WO2014124143A1 - Modified t lymphocytes having improved specificity - Google Patents

Modified t lymphocytes having improved specificity Download PDF

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Publication number
WO2014124143A1
WO2014124143A1 PCT/US2014/015113 US2014015113W WO2014124143A1 WO 2014124143 A1 WO2014124143 A1 WO 2014124143A1 US 2014015113 W US2014015113 W US 2014015113W WO 2014124143 A1 WO2014124143 A1 WO 2014124143A1
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WIPO (PCT)
Prior art keywords
antigen
modified
lymphocyte
polypeptide
domain
Prior art date
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Ceased
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PCT/US2014/015113
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English (en)
French (fr)
Inventor
Bitao Liang
Stewart Abbot
Wei Liu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Clarity Acquisition II LLC
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Anthrogenesis Corp
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Publication date
Priority to US14/765,896 priority Critical patent/US20150368360A1/en
Priority to AU2014214850A priority patent/AU2014214850C1/en
Priority to EP19182025.7A priority patent/EP3604500B1/en
Priority to ES14749306T priority patent/ES2748398T3/es
Priority to HK16106070.2A priority patent/HK1218049B/en
Priority to EP14749306.8A priority patent/EP2953475B1/en
Priority to EP21202957.3A priority patent/EP3985028A1/en
Priority to CN201480020004.4A priority patent/CN105101803A/zh
Priority to NZ711807A priority patent/NZ711807A/en
Priority to KR1020227022598A priority patent/KR20220100093A/ko
Priority to SG11201507026WA priority patent/SG11201507026WA/en
Priority to KR1020157024323A priority patent/KR102417657B1/ko
Application filed by Anthrogenesis Corp filed Critical Anthrogenesis Corp
Priority to CA2904014A priority patent/CA2904014C/en
Priority to JP2015557071A priority patent/JP6467661B2/ja
Publication of WO2014124143A1 publication Critical patent/WO2014124143A1/en
Anticipated expiration legal-status Critical
Priority to AU2018203558A priority patent/AU2018203558B2/en
Priority to US15/990,561 priority patent/US20180273640A1/en
Priority to AU2020294311A priority patent/AU2020294311A1/en
Ceased legal-status Critical Current

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Definitions

  • the disclosure herein relates to the field of immunology, and more specifically, to the modification of T lymphocytes or other immune cells.
  • T lymphocytes also referred to as T cells
  • T cells recognize and interact with specific antigens through receptors or receptor complexes which, upon recognition or an interaction with such antigens, cause activation of the cell.
  • An example of such a receptor is the antigen-specific T lymphocyte receptor complex (TCR/CD3), a complex of eight proteins.
  • TCR T cell receptor
  • CD3 One component, CD3, which has an invariant structure, is responsible for intracellular signaling following occupancy of the TCR by ligand.
  • the T lymphocyte receptor for antigen-CD3 complex recognizes antigenic peptides that are presented to it by the proteins of the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • TCR/CD3 complexes of MHC and peptide are expressed on the surface of antigen presenting cells and other T lymphocyte targets. Stimulation of the TCR/CD3 complex results in activation of the T lymphocyte and a consequent antigen-specific immune response.
  • the TCR/CD3 complex plays a central role in the effector function and regulation of the immune system.
  • T lymphocytes require a second, co-stimulatory signal to become fully active. Without such a signal, T lymphocytes are either non-responsive to antigen binding to the TCR, or become anergic.
  • a co -stimulatory signal for example, is provided by CD28, a T lymphocyte protein, which interacts with CD80 and CD86 on antigen-producing cells.
  • ICOS Inducible CO Stimulator
  • another T lymphocyte protein provides a co-stimulatory signal when bound to ICOS ligand.
  • CAR Chimeric Antigen Receptor
  • CARs effectively target T lymphocytes to specific tumor-associated antigen or tumor-specific antigen, and can effectively target T lymphocytes to kill tumor cells expressing such antigens
  • a design suffers the serious side effect of directing T lymphocytes to kill normal, healthy cells that also express such antigens.
  • use of such CAR-T lymphocytes is generally restricted to tumors expressing an antigen not expressed by any other cell in the body, a relatively rare circumstance.
  • Described herein are modified T lymphocytes comprising chimeric receptors that overcome this shortcoming of current CAR design.
  • modified lymphocytes e.g., modified T
  • lymphocytes that comprise at least two different polypeptides, e.g., chimeric receptors, in which the immune signal derived from binding of a first polypeptide, e.g., chimeric receptor, to a first antigen is separated from a costimulatory signal produced by a second polypeptide, e.g., chimeric receptors, and wherein the costimulatory signal is dependent on antigen binding of a second antigen by the second chimeric receptors.
  • first polypeptide indicates the polypeptide generating the primary antigen-binding immune signal
  • second polypeptide is the polypeptide generating the co-stimulatory immune signal.
  • the two polypeptides are introduced into the modified T lymphocytes using a single CAR construct, with the polypeptides are separated by a cleavable sequence (T2A or P2A) that allows for the expression of two, discrete polypeptides (at essentially equal amounts) from a single ORF.
  • T2A or P2A cleavable sequence
  • a modified T lymphocyte comprising a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and a first intracellular signaling domain, wherein said first polypeptide does not comprise a co-stimulatory domain; and a second polypeptide comprising a second extracellular antigen binding domain binding a second antigen, or a receptor that binds said second antigen; and a second intracellular signaling domain; wherein said modified lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • binding of said first antigen to said first antigen binding domain without binding of said second antigen to said second binding domain, or binding of said second antigen to said second antigen binding domain without binding of first second antigen to said first binding domain induces anergy of said modified T lymphocyte, or non-responsiveness of said T- lymphocyte to said first antigen or said second antigen.
  • said first antigen binding domain and said second antigen binding domain are independently an antigen-binding portion of a receptor, an antigen- binding portion of an antibody, or other peptide-based macromolecular antigen binding agent.
  • either or both of said first antigen binding domain or said second antigen binding domain are scFv antibody fragments.
  • either or both of said first polypeptide or said second polypeptide additionally comprise a transmembrane domain.
  • said first polypeptide or said second polypeptide comprises a T cell survival motif.
  • the T cell survival motif is a CD28 T cell survival motif.
  • said T cell survival motif is an intracellular signaling domain of IL-7 receptor (IL-7R), an intracellular signaling domain of IL-12 receptor, an intracellular signaling domain of IL-15 receptor, an intracellular signaling domain of IL-21 receptor, or an intracellular signaling domain of transforming growth factor ⁇ (TGFB) receptor.
  • said first polypeptide or said second polypeptide comprises a portion of a CD28 molecule that comprises a T cell survival motif.
  • said first polypeptide or said second polypeptide comprises a CD28 molecule that comprises a T cell survival motif.
  • said first intracellular signaling domain comprises a polypeptide sequence comprising an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • said polypeptide sequence is a CD3 ⁇ signaling domain.
  • said first antigen is an antigen on a tumor cell.
  • said tumor cell is a cell in a solid tumor.
  • said tumor cell is a blood cancer cell.
  • said antigen is a tumor-associated antigen or a tumor-specific antigen.
  • said tumor-associated antigen or tumor-specific antigen is Her2, prostate stem cell antigen (PSCA), PSMA (prostate-specific membrane antigen), B cell maturation antigen (BCMA), alpha- fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T
  • PSCA prostate stem cell antigen
  • BCMA B cell maturation antigen
  • AFP alpha- fetoprotein
  • CEA carcinoembryonic antigen
  • MART-1 myo-Dl
  • MSA muscle-specific actin
  • NSE neuron-specific enolase
  • transcription factor- 1 the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2- PK), CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EphA2, CSPG4, CD138, FAP (Fibroblast Activation Protein), CD171, kappa, lambda, 5T4, ⁇ ⁇ ⁇ 6 integrin, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD123, EGFR, EGP2, EGP40, EpCAM, fetal AchR, FRa, GD3, HL A- A 1 +M AGE 1 , HLA-Al+NY-ESO-1, IL- 1 IRa, IL-13Ra2, Lewis-Y, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, R
  • said first antigen is PSCA. In another specific embodiment, said first antigen is PSMA. In another specific embodiment, said first antigen is BCMA. In another specific embodiment, when said first antigen binding domain is specific for HER2, the antigen binding domain of the second polypeptide of the modified T cell is not specific for MUC-1.
  • said first antigen is integrin ⁇ 3 (CD61), galactin, K- Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene) or Ral-B.
  • said second antigen is a growth factor, cytokine, or interleukin.
  • the second antigen is a molecule, e.g., a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis.
  • said second antigen is vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8).
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • PDGF platelet-derived growth factor
  • HGF hepatocyte growth factor
  • IGF insulin-like growth factor
  • IL-8 interleukin-8
  • said second antigen binding domain can be, e.g., an antibody (or fragment thereof, e.g., scFv) specific to VEGF, bFGF, PDGF, HGF, IGF, or IL-8; or a receptor for VEGF, bFGF, PDGF, HGF, IGF, or IL-8.
  • said second antigen binding domain is a receptor for VEGF, bFGF, PDGF, HGF, IGF, TGF-beta, IL4, IL-10, IL13, or IL-8.
  • said second antigen binding domain is a receptor for VEGF, wherein said receptor for VEGF is VEGFR, e.g., VEGFRl, VEGFR2, or VEGFR3.
  • signal transduction activation provided by said second antigen is non-antigenic, but is associated with hypoxia.
  • said stimulus is induced by activation of hypoxia-inducible factor- la (HIF-la), HIF- ⁇ , HIF-2a, HIF- 2 ⁇ , HIF-3a, or HIF-3p.
  • said second antigen is an interleukin.
  • said second antigen is a damage associated molecular pattern molecule (DAMP; also known as an alarmin).
  • DAMP is a heat shock protein, chromatin-associated protein high mobility group box 1
  • HMGB1 S100A8 (also known as MRP8, or calgranulin A), S100A9 (also known as MRP 14, or calgranulin B), serum amyloid A (SAA), deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.
  • said second antigen is an antigen on an antibody that binds to an antigen presented by a tumor cell.
  • a modified T lymphocyte comprising a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and a first intracellular signaling domain, wherein said first polypeptide does not comprise a co-stimulatory domain; and a second polypeptide comprising a second extracellular antigen binding domain binding that binds VEGF, and a second intracellular signaling domain (costimulatory domain); wherein said modified lymphocyte becomes maximally cytotoxic when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • said first antigen is PSCA, PSMA, or BCMA.
  • said first extracellular antigen binding domain comprises an antibody or fragment thereof (e.g., scFv), e.g., an antibody or fragment thereof specific to PSCA, PSMA, or BCMA.
  • said antigen binding domain binding that binds VEGF is a receptor for VEGF, i.e., VEGFR.
  • said VEGFR is VEGFRl, VEGFR2, or VEGFR3.
  • said VEGFR is VEGFR2.
  • said second polypeptide comprises one or more co-stimulatory domains.
  • said one or more co- stimulatory domains comprises one or more of a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, a co-stimulatory OX40 (CD 134) polypeptide sequence, a co-stimulatory 4-1BB (CD137) polypeptide sequence, TILR2, TILR4, TILR7, TILR9, Fc receptor gamma chain, Fc receptor ⁇ chain, or a co-stimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence.
  • a co-stimulatory CD27 polypeptide sequence a co-stimulatory CD28 polypeptide sequence
  • a co-stimulatory OX40 (CD 134) polypeptide sequence a co-stimulatory 4-1BB (CD137) polypeptide sequence
  • TILR2, TILR4, TILR7, TILR9 Fc receptor gamma chain
  • said first polypeptide comprises an extracellular tumor antigen-binding domain and a CD3 ⁇ signaling domain
  • said second polypeptide comprises an antigen-binding domain wherein said antigen is an angiogenic or vasculogenic factor, and one or more co -stimulatory molecule signaling domains.
  • Said angiogenic factor can be, e.g., VEGF.
  • Said one or more co-stimulatory molecule signaling motifs can comprise, e.g., co-stimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4-1BB.
  • said first polypeptide comprises an extracellular tumor antigen-binding domain and a CD3 ⁇ signaling domain
  • said second polypeptide comprises an antigen-binding domain wherein said antigen is VEGF, and co-stimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4- 1BB.
  • said first polypeptide or said second polypeptide comprises a T cell survival motif.
