incubator and co-cultured with 1:1, 1:5 and 1:10 Target (tumor cells) : Effector (Transduced/Untransduced cell) ratios in triplicate. 96 well plate was centrifuged at 1400 rpm for 10 minutes and incubated overnight in 37°C incubator. Lactate Dehydrogenase (LDH) assay was performed using CytoTox 96 Non-Radioactive Cytotoxicity Assay. 50pl of cell supernatant was used for measuring LDH activity and CEA specific cytotoxicity was measured as follows:
CEA Specific Cytotoxicity % = Transduced Cytotoxicity - Untransduced Cytotoxicity x
100
Maximum Tumor Lysis-Spontaneous Tumor Lysis
Results are shown in FIG. 4.
Example 5 In Vivo Expression of nlrCEA-CAR T Cells at Tumor Site
[00194] C57BL6 SCID mice were injected IP with 2.5xl06 MC38CEA-luc cells on day 0 (FIG 5). Mice were treated IP with two rounds (T1 and T2) of 2.5xl06 nlrCEA CAR-T or UnTd cells on days 3 and 6, and given every other day until the end of the study. IL-2 (1000 IU per injection) was administered on a daily basis beginning with the first CAR-T injection on day 3. In vivo work was carried out over the span of 14 days, with CAR-T injections on days 3 and 6. Groups of control mice were treated with untransduced T cells on days 3 and 6 with IL- 2, or treated with IL-2 or antibodies alone. Mice were imaged on an IVIS 100 on even days during in vivo studies, after being injected with 200 pi of 15 mg/ml luciferin.
[00195] The peritoneal cavities were flushed with saline, and then the peritoneal lavage was collected. The saline taken from the peritoneal lavage was then spun down, and cells were then stained for CD3 and hnRNPM using the abeam hnRNPM antibody with FITC secondary antibody. Comparing the three groups, we see highest hnRNPM expression in the nlrCEA CAR-T treated groups. We are seeing distinct populations that are hnRNPM+ in all groups, so the antibody seems to have a lot of background binding. Looking at the WB, there is high expression of hnRNPM in the UnTD, with lower expression in both the TD cells and the VPCs, confirming background binding.
Example 6 - Panel of nlrCEA Constructs
[00196] Carcinoembryonic antigen (CEA, CEACAM5 or CD66e) is frequently expressed on colorectal cancer (CRC) tumors and has been implicated to enhance metastatic potential in CRC and other epithelial cancers such as breast, lung and gastric1. A majority of deaths from CRC are caused by liver metastasis (LM) and approximately 25% of patients have LM2’3. CEACAM5 is a large glycoprotein (-180 kD) and a glycosyl phosphatidyl inositol (GPI) cell-surface-anchored protein4. CEACAM5 exhibits one variable (V)-like domain, identified as the N domain, followed by three repeating units comprising, in total, six constant C2-like domains (termed Al, Bl, A2, B2, A3, and B3)5 7. It has been shown to affect several steps of LM from CRC and has been associated with liver cancer. In liver, CEA interacts with its receptor (CEAr), a protein which is related with hnRNPM on Kupffer cells8. HnRNPM belongs to a family of 20 heterogenous nuclear RNA-binding proteins (hnRNPs A-U)9. HnRNPM consists of two N-terminal RNA-recognition motifs (RRM1 and RRM2) followed by methionine/arginine/glycine rich region followed by one C-terminal RNA recognition motif (RRM3)10. RRM3 interacts with CEA antigen via PELPK motif present at the hinge region between CEA’s N and Al domains11.
[00197] Generated herein is a panel of chimeric antigen receptor (CAR) constructs with the following domains:
• CEA binding domain: RRM1 , RRM2, RRM3
• Hinge (optional): CD 8
• One or more costimulatory domain: CD28, 4-1BB
• Signaling domain: Oϋ3z
• Tag for detection (optional): Myc
CEA binding domain comprised of a combination of different RRMs as shown in FIG. 7.