  • said T cell survival motif is, or is derived from, an intracellular signaling domain of IL-7 receptor (IL-7R), an intracellular signaling domain of IL-12 receptor, an intracellular signaling domain of IL-15 receptor, an intracellular signaling domain of IL-21 receptor, or an intracellular signaling domain of transforming growth factor ⁇ (TGFB) receptor.
  • IL-7R IL-7 receptor
  • IL-12R intracellular signaling domain of IL-12 receptor
  • IL-15 receptor an intracellular signaling domain of IL-15 receptor
  • TGFB transforming growth factor ⁇
  • said first polypeptide comprises an extracellular tumor antigen-binding domain and a CD3 ⁇ signaling domain
  • said second polypeptide comprises an antigen-binding domain wherein said antigen is VEGF, an IL-7 receptor intracellular T cell survival motif, and co-stimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4- IBB.
  • said first antigen is a tumor-specific antigen or a tumor-associated antigen
  • said first intracellular signaling domain comprises a CD3 ⁇ signaling domain
  • said second polypeptide comprises an antigen- binding domain that binds said second antigen, and co-stimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4-1BB.
  • said second polypeptide further comprises an intracellular T cell survival motif, e.g., a T cell survival motif that is, or is derived from, an intracellular signaling domain of IL-7 receptor (IL-7R), an intracellular signaling domain of IL-12 receptor, an intracellular signaling domain of IL-15 receptor, an intracellular signaling domain of IL-21 receptor, or an intracellular signaling domain of transforming growth factor ⁇ (TGFB) receptor.
  • IL-7R IL-7 receptor
  • IL-12R intracellular signaling domain of IL-12 receptor
  • IL-15 receptor an intracellular signaling domain of IL-15 receptor
  • TGFB transforming growth factor ⁇
  • said second antigen is VEGF or IL-4.
  • only said first antigen binding domain or said second antigen binding domain binds to a tumor-associated antigen or a tumor-specific antigen, and the other of said first antigen binding domain or said second antigen binding domain binds to an antigen that is not a tumor-specific antigen or a tumor-associated antigen.
  • only one of the first or second stimulatory signals is generated by a tumor-specific antigen or tumor- associated antigen; the other of the first or second stimulatory signals is generated by another type of antigen, e.g., an antigen (e.g., protein or other biomolecule) associated with the tumor environment).
  • modified T lymphocytes that comprise the first and second polypeptides, and one or more additional polypeptides, e.g., one or more additional polypeptides that comprise an antigen-binding domain and a signaling domain.
  • only one of said polypeptides e.g., only one of said first polypeptide, said second polypeptide, or said one or more additional polypeptides
  • each of the remainder of said polypeptides comprises an antigen binding domain that binds to an antigen that is not a tumor-associated antigen or a tumor-specific antigen.
  • two or more of said first polypeptide, said second polypeptides, and said one or more additional polypeptides comprise antigen binding domains that bind to one or more tumor-associated antigens or tumor-specific antigens, wherein at least one of said polypeptides comprises an antigen binding domain that does not bind to a tumor-associated antigen or a tumor-specific antigen.
  • a modified T lymphocyte comprising a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and a first intracellular signaling domain; and a second polypeptide comprising a second extracellular antigen binding domain binding a second antigen, or a receptor that binds said second antigen; and a second intracellular signaling domain, wherein said second polypeptide does not comprise a co-stimulatory domain; wherein said modified lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • binding of said first antigen to said first antigen binding domain without binding of said second antigen to said second binding domain, or binding of said second antigen to said second antigen binding domain without binding of first second antigen to said first binding domain induces anergy of said modified T lymphocyte, or non-responsiveness of said modified T lymphocyte to said first antigen.
  • said first antigen-binding domain and said antigen-binding domain are independently an antigen-binding portion of a receptor or an antigen-binding portion of an antibody.
  • either or both of said first antigen binding domain or said second antigen binding domain are scFv antibody fragments.
  • said first polypeptide and/or said second polypeptide additionally comprises a transmembrane domain.
  • said first polypeptide or said second polypeptide comprises a T cell survival motif, e.g.., any of the T cell survival motifs described herein.
  • said first antigen is an antigen on a tumor cell, e.g., a cell in a solid tumor or a blood cancer cell.
  • said first antigen is a tumor-associated antigen or a tumor-specific antigen, e.g., Her2, prostate stem cell antigen (PSCA), PSMA (prostate-specific membrane antigen), B cell maturation antigen (BCMA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19- 9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen,
  • PSCA prostate stem cell antigen
  • MART-1 myo-Dl
  • MSA muscle-specific actin
  • NSE neuron-specific enolase
  • the tumor- associated antigen is CD 19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, or STEAP1 (six-transmembrane epithelial antigen of the prostate 1).
  • said first antigen is integrin ⁇ 3 (CD61), galactin, K- Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene) or Ral-B.
  • said second intracellular signaling domain comprises a polypeptide sequence comprising an immunoreceptor tyrosine-based activation motif (IT AM), e.g., a CD3 ⁇ signaling domain.
  • IT AM immunoreceptor tyrosine-based activation motif
  • said second antigen is a growth factor, cytokine, or interleukin.
  • said second antigen is a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis, e.g., VEGF, bFGF, PDGF, HGF, IGF, or IL-8.
  • signal transduction by said second chimeric receptor is induced by activation of a hypoxia-associated factor, e.g., HIF-l , HIF- ⁇ , HIF-2a, HIF-2p, HIF-3a, or HIF-3p.
  • said second antigen is an interleukin.
  • said second antigen is a DAMP, e.g., a heat shock protein, HMGB1, S100A8, S100A9, SAA, DNA, ATP, uric acid, or heparin sulfate.
  • said second antigen is an administered peptide, e.g., an antibody or a synthetic polypeptide.
  • said second antigen is an antigen on an antibody that binds to an antigen presented by a tumor cell.
  • said first polypeptide and or said second polypeptide comprises one or more co-stimulatory domains, e.g., one or more of a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, a co-stimulatory OX40 (CD 134) polypeptide sequence, a co- stimulatory 4-1BB (CD137) polypeptide sequence, TILR2, TILR4, TILR7, TILR9, Fc receptor gamma chain, Fc receptor ⁇ chain, or a co-stimulatory inducible T-cell co-stimulatory (ICOS) polypeptide sequence.
  • co-stimulatory CD27 polypeptide sequence e.g., one or more of a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, a co-stimulatory OX40 (CD 134)
  • said first polypeptide or said second polypeptide comprises a T cell survival motif, e.g., said T cell survival motif is, or is derived from, an intracellular signaling domain of IL-7 receptor (IL-7R), an intracellular signaling domain of IL-12 receptor, an intracellular signaling domain of IL-15 receptor, an intracellular signaling domain of IL-21 receptor, or an intracellular signaling domain of transforming growth factor ⁇ (TGFB) receptor.
  • IL-7R IL-7 receptor
  • IL-12R intracellular signaling domain of IL-12 receptor
  • IL-15 receptor an intracellular signaling domain of IL-15 receptor
  • TGFB transforming growth factor ⁇
  • a method of treating an individual having a disease or disorder wherein the disease or disorder is characterized, or is characterizable, by a first antigen, and is associated with a second antigen.
  • said first antigen is a tumor-associated antigen or a tumor-specific antigen.
  • FIG. 1 A schematic representation of four CARs.
  • SP signal peptide
  • EC extracellular
  • TM transmembrane
  • IC intracellular.
  • FIG. 2 A schematic representation of four CARs.
  • SP signal peptide
  • EC extracellular
  • TM transmembrane
  • IC intracellular.
  • T lymphocytes T lymphocytes
  • existing modified T lymphocytes are modified to express a polypeptide known as a Chimeric Antigen Receptor, or CAR. See, e.g., Eshhar, U.S. Patent No. 7,741 ,465.
  • CAR Chimeric Antigen Receptor
  • the general structure of a CAR comprises a single polypeptide chain that includes an extracellular portion and an intracellular portion; a transmembrane portion is optionally added to anchor the CAR in the T lymphocyte's cell membrane.
  • the extracellular portion includes a domain or motif that is able to bind an antigen of interest, e.g., an antigen on a cell, for example, a tumor-specific antigen or tumor-associated antigen.
  • the intracellular portion includes a domain or motif that is able to transmit a primary antigen-binding signal that is necessary for the activation of a T lymphocyte in response to the antigen's binding to the CAR's extracellular portion.
  • this domain or motif comprises, or is, an IT AM
  • ITAM-containing polypeptides suitable for CARs include, for example, the zeta CD3 chain (CD3Q or ITAM-containing portions thereof.
  • the intracellular portion further comprises one or more co- stimulatory motifs, typically a co-stimulatory portion of CD27, CD28 or CD137 (4-1BB).
  • co-stimulatory motifs typically a co-stimulatory portion of CD27, CD28 or CD137 (4-1BB).
  • lymphocyte to kill the antigen-bearing cell.
  • modified T lymphocytes can be highly effective at killing undesirable cells bearing a particular antigen, they are also effective at killing normal cells that express low, but detectable, amounts of such antigen.
  • off-tumor activity of modified T lymphocytes can result in severe damage to a recipient's normal tissues, and even death.
  • a metastatic colon cancer patient administered 10 10 CAR-T lymphocytes directed to ERBB2- overexpressing tumors, experienced pulmonary distress within 15 minutes after administration, and subsequently died of multiple organ failure and hemorrhage.
  • Such off-tumor effects mediated by CARs would, however, be reduced or eliminated by separating primary antigen-binding signal transduction from co-stimulation, so that antigen binding, alone, is not sufficient to activate the modified T lymphocytes, e.g., wherein both primary and secondary signals would be expected to be uniquely apparent within tumor-bearing, but not normal, tissues.
  • T lymphocytes modified to express a co-stimulatory polypeptide, e.g., chimeric receptor, or modified to express two or more artificial polypeptides, e.g., chimeric receptors, at least one of which comprises a primary antigen-binding signal transduction domain and does not also comprise a co-stimulatory motif, and at least one of which comprises a co-stimulatory domain or motif, but not a primary antigen-binding signal transduction domain, wherein the two or more chimeric receptors do not bind to the same antigen.
  • a co-stimulatory polypeptide e.g., chimeric receptor
  • two or more artificial polypeptides e.g., chimeric receptors
  • Non-binding of the co-stimulatory polypeptide in addition to the primary signal-transducing polypeptide results in non-responsiveness, anergy, or apoptosis of the modified T lymphocytes.
  • modified T lymphocytes that comprise a single artificial co-stimulatory polypeptide, e.g., chimeric receptor, which provides a co- stimulatory signal in response to a particular antigen; the primary antigen-binding signal, however, is transmitted through the native T cell receptor proteins.
  • This single polypeptide comprises, e.g., an antigen binding domain that binds to a first antigen, and one or more co- stimulatory domains, but lacks a primary antigen binding signal-generating domain, such as ITAM or CD3 ⁇ .
  • the modified T cells in this configuration, rely on the native T cell receptor and CD3 ⁇ signaling protein for primary antigen binding signaling.
  • the co-stimulatory polypeptide provides a co-stimulatory signal that enhances the T lymphocyte's response to the particular antigen.
  • the T cell natively recognizes a first antigen, e.g., a tumor-specific antigen (TSA) or tumor-associated antigen (TAA), and the artificial polypeptide (e.g., chimeric receptor), and the co-stimulatory polypeptide's antigen binding domain also binds said first antigen.
  • a first antigen e.g., a tumor-specific antigen (TSA) or tumor-associated antigen (TAA)
  • TAA tumor-specific antigen
  • TAA tumor-associated antigen
  • the artificial polypeptide e.g., chimeric receptor
  • the T cell natively recognizes a first antigen, e.g., a tumor-specific antigen (TSA) or tumor-associated antigen (TAA), and the artificial polypeptide (e.g., chimeric receptor), and the co-stimulatory polypeptide's antigen binding domain binds a second, different antigen, e.g., a different TSA or TAA.
  • a first antigen e.g., a tumor-specific antigen (TSA) or tumor-associated antigen (TAA)
  • TAA tumor-specific antigen
  • TAA tumor-associated antigen
  • the co-stimulatory polypeptide's antigen binding domain binds a second, different antigen, e.g., a different TSA or TAA.
  • the T cell natively recognizes a first antigen, e.g., a tumor-specific antigen (TSA) or tumor-associated antigen (TAA), and the artificial polypeptide (e.g., chimeric receptor), and the co -stimulatory polypeptide's antigen binding domain binds a second antigen that is not a TSA or TAA.
  • a first antigen e.g., a tumor-specific antigen (TSA) or tumor-associated antigen (TAA)
  • TAA tumor-specific antigen
  • TAA tumor-associated antigen
  • the antigen binding portion of the artificial polypeptide can be any polypeptide domain, motif or sequence that binds to an antigen.
  • the antigen binding domain is an antigen binding portion of a receptor or an antigen-binding portion of an antibody.
  • the antigen-binding domain can be receptors or an antigen binding portion thereof, e.g., a receptor for a ligand produced by a tumor cell, an antibody, antibody chain, or an antigen binding portion thereof, an Fc domain, a glycophosphatidylinositol anchor domain, or the like.
  • the antigen binding is an scFv antibody fragment.
  • the antigen-binding domain is another form of peptide - based macromolecular antigen binding agent, e.g., phage display protein.
  • the antigen binding domain does not bind the antigen directly, but binds to a modified protein that binds the antigen.
  • the antigen binding domain comprises a ligand, e.g., biotin, that binds to a ligand, e.g., avidin, on an antigen- binding polypeptide or macromolecule.
  • antigen binding by the antigen binding domain can be restricted to antigen presentation in association with major
  • MHC histocompatibility complexes
  • the one or more co -stimulatory domains within the artificial polypeptide comprises one or more of a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, a co-stimulatory OX40 (CD 134) polypeptide sequence, a co-stimulatory 4- IBB (CD 137) polypeptide sequence, or a co- stimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence.
  • the first antigen may be any antigen of interest, e.g., an antigen that is expressed on the surface of a cell.
  • said first antigen is an antigen on a tumor cell, e.g., a TAA or TSA.
  • the tumor cell can be a cell, e.g., of a solid tumor or a blood cancer.
  • said antigen is a tumor-associated antigen or a tumor-specific antigen, e.g., Her2, prostate stem cell antigen (PSCA), PSMA (prostate-specific membrane antigen), B cell maturation antigen (BCMA), ER 5, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA), neurofilament
  • the tumor-associated antigen is CD 19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, orSTEAPl (six-transmembrane epithelial antigen of the prostate 1).
  • the TAA or TSA is a cancer/testis (CT) antigen, e.g., BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-l, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE.
  • CT cancer/testis
  • the TAA or TSA is a carbohydrate or ganglioside, e.g., fuc-GMl, GM2 (oncofetal antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, and the like.
  • the TAA or TSA is alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA 125, CA 15-3 (CA 27.29 ⁇ BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein Barr virus antigens, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2,
  • HPV papillomavirus
  • said first antigen is integrin ⁇ 3 (CD61), galactin, K- Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene) or Ral-B.
  • tumor-associated and tumor-specific antigens are known to those in the art and may be targeted by the modified T lymphocytes provided herein.
  • the antigen can be, e.g., a growth factor, cytokine or interleukin, e.g., a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis.
  • growth factors, cytokines, or interleukins can include, e.g., vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet- derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8).
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • PDGF platelet- derived growth factor
  • HGF hepatocyte growth factor
  • IGF insulin-like growth factor
  • IL-8 interleukin-8
  • Tumors can also create a hypoxic environment local to the tumor.
  • signal transduction by said artificial polypeptide e.g., chimeric receptor
  • a hypoxia-associated factor e.g., HIF-la, HIF- ⁇ , HIF-2a, ⁇ 1 ⁇ -2 ⁇ , HIF-3a, or ⁇ 1 ⁇ -3 ⁇ , or is otherwise induced by hypoxic response element activation.
  • the second antigen is a DAMP, e.g., a heat shock protein, chromatin-associated protein high mobility group box 1 (HMGB1), S100A8 (MRP8, calgranulin A), S100A9 (MRP 14, calgranulin B), serum amyloid A (SAA), deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.
  • DAMP damage associated molecular pattern molecules
  • a modified T lymphocyte comprising a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and a first intracellular signaling domain, wherein said first polypeptide does not comprise a co- stimulatory domain; and a second polypeptide comprising a second extracellular antigen binding domain binding a second antigen, or a receptor that binds said second antigen; and a second intracellular signaling domain; wherein said modified lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • the two polypeptides are both chimeric receptors.
  • Binding of said first antigen to said first antigen binding domain without binding of said second antigen to said second binding domain, or binding of said second antigen to said second antigen binding domain without binding of first second antigen to said first binding domain either induces anergy of said modified T lymphocyte, or renders the modified T lymphocyte nonresponsive to the binding of the first antigen to the first antigen binding domain alone.
  • the antigen binding portions of the first polypeptide and the second polypeptide can, independently, be any polypeptide domain, motif or sequence that binds to an antigen, said first antigen binding domain and said second antigen binding domain are independently an antigen binding portion of a receptor or an antigen-binding portion of an antibody.
  • the first and second antigen-binding domains can be receptors or an antigen binding portion thereof, e.g., a receptor for a ligand produced by a tumor cell, an antibody, antibody chain, or an antigen binding portion thereof, an Fc domain, a glycophosphatidylinositol anchor domain, or the like.
  • first antigen binding domain or said second antigen binding domain are scFv antibody fragments.
  • the first and second antigen-binding domains can be another form of peptide-based macromolecular antigen binding agent, e.g., phage display proteins.
  • antigen binding by the antigen binding domains can be restricted to antigen presentation in association with major histocompatibility complexes (MHC), or can be MHC-unrestricted.
  • MHC major histocompatibility complexes
  • said first polypeptide and/or said second polypeptide preferably additionally comprise a transmembrane domain.
  • the transmembrane domain can be obtained or derived from the transmembrane domain of any transmembrane protein, and can include all or a portion of such transmembrane domain.
  • the transmembrane domain can be obtained or derived from, e.g., CD 16, a cytokine receptor, and interleukin receptor, or a growth factor receptor, or the like.
  • the first polypeptide or said second polypeptide may also comprise a T cell survival motif.
  • the T cell survival motif can be any polypeptide sequence or motif that facilitates the survival of the T lymphocyte after stimulation by an antigen.
  • the T cell survival motif is, or is derived from, CD3, CD28, an intracellular signaling domain of IL-7 receptor (IL-7R), an intracellular signaling domain of IL-12 receptor, an intracellular signaling domain of IL-15 receptor, an intracellular signaling domain of IL-21 receptor, or an intracellular signaling domain of transforming growth factor ⁇ (TGFB) receptor.
  • IL-7R intracellular signaling domain of IL-7 receptor
  • IL-12R intracellular signaling domain of IL-12 receptor
  • IL-15 receptor an intracellular signaling domain of IL-15 receptor
  • TGFB transforming growth factor ⁇
  • the first intracellular signaling domain of the first polypeptide can be any polypeptide that is capable of transmitting an antigen binding signal from the first antigen binding domain of the first polypeptide, e.g., in a manner similar to the CD3 ⁇ (CD3 zeta) chains of the native T lymphocyte receptor.
  • the first intracellular signaling domain comprises a polypeptide sequence comprising an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • the polypeptide sequence is a CD3 ⁇ signaling domain or a signal-transducing variant thereof.
  • the second polypeptide comprises one or more co-stimulatory domains, enabling the second polypeptide to provide co-stimulation when the second antigen binds to the second antigen-binding domain of the second polypeptide.
  • Any co-stimulatory motif, or functional portion thereof, may be used.
  • the one or more co-stimulatory domains comprises one or more of a co-stimulatory CD27 polypeptide sequence, a co- stimulatory CD28 polypeptide sequence, a co-stimulatory OX40 (CD 134) polypeptide sequence, a co-stimulatory 4- IBB (CD 137) polypeptide sequence, or a co-stimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence.
  • the first antigen may be any antigen of interest, e.g., an antigen that is expressed on the surface of a cell.
  • said first antigen is an antigen on a tumor cell, e.g., a TSA or TAA, e.g., any of the TSAs or TAAs disclosed in Section 5.1, above.
  • the tumor cell can be a cell, e.g., of a solid tumor or a blood cancer.
  • the antigen can be any antigen that is expressed on a cell of any tumor or cancer type, e.g., cells of a lymphoma, a lung cancer, a breast cancer, a prostate cancer, an adrenocortical carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, e.g., a malignant melanoma, a skin carcinoma, a colorectal carcinoma, a desmoid tumor, a desmoplastic small round cell tumor, an endocrine tumor, an Ewing sarcoma, a peripheral primitive neuroectodermal tumor, a solid germ cell tumor, a hepatoblastoma, a neuroblastoma, a non-rhabdomyosarcoma soft tissue sarcoma, an osteosarcoma, a retinoblastoma, a rhabdomyosarcoma, a Wilms tumor, a
  • said lymphoma can be chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, T lymphocyte prolymphocytic leukemia, T lymphocyte large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T lymphocyte leukemia/lymphoma, extranodal NK/T
  • hepatosplenic T lymphocyte lymphoma blastic NK cell lymphoma, mycosis fungoides, Sezary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T lymphocyte lymphoma, peripheral T lymphocyte lymphoma (unspecified), anaplastic large cell lymphoma, Hodgkin lymphoma, or a non-Hodgkin lymphoma.
  • the second antigen can be any antigen different from the first antigen, but is preferably related to the first antigen.
  • both the first and second antigens can be tumor- associated antigens or tumor-specific antigens that are present on the same tumor cell type.
  • the first antigen is a tumor-associated antigen or a tumor-specific antigens
  • said second antigen is not a tumor-associated antigen or a tumor-specific antigen.
  • the second antigen in certain embodiments, is related to an aspect of the tumor, e.g., the tumor environment.
  • a tumor can induce an inflammatory state in tissue surrounding the tumor, and can release angiogenic growth factors, interleukins, and/or cytokines that promote angiogenesis into and at the periphery of the tumor.
  • the second antigen is a growth factor, cytokine or interleukin, e.g., a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis.
  • growth factors, cytokines, or interleukins can include, e.g., vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulinlike growth factor (IGF), or interleukin-8 (IL-8).
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • PDGF platelet-derived growth factor
  • HGF hepatocyte growth factor
  • IGF insulinlike growth factor
  • IL-8 interleukin-8
  • Tumors can also create a hypoxic environment local to the tumor.
  • signal transduction by said second chimeric receptor is induced by activation of a hypoxia-associated factor, e.g., HIF-la, HIF- ⁇ , HIF-2a, HIF-2P, HIF-3a, or HIF- 3 ⁇ , or is otherwise induced by hypoxic response element activation.
  • a hypoxia-associated factor e.g., HIF-la, HIF- ⁇ , HIF-2a, HIF-2P, HIF-3a, or HIF- 3 ⁇
  • the second antigen is a DAMP, e.g., a heat shock protein, chromatin-associated protein high mobility group box 1 (HMGBl), S100A8 (MRP8, calgranulin A), S100A9 (MRP 14, calgranulin B), serum amyloid A (SAA), deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.
  • DAMP damage associated molecular pattern molecules
  • the polypeptide to an antigen that is not native to the antigen or to the surrounding tissue.
  • tumor cells or cells of surrounding normal tissue, may be contacted with an antibody that binds to at least one antigen on the cells.
  • any antigens on the antibody itself can bind to the second antigen-binding portion of the second chimeric receptor polypeptide.
  • the first antigen, that binds to the first antigen-binding portion of the first polypeptide is an antigen on an antibody that binds to an antigen presented by a tumor cell.
  • the second antigen that binds to the second antigen-binding portion of the second polypeptide, is an antigen on an antibody that binds to an antigen presented by a tumor cell.
  • the first antigen is an antigen on a first antibody
  • the second antigen is an antigen on a second antibody.