[00198] Virus producing cells (VPC) were generated by transiently transfecting PG13 with mR3-H-28 or mR123-28 or mR123-H-28 to produce high titer retrovirus for transducing activated human healthy donor T cells to generate CAR-T cells. Multiplicity of infection (MOI) was measured for the virus generated from the VPCs which ranged between 3.1e8-4.5e8/mL, as shown in FIGs. 2A-2B. Myc expression of the VPCs were also evaluated to assess the transfection efficiency (FIGs. 8A-8B).
[00199] Peripheral blood mononuclear cells (PBMC) were isolated from two healthy donors (#1, #2) and activated using anti-CD3 (OKT3; 50ng/mL) in AIM V + 5% human serum + 3000 IU/mL of IL-2 for two days. Viral supernatant isolated from PG13 VPCs (mR3-H-28, mR123-28, mR123-H-28) was used to perform three rounds of transductions over two days. CAR-T and untransduced (UnTd) cells were then expanded in the expansion medium (AIM V + 5% human serum + 3000 IU/ml IL-2). Transduction efficiency was measured by detecting myc expression on mR3-H-28, mR123-28 and mR123-H-28 CAR-T cells generated from both donors (#1, #2). UnTd cells from donors #1 and #2 were used as a control, as shown in FIG. 9A. CD4 and CD8 phenotyping was performed as well on UnTd and CAR-T cells from day of harvest (FIG. 9B). Cells were counted every other day using automated cell counter and the expansion curve and viabilities of mR3-H-28, mR123-28 and mR123-H-28 CAR-T cells were plotted, as shown in FIG. 10A. The cytolytic activity of CAR-T cells was measured by lactate dehydrogenase (LDH) release assay after 4 hrs. MC38-CEA cell line derived from a murine colon adenocarcinoma and engineered to express human carcinoembryonic antigen (CEA), was used as the target cells. The target (tumor) to effector (CAR-T) ratio used was 1:10 for LDH assay. Cytolytic killing of MC38 wild type (WT) cells were used as a control to determine CEA specific killing of tumor cells (FIG. 10B). CEA specific cytotoxicity for mR123-H-28 was significantly higher as compared to mR123-28 or mR3-H-28 (p<0.05). Since mR123-H-28 showed superior cytotoxic activity, a comparative in vitro analysis was performed for mR123- H-28 vs. R123-BB vs. R123-H-BB vs. R123-H-28-BB to evaluate the role of hinge and 4- IBB or combination of CD28 and 4- IBB costimulatory domains on the cytolytic activity of the CAR-T cells, as shown in FIG. 11 A.
[00200] PBMCs were isolated from two healthy donors (#5, #6) and activated using anti-CD3 (OKT3; 50ng/mL) in AIM V + 5% human serum + 3000 IU/ml of IL-2 for two days. Viral supernatant isolated from PG13 VPCs (mR123-H-28, R123-BB, R123-H-BB and R123- H-28-BB) and used to perform three rounds of transductions over two days and CAR-T cells were then expanded in the expansion medium (AIM V + 5% human serum + 3000 IU/mL IL- 2). Cells were counted every other day using automated cell counter and the expansion curve and viabilities of mR123-H-28, R123-BB, R123-H-BB and R123-H-28-BB CAR-T cells were plotted, as shown in FIG. 11B. The cytolytic activity of mR123-H-28, R123-BB, R123-H-BB and R123-H-28-BB CAR-T cells were measured by LDH release assay measured after 4 hours at a targeheffector ratio of 1 : 10. Cytolytic killing of MC38 cells were used as a control to determine CEA specific killing of tumor cells (FIG. 11C).
[00201] Since myc tag was not expressed by the CAR-T cells other than mR123-H- 28, transduction efficiency was measured by using PELPK short amino acid reagent, which is known to bind to RRM3 motif of CAR-T cells and CD3. UnTd cells from donors #5 and #6 were used as a control, as shown in FIG. 12. Since significantly higher cytotoxicity was observed for R123-H-28-BB as compared to mR123-H-28, R123-BB and R123-H-BB, its in vivo performance was tested in comparison to R123-28, which was previously tested in an established xenogeneic tumor model using a immunodeficient SCID mice, as shown in FIG. 13A.