  • the first antibody can be an antibody that binds to, e.g., a tumor-associated antigen or a tumor-specific antigen
  • the second antibody is an antibody to a cytokine, interleukin, growth factor, DAMP, or other non- TAA, non-TSA protein associated with the tumor.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and an intracellular CD3 ⁇ signaling domain; and b) a second polypeptide comprising an extracellular antigen binding domain that binds an angiogenic or vasculogenic factor, and a second intracellular signaling domain, wherein said first polypeptide does not comprise a co- stimulatory domain; wherein said modified lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising an scFv or antigen-binding portion thereof that binds a first antigen, and an intracellular CD3 ⁇ signaling domain, wherein said first polypeptide does not comprise a co-stimulatory domain; and b) a second polypeptide comprising an extracellular antigen binding domain that binds to an angiogenic or vasculogenic factor, and a second intracellular signaling domain; wherein said modified lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising an extracellular antigen binding domain that binds a first antigen, wherein said first antigen is a tumor-associated antigen (TAA) or tumor-specific antigen (TSA), and an intracellular CD3 ⁇ signaling domain, wherein said first polypeptide does not comprise a co-stimulatory domain; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds to a second antigen, wherein said second antigen is VEGF, bFGF, PDGF, HGF, IGF, or IL-8, and a second intracellular signaling domain;
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • said modified lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, wherein said first antigen is a TAA or TSA, and a first intracellular CD3 ⁇ signaling domain, wherein said first polypeptide does not comprise a co-stimulatory domain; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, wherein said second antigen is VEGF, bFGF, PDGF, HGF, IGF, or IL-8, and a second intracellular signaling domain comprising a co-stimulatory signaling domain from one or more of CD27, CD28, OX40, ICOS, and 4-1BB ; wherein said modified lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising an extracellular antigen binding domain that binds a first antigen, wherein said first antigen is Her2, PSCA, PSMA, BCMA, ERK5, AFP, CEA, CA-125, CA19-9, calretinin, MUC-1, EMA, ETA, tyrosinase, MAGE, CD34, CD45, CD99, CD1 17, chromogranin, cytokeratin, desmin, GFAP, GCDFP-15, HMB-45 antigen, MART-1, myo-Dl, MSA, neurofilament, NSE, placental alkaline phosphatase, synaptophysin,
  • thyroglobulin thyroid transcription factor- 1, tumor M2-PK, CD 19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAPl (six-transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, or an abnormal p53 protein; and an intracellular CD3 ⁇ signaling domain, wherein said first polypeptide does not comprise a co-stimulatory domain; and b) a second polypeptide comprising an extracellular antigen binding domain that binds to a second antigen, wherein said second antigen is VEGF, bFGF, PDGF, HGF, IGF, or IL-8, and a second antigen binding domain, wherein said
  • intracellular signaling domain comprising co-stimulatory signaling domains from one or more of CD27, CD28, OX40, ICOS, and 4-1BB ; wherein said modified lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising an extracellular antigen binding domain that binds a first antigen, wherein said first antigen is Her2, PSCA, PSMA, BCMA, ERK5, AFP, CEA, CA-125, CA19-9, calretinin, MUC-1, EMA, ETA, tyrosinase, MAGE, CD34, CD45, CD99, CD1 17, chromogranin, cytokeratin, desmin, GFAP, GCDFP-15, HMB-45 antigen, MART-1, myo-Dl, MSA, neurofilament, NSE, placental alkaline phosphatase, synaptophysin,
  • thyroglobulin thyroid transcription factor- 1, tumor M2-PK, CD 19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAPl (six-transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, or an abnormal p53 protein, and an intracellular CD3 ⁇ signaling domain, wherein said first polypeptide does not comprise a co-stimulatory domain; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds to a second antigen, wherein said second antigen is VEGF, bFGF, PDGF, HGF, IGF, or IL-8, and an intracellular signaling domain comprising co
  • modified T lymphocytes comprising: a) a first polypeptide comprising an scFv or antigen binding portion thereof that binds a first antigen, wherein said first antigen is Her2, PSCA, PSMA, BCMA, ERK5, AFP, CEA, CA-125, CA19-9, calretinin, MUC-1, EMA, ETA, tyrosinase, MAGE, CD34, CD45, CD99, CD 117, chromogranin, cytokeratin, desmin, GFAP, GCDFP-15, HMB-45 antigen, MAPvT-1, myo-Dl, MSA, neurofilament, NSE, placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor- 1, tumor M2-PK, CD 19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (e
  • intracellular signaling domain comprising co-stimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4-1BB; wherein said modified lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • either or both of said first polypeptide or said second polypeptide comprises a T cell survival motif, e.g., a T cell survival motif from CD28, IL-7R, IL-12R, IL-15R, IL-21R, TGFBR.
  • a T cell survival motif e.g., a T cell survival motif from CD28, IL-7R, IL-12R, IL-15R, IL-21R, TGFBR.
  • the two polypeptides (e.g., chimeric receptors) comprised within the modified T lymphocytes can be constructed so that binding of the first antigen, e.g., a TAA or TSA, to the first antigen binding domain of the first polypeptide produces not a primary antigen-binding signal, but a co-stimulatory signal, and binding of a second antigen produces the primary antigen-binding signal.
  • the first antigen e.g., a TAA or TSA
  • binding of a second antigen produces the primary antigen-binding signal.
  • a modified T lymphocyte comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and a first intracellular signaling domain; and b) a second polypeptide comprising a second extracellular antigen binding domain binding a second antigen; and a second intracellular signaling domain, wherein said second polypeptide does not comprise a co-stimulatory domain; wherein said modified lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • the first antigen and the second antigen are different antigens.
  • binding of said first antigen to said first antigen binding domain without binding of said second antigen to said second binding domain, or binding of said second antigen to said second antigen binding domain without binding of first second antigen to said first binding domain induces anergy of said modified T lymphocyte.
  • said first antigen binding domain and said second antigen binding domain are independently any antigen binding domain, e.g., any of the antigen binding domains disclosed in Section 5.2, above, e.g., an antigen-binding portion of a receptor or an antigen-binding portion of an antibody.
  • either or both of said first antigen binding domain or said second antigen binding domain are scFv antibody fragments.
  • the first polypeptide comprises one or more co-stimulatory domains, enabling the first polypeptide to provide co-stimulation when the first antigen binds to the first antigen-binding domain of the first polypeptide. Any co-stimulatory motif, or functional portion thereof, may be used.
  • the one or more co-stimulatory domains comprises one or more of a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, a co-stimulatory OX40 (CD 134) polypeptide sequence, a co-stimulatory 4- IBB (CD 137) polypeptide sequence, or a co-stimulatory inducible T-cell co-stimulatory (ICOS) polypeptide sequence.
  • the second intracellular signaling domain of the second polypeptide can be any polypeptide that is capable of transmitting an antigen binding signal from the second antigen binding domain of the second polypeptide, e.g., in a manner similar to the CD3 ⁇ (CD3 zeta) chains of the native T lymphocyte receptor.
  • the second intracellular signaling domain comprises a polypeptide sequence comprising an immunoreceptor tyrosine - based activation motif (IT AM).
  • the polypeptide sequence is a CD3 ⁇ signaling domain or a signal-transducing variant thereof.
  • either said first polypeptide or said second polypeptide additionally comprises a transmembrane domain.
  • the first polypeptide comprises one or more co-stimulatory domains, enabling the first polypeptide to provide co- stimulation when the first antigen binds to the first antigen-binding domain of the second polypeptide.
  • Any co-stimulatory domain, or functional portion thereof may be used, e.g., co- stimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4-1BB.
  • said first polypeptide or said second polypeptide comprises a T cell survival motif.
  • the first antigen may be any antigen of interest, e.g., an antigen that is expressed on the surface of a cell.
  • said first antigen is an antigen on a tumor cell.
  • the tumor cell can be a cell, e.g., of a solid tumor or a blood cancer, e.g., any of the cancer or tumor types disclosed in Section 5.2, above.
  • said antigen is a TAA or TSA, e.g., any of the TAAs or TSAs disclosed in Section 5.1, above.
  • the second antigen can be any antigen different from the first antigen, but is preferably related to the first antigen.
  • both the first and second antigens can be tumor- associated antigens or tumor-specific antigens that are present on the same tumor cell type.
  • the first antigen is a TAA or TSA
  • said second antigen is not a TAA or TSA
  • the second antigen in certain embodiments, can be related to an aspect of the tumor, e.g., the tumor environment.
  • a tumor can induce an inflammatory state in tissue surrounding the tumor, and can release angiogenic growth factors, interleukins, and/or cytokines that promote angiogenesis into and at the periphery of the tumor.
  • the second antigen is a growth factor, cytokine or interleukin, e.g., a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis, e.g., VEGF, bFGF, PDGF, HGF, IGF, or IL-8.
  • signal transduction by said second chimeric receptor is induced by activation of a hypoxia-associated factor, e.g., HIF-l , HIF- ⁇ , HIF-2a, HIF-2P, HIF-3a, or HIF-3p.
  • the second antigen is a DAMP, e.g., a heat shock protein, HMGB1, S100A8 (MRP 8, calgranulin A), S100A9 (MRP 14, calgranulin B), SAA, deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.
  • DAMP e.g., a heat shock protein, HMGB1, S100A8 (MRP 8, calgranulin A), S100A9 (MRP 14, calgranulin B), SAA, deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and a first intracellular signaling domain; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds an angiogenic or vasculogenic factor, and an intracellular CD3 ⁇ signaling domain, wherein said second polypeptide does not comprise a co-stimulatory domain; wherein said modified lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising an scFv or antigen-binding portion thereof, which binds a first antigen, and a first intracellular signaling domain; and b) a second polypeptide comprising a second extracellular antigen binding domain binding an angiogenic or vasculogenic factor, and an intracellular CD3 ⁇ signaling domain, wherein said second polypeptide does not comprise a co-stimulatory domain; wherein said modified lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, wherein said first antigen is a tumor-associated antigen (TAA) or tumor-specific antigen (TSA), and a first intracellular signaling domain; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, wherein said second antigen is VEGF, bFGF, PDGF, HGF, IGF, or IL-8, and an intracellular CD3 ⁇ signaling domain, wherein said second polypeptide does not comprise a co-stimulatory domain; wherein said modified lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, wherein said first antigen is a TAA or TSA, and a first intracellular signaling domain comprising co-stimulatory signaling domains from one or more of CD27, CD28, OX40, ICOS, and 4-1BB; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, wherein said second antigen is VEGF, bFGF, PDGF, HGF, IGF, or IL-8, and an intracellular CD3 ⁇ signaling domain, wherein said second polypeptide does not comprise a co-stimulatory domain; wherein said modified lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising an extracellular antigen binding domain that binds a first antigen, wherein said first antigen is Her2, PSCA, PSMA, BCMA, ERK5, AFP, CEA, CA-125, CA19-9, calretinin, MUC-1, EMA, ETA, tyrosinase, MAGE, CD34, CD45, CD99, CD1 17, chromogranin, cytokeratin, desmin, GFAP, GCDFP-15, HMB-45 antigen, MART-1, myo-Dl, MSA, neurofilament, NSE, placental alkaline phosphatase, synaptophysin,
  • thyroglobulin thyroid transcription factor- 1, tumor M2-PK, CD 19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAPl (six-transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, or an abnormal p53 protein; and an intracellular signaling domain comprising co -stimulatory signaling domains from one or more of CD27, CD28, OX40, ICOS, and 4- IBB; and b) a second polypeptide comprising an extracellular antigen binding domain that binds a second antigen, wherein said second antigen is VEGF, bFGF, PDGF, HGF, IGF, or IL-8,
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, wherein said first antigen is Her2, PSCA, PSMA, BCMA, ERK5, AFP, CEA, CA-125, CA19-9, calretinin, MUC-1, EMA, ETA, tyrosinase, MAGE, CD34, CD45, CD99, CD 117, chromogranin, cytokeratin, desmin, GFAP, GCDFP-15, HMB-45 antigen, MAPvT-1, myo-Dl, MSA, neurofilament, NSE, placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor- 1, tumor M2-PK, CD 19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal)
  • modified T lymphocytes comprising a first polypeptide comprising: a) a first extracellular antigen binding domain that binds a first antigen, wherein said first extracellular antigen binding domain is an csFv or antigen binding portion thereof, and wherein said first antigen is Her2, PSCA, PSMA, BCMA, ERK5, AFP, CEA, CA-125, CA19-9, calretinin, MUC-1, EMA, ETA, tyrosinase, MAGE, CD34, CD45, CD99, CD 117, chromogranin, cytokeratin, desmin, GFAP, GCDFP-15, HMB-45 antigen, MART-1, myo-Dl, MSA, neurofilament, NSE, placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor- 1, tumor M2-PK, CD 19, CD22, CD27, CD30, CD
  • either or both of said first polypeptide or said second polypeptide comprises a T cell survival motif, e.g., a T cell survival motif from CD28, IL-7R, IL-12R, IL-15R, IL-21R, TGFBR.
  • a T cell survival motif e.g., a T cell survival motif from CD28, IL-7R, IL-12R, IL-15R, IL-21R, TGFBR.
  • the two chimeric receptors contained within the modified T lymphocytes can be constructed so that a first chimeric receptor directed to a first antigen comprises both primary, antigen binding signaling domains and co-stimulatory domains, but no polypeptide sequence comprising a T cell survival motif, while a second chimeric receptor, directed to a second antigen, comprises a polypeptide sequence comprising a T cell survival motif.
  • T lymphocytes are directed to cells expressing a desired antigen, and, upon antigen binding, antigen binding and co-stimulatory signals are generated; however, in the absence of the binding of the second chimeric receptor to a second antigen, T lymphocytes are not directed to survive. As such, off-tumor effects are, again, eliminated or mitigated.
  • a modified T lymphocyte comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, a signaling domain, and one or more co-stimulatory motifs; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, and a T cell survival motif, wherein said second polypeptide does not comprise a co-stimulatory motif; wherein said modified lymphocyte survives only when said signaling domain and said T cell survival motif are both activated by said first antigen and said second antigen, respectively.
  • said T cell survival motif is an IL-7 receptor intracellular T cell survival motif is a T cell survival motif from CD28, IL-7R, IL-12R, IL-15R, IL-21R, or TGFBR.
  • either or both of said first polypeptide or said second polypeptide comprise a transmembrane domain.
  • said second polypeptide comprises a domain of CD27, CD28, IL-7R, IL-12R, IL-15R, IL-21R, or TGFBR that comprises a T cell survival motif.
  • first antigen binding domain and said second antigen binding domain are independently any antigen binding domain, e.g., any of the antigen binding domains disclosed in Section 5.2, above, e.g., an antigen-binding portion of a receptor or an antigen- binding portion of an antibody.
  • first antigen binding domain or said second antigen binding domain are scFv antibody fragments.
  • the first antigen may be any antigen of interest, e.g., an antigen that is expressed on the surface of a cell.
  • said first antigen is an antigen on a tumor cell.
  • the tumor cell can be a cell, e.g., of a solid tumor or a blood cancer, e.g., any of the cancer or tumor types disclosed in Section 5.2, above.
  • said antigen is a TAA or TSA, e.g., any of the TAAs or TSAs disclosed in Section 5.1, above.
  • the second antigen which is different from the first antigen, can be an angiogenic or vasculogenic factor, e.g., any of the angiogenic or vasculogenic factors disclosed in Section 5.1, above; or any DAMP, e.g., the DAMPs disclosed in Section 5.1, above.
  • signal transduction by said second chimeric receptor is induced by activation of a hypoxia-associated factor, e.g., HIF- la, HIF- ⁇ ⁇ , HIF-2a, HIF-2p, HIF-3a, or HIF-3p.
  • a hypoxia-associated factor e.g., HIF- la, HIF- ⁇ ⁇ , HIF-2a, HIF-2p, HIF-3a, or HIF-3p.
  • the first intracellular signaling domain of the first polypeptide can be any polypeptide that is capable of transmitting an antigen binding signal from the first antigen binding domain of the first polypeptide, e.g., in a manner similar to the CD3 ⁇ (CD3 zeta) chains of the native T lymphocyte receptor.
  • the second intracellular signaling domain comprises a polypeptide sequence comprising an immunoreceptor tyrosine-based activation motif (IT AM).
  • the polypeptide sequence is a CD3 ⁇ signaling domain or a signal- transducing variant thereof.
  • the first polypeptide additionally comprises one or more co- stimulatory domains, e.g., any co-stimulatory motif or functional portion thereof, e.g., a co- stimulatory CD27, CD28, OX40 (CD134), 4-1BB (CD137), or ICOS polypeptide sequence.
  • co-stimulatory motif or functional portion thereof e.g., a co- stimulatory CD27, CD28, OX40 (CD134), 4-1BB (CD137), or ICOS polypeptide sequence.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, an intracellular CD3 ⁇ signaling domain, and one or more co-stimulatory motifs; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, and a T cell survival motif, wherein said second polypeptide does not comprise a co- stimulatory motif; wherein said modified lymphocyte survives only when said signaling domain and said T cell survival motif are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, an intracellular CD3 ⁇ signaling domain, and a co-stimulatory polypeptide sequence from CD27, CD28, OX40 (CD134), 4-1BB (CD137), or ICOS; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, and a T cell survival motif, wherein said second polypeptide does not comprise a co-stimulatory motif; wherein said modified lymphocyte survives only when said signaling domain and said T cell survival motif are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, an intracellular CD3 ⁇ signaling domain, and a co-stimulatory polypeptide sequence from CD27, CD28, OX40 (CD134), 4-1BB (CD137), or ICOS; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, wherein said second antigen is an angiogenic or vasculogenic factor, or a DAMP, and a T cell survival motif, wherein said second polypeptide does not comprise a co-stimulatory motif; wherein said modified lymphocyte survives only when said signaling domain and said T cell survival motif are both activated by said first antigen and said second antigen, respectively.
  • signal transduction by said second chimeric receptor is induced by activation of a hypoxia-associated factor, e.g., HIF-la, HIF- ⁇ , HIF-2a, HIF-2p, HIF-3a, or HIF-3p.
  • a hypoxia-associated factor e.g., HIF-la, HIF- ⁇ , HIF-2a, HIF-2p, HIF-3a, or HIF-3p.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, an intracellular CD3 ⁇ signaling domain, and a co-stimulatory polypeptide sequence from CD27, CD28, OX40 (CD134), 4-1BB (CD137), or ICOS; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, wherein said second antigen is VEGF, bFGF, PDGF, HGF, IGF, or IL-8, and a T cell survival motif, wherein said second polypeptide does not comprise a co-stimulatory motif; wherein said modified lymphocyte survives only when said signaling domain and said T cell survival motif are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, an intracellular CD3 ⁇ signaling domain, and a co-stimulatory polypeptide sequence from CD28, OX40 and 4- IBB; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, and a T cell survival motif, wherein said second polypeptide does not comprise a co-stimulatory motif; wherein said modified lymphocyte survives only when said signaling domain and said T cell survival motif are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, wherein said first antigen is a TAA or TSA, an intracellular CD3 ⁇ signaling domain, and a co- stimulatory polypeptide sequence from CD27, CD28, OX40 (CD134), 4-1BB (CD137), or ICOS; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, and a T cell survival motif, wherein said second polypeptide does not comprise a co-stimulatory motif; wherein said modified lymphocyte survives only when said signaling domain and said T cell survival motif are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, wherein said first antigen is a TAA or TSA, an intracellular CD3 ⁇ signaling domain, and a co- stimulatory polypeptide sequence from CD27, CD28, OX40 (CD134), 4-1BB (CD137), or ICOS; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, wherein said second antigen is VEGF, bFGF, PDGF, HGF, IGF, or IL-8, and a T cell survival motif, wherein said second polypeptide does not comprise a co-stimulatory motif; wherein said modified lymphocyte survives only when said signaling domain and said T cell survival motif are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, wherein said first antigen is Her2, PSCA, PSMA, BCMA, ERK5, AFP, CEA, CA-125, CA19-9, calretinin, MUC-1 , EMA, ETA, tyrosinase, MAGE, CD34, CD45, CD99, CD1 17, chromogranin, cytokeratin, desmin, GFAP, GCDFP-15, HMB-45 antigen, MART-1 , myo-D l , MSA,
  • NSE neurofilament
  • placental alkaline phosphatase synaptophysin, thyroglobulin, thyroid transcription factor- 1 , tumor M2-PK, CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFPvvIII (epidermal growth factor variant III), sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, or an abnormal p53 protein; an intracellular CD3 ⁇ signaling domain; and a co- stimulatory polypeptide sequence from CD27, CD28, OX40 (CD134), 4-lBB (CD137), or ICOS; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, wherein said first antigen is Her2, PSCA, PSMA, BCMA, ERK5, AFP, CEA, CA-125, CA19-9, calretinin, MUC-1 , EMA, ETA, tyrosinase, MAGE, CD34, CD45, CD99, CD1 17, chromogranin, cytokeratin, desmin, GFAP, GCDFP-15, HMB-45 antigen, MART-1 , myo-D l , MSA,
  • NSE neurofilament
  • placental alkaline phosphatase synaptophysin, thyroglobulin, thyroid transcription factor- 1 , tumor M2-PK, CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, or an abnormal p53 protein; an intracellular CD3 ⁇ signaling domain; and a co- stimulatory polypeptide sequence from each of CD27, CD28, OX40 and 4-lBB; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, wherein said second
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds Her2, PSCA, PSMA, BCMA, ER 5, AFP, CEA, CA-125, CA19-9, calretinin, MUC-1, EMA, ETA, tyrosinase, MAGE, CD34, CD45, CD99, CD 117, chromogranin, cytokeratin, desmin, GFAP, GCDFP-15, HMB-45 antigen, MART-1, myo-Dl, MSA, neurofilament, NSE, placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor- 1, tumor M2-PK, CD 19, CD22, CD27, CD30, CD70, GD
  • the two chimeric receptors contained within the modified T lymphocytes can be constructed so that a first chimeric receptor directed to a first antigen comprises a first extracellular antigen-binding domain and an T cell survival motif, e.g., an intracellular T cell survival motif, but no primary antigen binding signaling domain (e.g., CD3 ⁇ ), and no co-stimulatory domains, while a second chimeric receptor, directed to a second antigen, comprises a polypeptide sequence comprising a primary antigen binding signaling domain (e.g., CD3 ⁇ ), and one or more co-stimulatory domains.
  • a first chimeric receptor directed to a first antigen comprises a first extracellular antigen-binding domain and an T cell survival motif, e.g., an intracellular T cell survival motif, but no primary antigen binding signaling domain (e.g., CD3 ⁇ ), and no co-stimulatory domains
  • a second chimeric receptor, directed to a second antigen comprises a polypeptide sequence
  • T lymphocytes are directed to cells expressing a desired antigen, and, upon antigen binding, a T lymphocyte survival signal is generated; however, in the absence of the binding of the second chimeric receptor to a second antigen, because no antigen binding and co-stimulatory signals are generated, T lymphocytes are not activated. As such, off-tumor effects are, again, eliminated or mitigated.
  • modified T lymphocyte comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and a T cell survival motif, wherein said first polypeptide does not comprise a primary antigen binding signaling domain or a co-stimulatory motif; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, and one or more co-stimulatory motifs; wherein said modified lymphocyte survives and is activated only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively.
  • said T cell survival motif is an IL-7 receptor intracellular T cell survival motif is a T cell survival motif from CD28, IL-7R, IL-12R, IL-15R, IL-21R, or TGFBR.
  • either or both of said first polypeptide or said second polypeptide comprise a transmembrane domain.
  • said first polypeptide comprises a domain of CD27, CD28, IL-7R, IL-12R, IL-15R, IL-21R, or TGFBR that comprises a T cell survival motif.
  • first antigen binding domain and said second antigen binding domain are independently any antigen binding domain, e.g., any of the types of antigen binding domains disclosed in Section 5.2, above, e.g., an antigen-binding portion of a receptor or an antigen-binding portion of an antibody.
  • first antigen binding domain or said second antigen binding domain are scFv antibody fragments.
  • the first antigen may be any antigen of interest, e.g., an antigen that is expressed on the surface of a cell.
  • said first antigen is an antigen on a tumor cell.
  • the tumor cell can be a cell, e.g., of a solid tumor or a blood cancer, e.g., any of the cancer or tumor types disclosed in Section 5.1, above.
  • said antigen is a TAA or TSA, e.g., any of the TAAs or TSAs disclosed in Section 5.1, above.
  • the second antigen which is different from the first antigen, can be an angiogenic or vasculogenic factor, e.g., any of the angiogenic or vasculogenic factors disclosed in Section 5.1, above; or any DAMP, e.g., the DAMPs disclosed in Section 5.1, above.
  • signal e.g., any of the angiogenic or vasculogenic factors disclosed in Section 5.1, above.
  • transduction by said second chimeric receptor is induced by activation of a hypoxia-associated factor, e.g., HIF-la, HIF- ⁇ ⁇ , HIF-2a, HIF-2P, HIF-3a, or HIF-3p.