[00202] MC38 CEA tumor cells (2.5e6) expressing firefly luciferase were injected intraperitoneally (IP) on day 0 and on days 3 and 6, 2.5e6 of R123-28 or R123-H-28-BB CAR- T cells or UnTd cells or PBS (vehicle control) were injected IP. Tumor growth was assessed using IVIS live imaging on days 3, 4, 6, 7, 9, 11,13 and 16 as shown by the arrows in FIG. 13A.On days 3 and 6, imaging was performed prior to PBS or CAR-T or UnTd cell infusions. Bioluminescence was expressed as foldover day 3. On day 11 both R123-28 and R123-H-28- BB significantly over PBS (p<0.001) while R123-28 outperformed R123-H-28-BB as compared to UnTd group (p=0.005) as shown in FIGs. 13B-13C. Bioluminescence data obtained from previous experiments performed for PBS (n=5), R123-28 (n=10), UnTd (n=5) were combined in this data set. Previous experiment was terminated at day 11 and hence there were no bioluminescence data for days 13 and 16 for those groups.
[00203] Mice from UnTd (n=3), R123-28 (n=2) and R123-H-28-BB (n=5) were sacrificed on day 17 of the study and cells isolated from intraperitoneal lavage was evaluated for CAR-T persistence, as per the gating strategy shown in FIG. 14A.
[00204] CAR-T persistence was further confirmed by immunofluorescence using paraffin sections of tumors from UnTd, R123-28 and R123-H-28-BB mice and analysed for presence of human T cells using human CD3. Furthermore, CAR-T cell tumor killing was assessed by staining tumor sections with human CD3 (T cell), murine cleaved caspase-3 (apoptosis marker) and human CD66 (tumor marker), as shown in top panel of FIG. 15 while bottom panel shows proliferation by staining tumor sections with human CD3 (T cell), murine Ki67 (proliefration marker) and human CD66 (tumor marker).
References:
1. Hostetter RB, Augustus LB, Mankarious R, et al. Carcinoembryonic antigen as a selective enhancer of colorectal cancer metastasis. J Natl Cancer Inst 1990;82:380-5.
2. Millikan KW, Staren ED, Doolas A. Invasive therapy of metastatic colorectal cancer to the liver. Surg Clin North Am 1997;77:27-48.
3. Bird NC, Mangnall D, Majeed AW. Biology of colorectal liver metastases: A review. J Surg Oncol 2006;94:68-80.
4. Paulick MG, Bertozzi CR. The glycosylphosphatidylinositol anchor: a complex membrane-anchoring structure for proteins. Biochemistry 2008;47:6991-7000.
5. Zimmermann W, Ortlieb B, Friedrich R, von Kleist S. Isolation and characterization of cDNA clones encoding the human carcinoembryonic antigen reveal a highly conserved repeating structure. Proc Natl Acad Sci U S A 1987;84:2960-4.
6. Thompson JA, Pande H, Paxton RJ, et al. Molecular cloning of a gene belonging to the carcinoembryonic antigen gene family and discussion of a domain model. Proc Natl Acad Sci U S A 1987;84:2965-9. 7. Beauchemin N, Benchimol S, Cournoyer D, Fuks A, Stanners CP. Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen. Mol Cell Biol 1987;7:3221-30.
8. Thomas P, Forse RA, Bajenova O. Carcinoembryonic antigen (CEA) and its receptor hnRNP M are mediators of metastasis and the inflammatory response in the liver. Clin Exp Metastasis 2011;28:923-32.
9. Krecic AM, Swanson MS. hnRNP complexes: composition, structure, and function. Curr Opin Cell Biol 1999;11:363-71.
10. Palermo NY, Thomas P, Murphy RF, Lovas S. Hexapeptide fragment of carcinoembryonic antigen which acts as an agonist of heterogeneous ribonucleoprotein M. J Pept Sci 2012;18:252-60.
11. Beauchemin N, Arabzadeh A. Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) in cancer progression and metastasis. Cancer Metastasis Rev 2013;32:643-71.