  • a hypoxia-associated factor e.g., HIF-la, HIF- ⁇ ⁇ , HIF-2a, HIF-2P, HIF-3a, or HIF-3p.
  • the intracellular signaling domain of the second polypeptide can be any polypeptide that is capable of transmitting an antigen binding signal from the antigen binding domain of the second polypeptide, e.g., in a manner similar to the CD3 ⁇ (CD3 zeta) chains of the native T lymphocyte receptor.
  • the intracellular signaling domain comprises a polypeptide sequence comprising an immunoreceptor tyrosine-based activation motif (IT AM).
  • the polypeptide sequence is a CD3 ⁇ signaling domain or a signal-transducing variant thereof.
  • the second polypeptide additionally comprises one or more co-stimulatory domains, e.g., any co-stimulatory motif or functional portion thereof, e.g., a co-stimulatory CD27, CD28, OX40 (CD134), 4- IBB (CD137), or ICOS polypeptide sequence.
  • co-stimulatory domains e.g., any co-stimulatory motif or functional portion thereof, e.g., a co-stimulatory CD27, CD28, OX40 (CD134), 4- IBB (CD137), or ICOS polypeptide sequence.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and a T cell survival motif, wherein said first polypeptide does not comprise a primary antigen binding signaling domain and does not comprise a co-stimulatory motif; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, an intracellular CD3 ⁇ signaling domain, and one or more co-stimulatory motifs; wherein said modified lymphocyte is activated and survives only when said signaling domain and said T cell survival motif are activated by said second antigen and said first antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and a T cell survival motif, wherein said first polypeptide does not comprise a primary antigen binding signaling domain and does not comprise a co-stimulatory motif; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, an intracellular CD3 ⁇ signaling domain, and a co-stimulatory polypeptide sequence from CD27, CD28, OX40 (CD134), 4- IBB (CD137), or ICOS; wherein said modified lymphocyte is activated and survives only when said T cell survival motif and said signaling domain are activated by said first antigen and said second antigen, respectively.
  • a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and a T cell survival motif, wherein said first polypeptide does not comprise a primary antigen binding signaling domain and
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and a T cell survival motif, wherein said first polypeptide does not comprise a primary antigen binding signaling domain and does not comprise a co-stimulatory motif; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, wherein said second antigen is an angiogenic or vasculogenic factor, or a DAMP, an intracellular CD3 ⁇ signaling domain, and a co-stimulatory polypeptide sequence from CD27, CD28, OX40 (CD134), 4-1BB (CD137), or ICOS; wherein said second polypeptide does not comprise a primary antigen binding signaling domain and does not comprise a co-stimulatory motif; wherein said modified lymphocyte survives only when said T cell survival motif and said signaling domain are both activated by said first antigen
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and a T cell survival motif, wherein said first polypeptide does not comprise a primary antigen binding signaling domain and does not comprise a co-stimulatory motif; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, wherein said second antigen is VEGF, bFGF, PDGF, HGF, IGF, or IL-8, an intracellular CD3 ⁇ signaling domain, and a co-stimulatory polypeptide sequence from CD27, CD28, OX40 (CD134), 4-1BB (CD137), or ICOS; wherein said modified lymphocyte survives only when said T cell survival motif said signaling domain are both activated by said first antigen and said second antigen, respectively.
  • a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, and a T cell survival motif, wherein said first polypeptide does not comprise a primary antigen binding signaling domain and does not comprise a co-stimulatory motif; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, an intracellular CD3 ⁇ signaling domain, and a co-stimulatory polypeptide sequence from CD28, OX40 and 4-1BB; wherein said modified lymphocyte survives only when said T cell survival motif and said signaling domain are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, wherein said first antigen is a TAA or TSA, and a T cell survival motif, wherein said first polypeptide does not comprise a primary antigen binding signaling domain and does not comprise a co-stimulatory motif; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, an intracellular CD3 ⁇ signaling domain, and a co-stimulatory polypeptide sequence from CD27, CD28, OX40 (CD134), 4-1BB (CD137), or ICOS; wherein said modified lymphocyte survives only when said T cell survival motif and said signaling domain are both activated by said first antigen and said second antigen, respectively.
  • a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, wherein said first antigen is a TAA or TSA, and
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, wherein said first antigen is a TAA or TSA, and a T cell survival motif, wherein said first polypeptide does not comprise a primary antigen binding signaling domain and does not comprise a co-stimulatory motif; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds VEGF, bFGF, PDGF, HGF, IGF, or IL-8, an intracellular CD3 ⁇ signaling domain, and a co-stimulatory polypeptide sequence from CD27, CD28, OX40 (CD134), 4-1BB (CD137), or ICOS; wherein said modified lymphocyte survives only when said T cell survival motif and said signaling domain are both activated by said first antigen and said second antigen, respectively.
  • a first polypeptide comprising a first extracellular antigen binding domain that binds a first anti
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, wherein said first antigen is Her2, PSCA, PSMA, BCMA, ER 5, AFP, CEA, CA-125, CA19-9, calretinin, MUC-1, EMA, ETA, tyrosinase, MAGE, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, GFAP, GCDFP-15, HMB-45 antigen, MART-1, myo-Dl, MSA, neurofilament, NSE, placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, tumor M2-PK, CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III),
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds a first antigen, wherein said first antigen is Her2, PSCA, PSMA, BCMA, ER 5, AFP, CEA, CA-125, CA19-9, calretinin, MUC-1, EMA, ETA, tyrosinase, MAGE, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, GFAP, GCDFP-15, HMB-45 antigen, MART-1 , myo-Dl , MSA, neurofilament, NSE, placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor- 1 , tumor M2-PK, CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor- 1 , tumor M2-PK, CD
  • polypeptide does not comprise a primary antigen binding signaling domain and does not comprise a co-stimulatory motif; and b) a second polypeptide comprising a second extracellular antigen binding domain that binds a second antigen, wherein said second antigen is VEGF, bFGF, PDGF, HGF, IGF, or IL-8; an intracellular CD3 ⁇ signaling domain; and a co-stimulatory polypeptide sequence from each of CD27, CD28, OX40 and 4-1BB; wherein said modified lymphocyte survives only when said T cell survival motif and said signaling domain are both activated by said first antigen and said second antigen, respectively.
  • modified T lymphocytes comprising: a) a first polypeptide comprising a first extracellular antigen binding domain that binds Her2, PSCA, PSMA, BCMA, ERK5, AFP, CEA, CA-125, CA19-9, calretinin, MUC-1 , EMA, ETA, tyrosinase, MAGE, CD34, CD45, CD99, CD 1 17, chromogranin, cytokeratin, desmin, GFAP, GCDFP-15, HMB-45 antigen, MART-1 , myo-Dl , MSA, neurofilament, NSE, placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor- 1 , tumor M2-PK, CD 19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp
  • the modified T lymphocyte comprises a modified TCR as a first polypeptide, and an artificial second polypeptide that produces a co-stimulatory signal.
  • the modified TCR is modified, e.g., by replacement of the native antigen- binding domain with a domain that binds to a specific antigen.
  • the T lymphocytes are transformed with polynucleotides encoding alpha and beta chains of the anti- MART-1 TCR. T lymphocytes may similarly be transformed with polynucleotides encoding alpha and beta TCR subunits, where the TCR is directed to an antigen, e.g., a TSA or TAA.
  • the modified T lymphocyte is additionally transformed with a polynucleotide that encodes an artificial co-stimulatory polypeptide, e.g., the co-stimulatory polypeptide disclosed in Section 5.1, above, or one of the second polypeptides disclosed in Section 5.2, above.
  • an artificial co-stimulatory polypeptide e.g., the co-stimulatory polypeptide disclosed in Section 5.1, above, or one of the second polypeptides disclosed in Section 5.2, above.
  • modified T lymphocytes comprise two polypeptides, e.g., chimeric receptors
  • a first polypeptide comprises a first antigen binding domain, a primary antigen binding signal transduction domain (e.g., CD3 ⁇ ), and a single co-stimulatory domain (e.g., CD28 or a co-stimulatory polypeptide sequence therefrom), and a second polypeptide that comprises a second antigen binding domain and at least one co-stimulatory domain, e.g., a co- stimulatory domain from CD27, 4- IBB, OX40, IL-7R or the like.
  • a co-stimulatory domain from CD27, 4- IBB, OX40, IL-7R or the like.
  • the second polypeptide comprises at least two, or at least three co-stimulatory domains.
  • the modified T lymphocytes provided herein comprise a first polypeptide (e.g., chimeric receptor) comprising a first antigen binding domain, a primary antigen binding signal transduction domain (e.g., CD3 ⁇ ), and no co-stimulatory domain (e.g., CD28 or a co-stimulatory polypeptide sequence therefrom); a second polypeptide (chimeric receptor) comprising a second antigen-binding domain and at least one co-stimulatory domain; and a third polypeptide comprising an antigen-binding domain and at least one other co- stimulatory domain.
  • the total number of co-stimulatory domains is divided between at least two separate chimeric receptors.
  • the at least two different chimeric receptors can comprise antigen binding domains that bind to the same antigen, or different antigens.
  • the first and second polypeptides provided herein, useful for producing the modified T lymphocytes provided herein, may be modified by, e.g., acylation, amidation, glycosylation, methylation, phosphorylation, sulfation, sumoylation, ubiquitylation, or the like.
  • polypeptides may be labeled with a label capable of providing a detectable signal, e.g., with radioisotopes and fluorescent compounds.
  • One or more side chains of the first or second polypeptides may be derivatized, e.g., derivatization of lysinyl and amino terminal residues with succinic or other carboxylic acid anhydrides, or derivatization with, e.g., imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;
  • polypeptides e.g., chimeric receptors
  • the polynucleotides may be contained within any polynucleotide vector suitable for the transformation of immune cells, e.g., T lymphocytes.
  • T lymphocytes may be transformed using synthetic vectors, lentiviral or retroviral vectors, autonomously replicating plasmids, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or the like, containing polynucleotides encoding the first and second polypeptides (e.g., chimeric receptors).
  • Lentiviral vectors suitable for transformation of T lymphocytes include, but are not limited to, e.g., the lentiviral vectors described in U.S. Patent Nos. 5,994,136; 6,165,782; 6,428,953; 7,083,981; and 7,250,299.
  • HIV vectors suitable for transformation of T lymphocytes include, but are not limited to, e.g., the lentiviral vectors described in U.S. Patent No. 5,665,577.
  • Nucleic acids useful in the production of the first and second polypeptides include DNA, RNA, or nucleic acid analogs.
  • Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone, and can include deoxyuridine substitution for deoxythymidine, 5-methyl-2'-deoxycytidine or 5-bromo-2'- deoxycytidine substitution for deoxycytidine.
  • Modifications of the sugar moiety can include modification of the 2' hydroxyl of the ribose sugar to form 2'-0-methyl or 2'-0-allyl sugars.
  • the deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7: 187-195; and Hyrup et al. (1996) Bioorgan. Med. Chain. 4:5-23. In addition, the
  • deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or
  • phosphorodithioate backbone a phosphoroamidite, or an alkyl phosphotriester backbone.
  • the T lymphocytes used in the compositions and methods provided herein may be naive T lymphocytes or MHC-restricted T lymphocytes.
  • the T lymphocytes are tumor infiltrating lymphocytes (TILs).
  • TILs tumor infiltrating lymphocytes
  • lymphocytes have been isolated from a tumor biopsy, or have been expanded from T
  • lymphocytes isolated from a tumor biopsy In certain other embodiments, the T cells have been isolated from, or are expanded from T lymphocytes expanded from, peripheral blood, cord blood, or lymph.
  • the immune cells e.g., modified T lymphocytes, used in the present methods are preferably autologous to an individual to whom the modified T lymphocytes are to be
  • the modified T lymphocytes are allogeneic to an individual to whom the modified T lymphocytes are to be administered. Where allogeneic T lymphocytes are used to prepare modified T lymphocytes, it is preferable to select T
  • virus-specific T lymphocytes are selected for preparation of modified T lymphocytes; such lymphocytes will be expected to have a greatly reduced native capacity to bind to, and thus become activated by, any recipient antigens.
  • recipient-mediated rejection of allogeneic T lymphocytes can be reduced by co-administration to the host of one or more immunosuppressive agents, e.g., cyclosporine, tacrolimus, sirolimus, cyclophosphamide, or the like.
  • T lymphocytes are obtained from an individual, optionally then expanded, and then transformed with a first polynucleotide encoding the first polypeptide and a second polynucleotide encoding the second polypeptide, and optionally then expanded. Double transformants may be selected using, e.g., a selectable marker unique to each of the vectors.
  • T lymphocytes are obtained from an individual, optionally then expanded, and then transformed with a polynucleotide encoding the first polypeptide and the second polypeptide, and optionally then expanding. Cells containing the polynucleotide are selected using a selectable marker.
  • the modified T lymphocytes comprise native TCR proteins, e.g., TCR-a and TCR- ⁇ that are capable of forming native TCR complexes, in addition to the artificial co-stimulatory polypeptide (in embodiments in which a single co-stimulatory polypeptide is used), or in addition to the first polypeptide and second polypeptide (in embodiments in which the modified T lymphocytes comprise polypeptides separating the antigen binding signaling and co-stimulatory signaling).
  • the native genes encoding TCR-a and TCR- ⁇ in the modified T lymphocytes are modified to be non- functional, e.g., a portion or all are deleted, a mutation is inserted, etc.
  • the T lymphocytes are isolated from a tumor lesion, e.g., are tumor-infiltrating lymphocytes; such T lymphocytes are expected to be specific for a TSA or TAA.
  • the signaling motifs of the first polypeptide and the second polypeptide can be used to promote proliferation and expansion of the modified T lymphocytes.
  • unmodified T lymphocytes, and T lymphocytes comprising a polypeptide comprising a CD3 ⁇ signaling domain and a CD28 co-stimulatory domain can be expanded using antibodies to CD3 and CD28, e.g., antibodies attached to beads; see, e.g., U.S. Patent Nos.
  • antibodies to a signaling motif on the first polypeptide and antibodies to a signaling motif on the second polypeptide can be used to stimulate proliferation of T lymphocytes comprising both the first and second polypeptides.
  • the first and second antigens to which the first polypeptide and second polypeptide bind, respectively can be used to promote selective expansion of T lymphocytes expressing both the first polypeptide and the second polypeptide.
  • the T lymphocytes comprising the first polypeptide and second polypeptide are cultured in the presence of the TSA and the angiogenic factor, resulting in increased proliferation as compared to culturing in the presence of the first or second antigens, alone, or in the absence of either.
  • the T lymphocytes comprising the first and second polypeptides are stimulated to proliferate using an antibody that binds to a signaling domain on the first polypeptide coupled with an antigen that can be bound by the second polypeptide.
  • an antibody that binds to a signaling domain on the first polypeptide coupled with an antigen that can be bound by the second polypeptide For example, in embodiments in which the first polypeptide's signaling domain is CD3 ⁇ and the antigen that binds to the second polypeptide is VEGF, T lymphocytes comprising the first and second polypeptides are stimulated to proliferate by culturing the cells in the presence of VEGF in combination with an antibody that binds to CD3 ⁇ .
  • the T lymphocytes comprising the first and second polypeptides are stimulated to proliferate using an antigen that can be bound by the first polypeptide and a co-stimulatory motif on the second peptide.
  • an antigen that can be bound by the first polypeptide is HER2 and the co-stimulatory motif on the second polypeptide is obtained from CD28
  • T lymphocytes comprising the first and second polypeptides are stimulated to proliferate by culturing the cells in the presence of HER2 protein and an antibody that binds to CD28.
  • the antigen and/or antibody can exist free in the medium in which the T lymphocytes are cultures, or either or both can be attached to a solid support, e.g., tissue culture plastic surface, beads, or the like.
  • the modified T lymphocytes can optionally comprise a "suicide gene” or "safety switch” that enables killing of substantially all of the modified T lymphocytes when desired.
  • the modified T lymphocytes in certain embodiments, can comprise an HSV thymidine kinase gene (HSV-TK), which causes death of the modified T lymphocytes upon contact with gancyclovir.
  • the modified T lymphocytes comprise an inducible caspase, e.g., an inducible caspase 9 (icaspase9), e.g., a fusion protein between caspase 9 and human FK506 binding protein allowing for dimerization using a specific small molecule pharmaceutical. See Straathof et al, Blood 105(11):4247-4254 (2005).
  • the modified immune cells can be used to treat an individual having one or more types of cells desired to be targeted by T lymphocytes, e.g., to be killed.
  • the cells to be killed are cancer cells, e.g., tumor cells.
  • the cancer cells are cells of a solid tumor.
  • the cells are cells of a lymphoma, a lung cancer, a breast cancer, a prostate cancer, an adrenocortical carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, e.g., a malignant melanoma, a skin carcinoma, a colorectal carcinoma, a desmoid tumor, a desmoplastic small round cell tumor, an endocrine tumor, an Ewing sarcoma, a peripheral primitive neuroectodermal tumor, a solid germ cell tumor, a hepatoblastoma, a neuroblastoma, a non-rhabdomyosarcoma soft tissue sarcoma, an osteosarcoma, a retinoblastoma, a rhabdomyosarcoma, a Wilms tumor, a glioblastoma, a myxoma, a fibroma,
  • said lymphoma can be chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, T lymphocyte prolymphocytic leukemia, T lymphocyte large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T lymphocyte leukemia/lymphoma, extranodal NK/T
  • Efficacy of the modified T lymphocytes after administration to an individual having a disease or disorder remediable by T lymphocytes, e.g., an individual having cancer, can be assessed by one or more criteria, specific to the particular disease or disorder, known to those of ordinary skill in the art, to be indicative of progress of the disease or disorder.
  • administration of the modified T lymphocytes to such an individual is effective when one or more of said criteria detectably, e.g., significantly, moves from a disease state value or range to, or towards, a normal value or range.
  • the modified T lymphocytes may be formulated in any pharmaceutically-acceptable solution, preferably a solution suitable for the delivery of living cells, e.g., saline solution (such as Ringer's solution), gelatins, carbohydrates (e.g., lactose, amylose, starch, or the like), fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidine, etc.
  • saline solution such as Ringer's solution
  • Such preparations are preferably sterilized prior to addition of the modified T lymphocytes, and may be mixed with auxiliary agents such as lubricants, preservatives, stabilizers, emulsifiers, salts for influencing osmotic pressure, buffers, and coloring.
  • Pharmaceutical carriers suitable for use in formulating the modified T lymphocytes are known in the art and are described, for example, in WO 96/05309.
  • the modified T lymphocytes are formulated into individual doses, wherein said individual doses comprise at least, at most, or about lxlO 4 , 5x l0 4 , lx lO 5 , 5x l0 5 , lx lO 6 , 5x l0 6 , lxlO 7 , 5xl0 7 , lxlO 8 , 5xl0 8 , lxlO 9 , 5xl0 9 , lx lO 10 , 5xl0 10 , or lxlO 11 modified T lymphocytes.
  • the modified T lymphocytes are formulated for intravenous, intraarterial, parenteral, intramuscular, subcutaneous, intrathecal, or intraocular administration, or administration within a particular organ or tissue.
  • An individual presents with stage T2 prostate cancer, with no spread to regional or other lymph nodes (NO, M0). Histological grade is determined to be G2. Overall, the individual is determined to have Stage II prostate cancer.
  • the individual is administered between 10 9 and 10 10 modified T lymphocytes that comprise a single chimeric receptor, in 200 mL saline solution by intravenous infusion over 30 minutes.
  • the chimeric receptor comprises an extracellular antigen-binding region that binds to PSCA, a transmembrane domain, and intracellular co- stimulatory domains from each of CD27, CD28, 4-1BB, and OX40.
  • the individual is reassessed for prostate cancer stage and spread to lymph nodes, and histology of biopsied prostate tissue is performed, at 30, 60 and 90 days post-administration.
  • An individual presents with stage T2 prostate cancer, with no spread to regional or other lymph nodes (NO, M0). Histological grade is determined to be G2. Overall, the individual is determined to have Stage II prostate cancer.
  • the individual is administered between 10 9 and 10 10 modified T lymphocytes that comprise a first and second chimeric receptor, in 200 mL saline solution by intravenous infusion over 30 minutes.
  • the first chimeric receptor comprises an extracellular antigen-binding region that binds to PSCA, a transmembrane domain, and a signal transduction domain derived from CD3 ⁇ .
  • the second chimeric receptor comprises an antigen-binding domain that binds to the protein ER 5, a transmembrane domain, and intracellular co-stimulatory domains from each of CD27, CD28, 4-1BB, and OX40.
  • the individual is re-assessed for prostate cancer stage and spread to lymph nodes, and histology of biopsied prostate tissue is performed, at 30, 60 and 90 days post-administration.
  • An individual presents with stage 3 breast cancer that has spread to at least one regional lymph node. After surgery to remove cancerous tissue, the individual is administered between 10 9 and 10 10 modified T lymphocytes that comprise a first and second chimeric receptor, in 200 mL saline solution by intravenous infusion over 30 minutes.
  • the first chimeric receptor comprises an extracellular antigen-binding region that binds to HER2, a transmembrane domain, and a signal transduction domain derived from CD3 ⁇ .
  • the second chimeric receptor comprises an antigen-binding domain that binds to the estrogen receptor (ER), a transmembrane domain, and intracellular co-stimulatory domains from each of CD27, CD28, 4-1BB, and OX40.
  • the individual is assessed for breast cancer in remaining breast tissue, and spread to other lymph nodes, 30, 60, 90 and 180 days post-administration.
  • This Example describes the generation of modified T lymphocytes comprising two chimeric antigen receptors (CARs), wherein the first CAR comprises an antigen binding domain specific to a tumor-specific antigen and wherein the second CAR comprises an antigen binding domain specific to an antigen that is not a tumor-specific antigen, but that is associated with tumorigenesis.
  • CARs chimeric antigen receptors
  • the CARs depicted in Figure 1 were prepared using standard methodology.
  • An anti- HER2 -based CAR, "HER2-CAR ⁇ ,” consisting of a scFv of an anti-HER2 antibody linked to a CD28 hinge, a CD28 transmembrane (TM) domain, a CD3 zeta chain, and a tdTomato reporter gene was constructed and cloned into a lentivirus vector.
  • This CAR represents a CAR
  • an antigen binding domain specific to a tumor-specific antigen comprising an antigen binding domain specific to a tumor-specific antigen, and a stimulatory domain.
  • VEGFR2-CD28 comprises the human VEGFR2 extracellular (EC) domain followed by a VEGFR2 TM domain, a CD28 IC domain, a T2A sequence (thosea asigna virus 2A peptide), and GFP (for use as a reporter gene).
  • VEGFR2-28TM-CD28 comprises the human VEGFR2 extracellular (EC) domain followed by a CD28 TM domain, a CD28 intracellular (IC) domain, a T2A sequence, and GFP.
  • a control construct for VEGF recognition also was generated.
  • the control construct designated as "VEGFR2,” comprises the human VEGFR2 extracellular (EC) domain followed by a VEGFR2 TM domain, a T2A sequence, and GFP.
  • the control construct thus lacks the CD28 IC domain present in the constructs designated VEGFR2-CD28 and VEGFR2-28TM- CD28.
  • PBMC peripheral blood mononuclear cells
  • Ficoll-Paque PlusTM density gradient centrifugation GE Healthcare, Piscataway, NJ
  • Pan T cells were negatively selected from PBMCs using the Pan T Isolation Kit II (Miltenyi Biotec, Cambridge, MA), according to the manufacturer's instructions.
  • Plasmids comprising the HER2-CAR ⁇ , VEGFR2-CD28, VEGFR2-28TM-CD28, or VEGFR2 CAR constructs were electroporated into primary T cells, and the electroporated T cells were cultured in RPMI-10 media overnight. T cells were harvested at 24 hours post electroproation and stained with HER2 -human IgG-Fc chimera protein, followed by staining with a goat anti-human IgG-Fc polyclonal antibody conjugated with APC (for anti-HER2 detection); or a mouse anti-human VEGFR2 monoclonal antibody (mAb; for VEGFR2 detection). The stained cells were analyzed by flow cytometry.
  • VEGF Production and VEGFR2 Expression by Activated T cells [00139] To ensure that endogenous VEGFR2 expression does not compete with expression of the VEGFR2 extracellular domain in the VEGFR2 EC domain containing constructs for binding to the VEGF, preliminary experiments were carried out to assess levels of VEGF production and VEGFR2 expression by activated T cells.
  • Human primary T cells were isolated as described above and stimulated with anti- CD3/CD28 DynaBeads® at a ratio of 3 to 1 beads per cell.