Claims

CLAIMS What is claimed is:
1. A chimeric receptor comprising: (a) an extracellular binding domain comprising a CEA binding domain, wherein the CEA binding domain is derived from a naturally occurring CEA receptor or a fragment thereof; (b) a hinge domain, (c) a transmembrane domain; and (d) a cytoplasmic domain, said cytoplasmic domain comprising a CD28 signaling domain and a CD3 activating domain.
2. The chimeric receptor of claim 1, wherein the activating CD3 activating domain is CD3 zeta.
3. The chimeric receptor of claim 2, wherein the activating CD3 zeta domain comprises the amino acid sequence of SEQ ID NO. 14.
4. The chimeric receptor of claim 3, wherein in the CEA binding domain is a Kupffer cell receptor or a fragment thereof.
5. The chimeric receptor of claim 4, wherein the CEA binding domain is hnRNP M4 or a fragment thereof.
6. The chimeric receptor of claim 5, wherein the CEA binding domain is comprised of an amino acid sequence selected from the group consisting of SEQ ID Nos. 2, 67, 67, 69, 71, 73 and 75.
7. The chimeric receptor of claim 1, wherein the CEA binding domain comprises the amino acid sequence of SEQ ID NO. 2.
8. The chimeric receptor of claim 1 wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO. 4.
9. The chimeric receptor of claim 1 wherein the CD28 signaling domain comprises the amino acid sequence of SEQ ID NO. 6.
10. The chimeric receptor of claim 1 comprising the amino acid sequence of SEQ ID NO. 15.
11. A polynucleotide encoding the chimeric receptor of claim 10.
12. A cell comprising the chimeric receptor of claim 1.
13. The cell according to claim 12 wherein the cell is a T cell.
14. The cell according to claim 12 wherein the cell is an NK cell.
15. The cell according to claim 12 wherein the cell is a stem cell.
16. The cell according to claim 12 wherein the cell is a red blood cell.
17. A polypeptide comprising the amino acid sequence of SEQ ID NO. 15.
18. A polynucleotide encoding the polypeptide of claim 16.
19. A polypeptide comprising the CEA binding domain of SEQ ID NO. 2, a hinge domain, a transmembrane domain, a costimulatory domain, and a CD3 domain.
20. The polypeptide according to claim 19 wherein the costimulatory domain is a signaling region of CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death- 1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen- 1 (LFA-1 (CD1 1 a/CD 18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof.
21. The chimeric antigen receptor according to claim 19, wherein the costimulatory domain comprises CD28.
22. The chimeric antigen receptor according to claim 21 wherein the CD28 costimulatory domain comprises a sequence that differs at no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residues from the sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.
23. A method of enhancing T cell or an NK cell activity in a mammal comprising introducing into the mammal a T cell or NK cell, which T cell or NK cell comprises a chimeric receptor according to claim 1.
24. A method for treating a mammal suffering from cancer comprising introducing into the mammal a T cell or an NK cell, which T cell or NK cell comprises a chimeric receptor according to claim 1.
25. A method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal comprising administering to a mammal an effective amount of a cell genetically modified to express a chimeric receptor according to claim 1.
26. The method of any one of claims 23 to 25, wherein the mammal is suffering from a cancer metastasized from a primary tumor in the liver, breast, bladder, lymphoma, kidney, endometrial, cervical, ovarian, colon, rectum, esophagus, lung, pancreas and/or stomach.
27. A method of treating a patient suffering from cancer comprising: a. obtaining a plurality of cells from said patient; b. pre-treating said patient with an agent capable of reducing the endogenous cell population in said patient; c. transducing said plurality of cells with a chimeric receptor according to claim l; d. expanding said transduced calls (should have disclosure in the spec that says this can be done by exposing to e.g., IL-7, IL-15, etc.) e. obtaining a population of said cells in the range of lxlO6 to lxlO9. f. administering said cells to said patient.
28. The method of claim 27, wherein said patient is administered an effective amount of one or more steroid compound.
29. A polypeptide comprising the amino acid sequence selected from the group consisting of of SEQ ID NOs. 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, and 63.
30. A polynucleotide encoding the polypeptide of claim 29.
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