  • the cells were cultured in RPMI-10 media in the presence of IL-2 at 50IU/ml.
  • VEGFR2 expression by the stimulated T cells was assessed via flow cytometry for 25 days post stimulation (every day for the first 4 days, then every two days after the first week).
  • VEGFR2 expression by activated T cells was not observed until day 2 post stimualtion, followed by a dramatic decrease in expression by day 3 and disappearance of expression by day 4. After day 4, VEGFR2 expression was not observed.
  • VEGF-A in the supernant of T cells after the DynaBead activation described above was measured via Cytometric Bead Array (CBA). Minimal VEGF secretion ( ⁇ 10pg/ml) was detected.
  • human primary pan T cells were transfected with either the VEGFR2-CD28, VEGFR2-28TM-CD28, or VEGFR2 lentivectors followed by stimulation with immobilized anti- human CD3 and anti-human VEGFR2 mAb or soluble VEGF.
  • Anti-human CD3 was selected as a ligand to trigger the first "signal" in the dual signaling system (that is, binding of a tumor antigen by a CAR comprising an antigen binding domain specific to a tumor antigen and an activation domain (e.g., a CD3 zeta chain)).
  • Culturing the T cells with either anti-VEGFR2 mAb or soluble VEGF resulted in stimulation of T cells that had been transfected with either
  • VEGFR2-CD28 lentivector or VEGFR2-28TM-CD28 lentivector but not T cells transfected with VEGFR2 lentivector, as evidenced by upregulation of the activation markers CD69 and 4- 1BB.
  • VEGFR2-CD28 lentivector and VEGFR2-28TM- CD28 lentivector transfected T cells secrete elevated levels of IL-2, granzyme B, and IFN- ⁇ upon anti-VEGFR2 mAb treatment or VEGF treatment.
  • the results indicate that expression of the VEGFR2 EC domain in transfected T cells can mediate intracellular CD28 signaling when VEGFR2 EC is expressed as part of a CAR comprising a CD28 IC domain.
  • HER2-CAR28 ⁇ Functional validation of HER2-CAR ⁇ was determined in transfected T cells by stimulation of the T cells with immobilized HER2-Fc chimera protein.
  • HER2-CAR28 ⁇ As a positive control for CD28 costimulation, another construct, "HER2-CAR28 ⁇ ,” which is identical to the CAR construct designated HER2-CAR ⁇ with the exception of the inclusion of a CD28 intracellular domain between the CD28 transmembrane (TM) domain and the CD3 zeta chain, was generated.
  • T cell activation markers CD69 and CD71 were examined 48 hours post-stimulation. Only tdTomato-positive cells showed CD69 and CD71 up-regulation in both HER2-CAR ⁇ and HER2- CAR28 ⁇ transfected cells.
  • T cells were treated with HER2-Fc and VEGFR2 mAb or VEGF for 48 hours, followed by flow cytometric analysis to assess surface expression of CD69 and CD71. Only very minimal enhancement of both CD69 and CD71 expression relative to that described above was observed.
  • HER2-CAR ⁇ and VEGFR2- CD28IC dual signaling were assessed.
  • T cells were isolated as described above and transfected with both (i) a CAR that comprises an anti-HER2 domain (i.e., HER2-CAR ⁇ ); and (ii) a CAR that comprises a VEGFR2 receptor extracellular domain (i.e., the CAR designated VEGFR2-CD28, VEGFR2-28TM-CD28, or VEGFR2).
  • Expression of each CAR by the transfected T cells was confirmed using flow cytometry by measurement of reporter gene expression (i.e., tdTomato or GFP), as described above.
  • T cell activation markers CD69 and CD71 by T cells transfected with both HER2-CAR ⁇ and one of of the three VEGFR2 CAR constructs (i.e., the CAR construct designated VEGFR2-CD28, VEGFR2-28TM-CD28, or VEGFR2) was examined following stimulation of the T cells with HER2-Fc and either anti-VEGFR2 mAb or VEGF.
  • T cells expressing GFP i.e., T cells expressing a CAR construct comprising a VEGFR2 EC domain
  • Stimulation with VEGF also showed amplification of CD69 and CD71 expression in T cells expressing GFP (i.e., T cells expressing a CAR construct comprising a VEGFR2 EC domain).
  • CAR T cells can be generated that comprise two CARs, with the signaling domain present in a first CAR, and the costimulatory domains present in a second CAR.
  • Such CAR T cells are useful in the treatment of diseases, e.g., cancer, where it is desirable to utilize a dual signaling approach that relies on the recognition of two separate antigens by the two CARs.
  • This Example describes the generation of modified T lymphocytes comprising CARs that can be used in the dual signaling approach described in the present application.
  • the modified T lymphocytes comprise a first chimeric antigen receptor that comprises an antigen binding domain specific to a tumor-specific antigen and a second chimeric antigen receptor that comprises an antigen binding domain specific to an antigen that is not a tumor-specific antigen, but that is associated with tumorigenesis.
  • the two CARs are introduced into the modified T cells using a single CAR construct, with the CARs separated by P2A, which allows for the expression of two, discrete CARs (at essentially equal amounts) from a single ORF.
  • Constructs comprising CARs are depicted in Figure 4.
  • CAR1 comprises an anti-HER2 scFv, followed by a CD28 hinge, a CD28 transmembrane (TM) domain, a CD3 zeta chain, a T2A sequence, and a tdTomato reporter gene.
  • CAR2 comprises an anti- HER2 scFv, followed by a CD28 hinge, a CD28 transmembrane (TM) domain, a CD28 IC domain, a CD3 zeta chain, a T2A sequence, and a tdTomato reporter gene.
  • CAR3 comprises the human VEGFR2 extracellular (EC) domain, followed by a VEGFR2 TM domain, a P2A sequence, an anti-HER2 scFv, a CD28 hinge, a CD28 transmembrane (TM) domain, and a CD3 zeta chain.
  • CAR4 comprises the human VEGFR2 extracellular (EC) domain, followed by a CD28 transmembrane (TM) domain, a CD28 IC domain, an anti-HER2 scFv, a CD28 hinge, a CD28 transmembrane (TM) domain, and a CD3 zeta chain.
  • CAR1 represents a first generation anti-HER2 CAR that comprises a primary signaling domain (CD3 zeta chain), but lacks a costimulatory domain.
  • CAR 2 represents a second generation anti-HER2 CAR that comprises both a primary signaling domain (CD3 zeta chain) and a costimulatory domain (CD28 IC domain).
  • CAR3 is a dual CAR control construct; it comprises a HER2 primary signaling portion (HER2 scFV and CD3 zeta chain) and also comprises a VEGFR2 secondary signaling domain, but the secondary signaling domain lacks a costimulatory domain.
  • CAR4 is a dual CAR construct; it comprises a HER2 primary signaling portion (HER2 scFV and CD3 zeta chain) and also comprises a VEGFR2 secondary signaling domain with a costimulatory domain (CD28 IC).
  • Pan T cells were isolated as described above, transfected with the CAR constructs (CAR1-CAR4) described above and analyzed 24-hours post-transduction. Expression of anti- HER2 was detected in T cells transfected with all of the CAR constructs. Expression of both anti-HER2 and VEGFR2 was detected in T cells transfected with CAR3 or CAR4. Thus, proper expression of the CAR constructs described above by T cells was confirmed.
  • T cells expressing the constructs were cultured with HER2-Fc (0.25 ug/ml or 1.0 ug/ml) - to induce stimulation the HER2-scFv containing constructs (primary signaling) - alone or in combination with either an anti-VEGFR2 antibody (0.25 ug/ml or 1.0 ug/ml) or VEGF (1, 10, or 100 ng/ml) - to induce stimulation of VEGFR2 containing constructs (costimulation).
  • VEGFR2 activation was found not to alter surface marker expression T cell activation markers CD69 or CD71 (as assessed by flow cytometry) in T cells transfected with either CARl or CAR2 over stimulation observed with anti-HER2 activation alone.
  • CD71 expression was analyzed: 45.2% and 50.7% of CAR4-expressing CAR T cells expressed CD71 when stimulated with doses of 1.0 ug/ml HER2-Fc/0.25 ug/ml anti-VEGFR2 and of 1.0 ug/ml HER2-Fc/1.0 ug/ml anti-VEGFR2, respectively, as compared to 22.8% of CD71 + CAR4- expressing CAR T cells stimulated with 1.0 ⁇ g/ml HER2-Fc alone ; whereas only 7.80%> and 7.89%) of CAR3 -expressing CAR T cells expressed CD71 when stimulated with HER2-Fc and anti-VEGFR2 at the same doses, respectively, as compared to 10.3% of CD71 + CAR3- expressing CAR T cells stimulated with 1.0 ⁇ g/ml HER2-Fc alone.
  • CAR4-expressing CAR T cells expressed CD69 when stimulated with doses of 0.25 ug/ml HER2-Fc/1 ng/ml VEGF, 0.25 ug/ml HER2-Fc/10 ng/ml VEGF, and 0.25 ug/ml HER2- Fc/100 ng/ml VEGF, respectively, as compared to 33.4% of CD69 + CAR4-expressing CAR T cells when stimulated with 0.25 ⁇ HER2-Fc alone; whereas only 3.40%, 2.69%, and 2.55% of CAR3 -expressing CAR T cells expressed CD69 when stimulated with HER2-Fc and VEGF at the same doses, respectively, as compared to 4.9% of CD69 CAR3 -expressing CAR T cells stimulated with 0.25 ⁇ g/ml HER2-Fc alone .
  • CD71 expression it was determined that 30.10%, 42.30%, and 47.30% of C AR4-expressing CAR T cells expressed CD71 when stimulated with doses of 1.0 ug/ml HER2-Fc/1 ng/ml VEGF, 1.0 ug/ml HER2-Fc/10 ng/ml VEGF, and 1.0 ug/ml HER2-Fc/100 ng/ml VEGF, respectively, as compared to 22.8% of CD71 + CAR4-expressing CAR T cells stimulated with 1.0 ⁇ g/ml HER2-Fc alone; whereas only 10.70%), 4.81%), and 7.33% of CAR3 -expressing CAR T cells expressed CD69 when stimulated with
  • HER2-FC and VEGF at the same doses, respectively, as compared to 10.3% of CD71 + CAR3- expressing CAR T cells stimulated with 1.0 ⁇ g/ml HER2-Fc alone.
  • Granzyme B is an enzyme enzyme present in cytotoxic T lymphocyte granules.
  • T cells transfected with either CARl, CAR3, or CAR4 were assessed.
  • T cells transfected with CAR4 expressed increased levels of granzyme B when stimulated with HER2-Fc and anti-VEGFR2 at doses of .25 ug/ml HER2-Fc/0.25 ug/ml anti-VEGFR2 and of 0.25 ug/ml HER2-Fc/1.0 ug/ml anti-VEGFR2 as compared to T cells transfected with either control CAR (CARl or CAR3).
  • T cells transfected with CAR4 expressed increased levels of granzyme B when stimulated with HER2-Fc and VEGF at doses of .25 ug/ml HER2- Fc/lng/ml VEGF and of 0.25 ug/ml HER2-Fc/100 ng/ml VEGF as compared to T cells transfected with either control CAR (CARl or CAR3).
  • T cells transfected with CARl, CAR3, or CAR4 following stimulation with HER2-Fc in combination with either an anti-VEGFR2 antibody or VEGF was assessed.
  • Pan T cells were isolated as described. After 24 hours of culture, the T cells were stimulated with HER2-Fc (1.0 ug/ml) and either anti-VEGFR2 antibody (0.25 ug/ml or 1.0 ug/ml) or VEGF (1 ng/ml or 100 ng/ml) for 48 hours. After 13 total days of culture, viability of the T cells was determined. In each case, T cells transfected with the dual stimulation CAR (i.e., CAR4) showed increased viability over T cells transfected with the dual control CAR (i.e., CAR3) or the control CAR designated CARl .
  • Example 4 confirms the result of Example 4 - that functional CAR T cells can be generated that comprise two CARs, with the signaling domain present in a first CAR the costimulatory domains present in a second CAR - and further demonstrates that the two CAR constructs can be expressed in the CAR T cells as a single construct (e.g., can be the T cells can be transfected with a single CAR construct that comprises both of the CARs).

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AU2014214850A1 (en) 2015-08-13
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