WO2024091669A1 - Récepteurs antigéniques chimériques comprenant une paire de domaines intracellulaires - Google Patents

Récepteurs antigéniques chimériques comprenant une paire de domaines intracellulaires Download PDF

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WO2024091669A1
WO2024091669A1 PCT/US2023/036126 US2023036126W WO2024091669A1 WO 2024091669 A1 WO2024091669 A1 WO 2024091669A1 US 2023036126 W US2023036126 W US 2023036126W WO 2024091669 A1 WO2024091669 A1 WO 2024091669A1
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cell
domain
car
cells
amino acid
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PCT/US2023/036126
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Taeyoon KYUNG
Shawdee ESHGHI
Joshua MACE
Siqi ZHAO
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Ginkgo Bioworks, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464469—Tumor associated carbohydrates
    • A61K39/464471—Gangliosides, e.g. GM2, GD2 or GD3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/17—Hinge-spacer domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/22—Intracellular domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • T cell exhaustion is a state of dysfunction arising from repeated or excessive cellular activation. Normally, T cell exhaustion is a compensatory mechanism to prevent excessive immune activation, but it has also been shown to be a limiting factor in efficacy in the context of adoptive T cell therapies.
  • Chimeric antigen receptors are comprised of several domains, including a targeting domain that determines its antigen specificity (e.g., an antigen-binding domain) and an intracellular signaling region, which comprises intracellular domains (ICDs) that activate cell signaling.
  • ICDs intracellular domains
  • Signaling cascades triggered by ICDs of CARs in immune cells drive cell behaviors that correspond to different therapeutic outcomes.
  • Currently approved cell therapies contain one of the two combinations of ICDs: a 4-1BB costimulatory domain combined with a CD3-zeta activation domain (BBz) or a CD28 costimulatory domain combined with a CD3-zeta activation domain (28z).
  • CAR-based cell therapies have not yet been 1 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 successful in other indications, such as the treatment of solid tumors.
  • CARs with different intracellular domains that drive more favorable T cell phenotypes, such as more favorable exhaustion profiles.
  • ICDs intracellular domains
  • immune cells expressing the CARs provided herein have favorable therapeutic properties, such as reduced cellular exhaustion and/or improved effector function against solid tumors.
  • Chimeric antigen receptors are comprised of several domains, including a targeting domain that determines its antigen specificity (e.g., an antigen-binding domain) and an intracellular signaling region, which comprises intracellular domains (ICDs) that activate cell signaling. Signaling cascades triggered by ICDs of CARs in immune cells drive cell behaviors that correspond to different therapeutic outcomes.
  • CAR-based cell therapies contain one of two combinations of ICDs: a 4-1BB costimulatory domain combined with a CD3-zeta activation domain (BBz) or a CD28 costimulatory domain combined with a CD3-zeta activation domain (28z). While these canonical CAR-T ICD combinations have achieved clinical success in a limited number of hematologic cancer indications, CAR-based cell therapies have not yet been successful in other indications, such as the treatment of solid tumors. Thus, there is a need for CARs with different intracellular domains that drive more favorable T cell phenotypes, such as more favorable exhaustion profiles.
  • a CAR polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein the intracellular domain pair is selected from the intracellular domain pairs listed in Table 1.
  • the ICD1 and/or ICD2 comprise or consist of an amino acid sequence set forth in Table 4.
  • the intracellular signaling region is comprises an amino acid sequence set forth in Table 5.
  • the CAR peptide provided herein is a CAR4 polypeptide.
  • CAR4 refers to a CAR polypeptide comprising an intracellular signaling region comprising amino acid sequences listed as CD3z_X23mut (SEQ ID NO: 22) as ICD1and CD40 (SEQ ID NO: 21) as ICD2, herein. Therefore, in some embodiments, the CAR4 intracellular signaling region is or comprises SEQ ID NO: 23 (i.e., CD3z_X23mut (SEQ ID NO: 22) and CD40 (SEQ ID NO: 21), combined).
  • the CAR polypeptide comprises an intracellular signaling region comprising or consisting of an amino acid sequence at least 90% identical to a CAR4 polypeptide intracellular signaling region (e.g., at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 23).
  • a CAR polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein the intracellular domain pair is selected from the intracellular domain pairs listed in Table 2.
  • the ICD1 and/or ICD2 is or comprises an amino acid sequence set forth in Table 4.
  • the intracellular signaling region is or comprises an amino acid sequence set forth in Table 6.
  • a CAR polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein the intracellular domain pair is selected from the intracellular domain pairs listed in Table 3.
  • the ICD1 and/or ICD2 is or comprises an amino acid sequence set forth in Table 4.
  • the intracellular signaling region is or comprises an amino acid sequence set forth in Table 7.
  • a CAR polypeptide comprising an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 has a sequence of SAPHR (SEQ ID NO: 90).
  • the ICD1 has a sequence of SAPHR and the ICD2 is or comprises an amino acid sequence set forth in Table 4.
  • a CAR polypeptide comprising an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD2 has a sequence of SAPHR (SEQ ID NO: 90).
  • the ICD2 has a sequence of SAPHR and the ICD1 is or comprises an amino acid sequence set forth in Table 4.
  • the CAR polypeptide comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 comprises or consists of an amino acid sequence at least 90% identical to SAPHR (SEQ ID NO: 90) (e.g., at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 90).
  • SAPHR SAPHR
  • the CAR polypeptide comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD2 comprises or consists of an amino acid sequence at least 90% identical to SAPHR (SEQ ID NO: 90) (e.g., at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 90).
  • SAPHR SAPHR
  • chimeric antigen receptor (CAR) polypeptides comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein ICD1 is CD3z_X23mut and ICD2 is CD40.
  • the ICD1 is or comprises an amino acid sequence of SEQ ID NO: 22.
  • the ICD2 is or comprises an amino acid sequence of SEQ ID NO: 21.
  • the CAR polypeptide comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 comprises or consists of an amino acid sequence at least 90% identical to CD3z_X23mut (SEQ ID NO: 22) (e.g., at least 91% identical, at least 92% identical, at least FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 22).
  • ICD1 comprises or consists of an amino acid sequence at least 90% identical to CD3z_X23mut (SEQ ID NO: 22) (e.g., at least 91% identical, at least 92% identical, at least FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 93% identical, at least 94% identical, at least 95%
  • the CAR polypeptide comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD2 comprises or consists of an amino acid sequence at least 90% identical to CD40 (SEQ ID NO: 21) (e.g., at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 21).
  • SEQ ID NO: 21 amino acid sequence at least 90% identical to CD40
  • the intracellular domain pair is or comprises an ICD1 comprising or consisting of an amino acid sequence at least 90% identical to CD3z_X23mut (SEQ ID NO: 22) (e.g., at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 22) and an ICD2 comprising or consisting of an amino acid sequence at least 90% identical to CD40 (SEQ ID NO: 21) (e.g., at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 21).
  • ICD1 comprising or consisting of an amino acid sequence at least 90% identical to CD3z_X23mut (SEQ ID NO: 22) (e.g.
  • chimeric antigen receptor (CAR) polypeptides comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein ICD1 is CD40 and ICD2 is CD3z_X23mut.
  • the ICD1 is or comprises an amino acid sequence of SEQ ID NO: 21.
  • ICD2 is or comprises an amino acid sequence of SEQ ID NO: 22.
  • chimeric antigen receptor (CAR) polypeptides comprising an intracellular signaling region comprising or consisting of an amino acid sequence of SEQ ID NO: 23.
  • the ICD1 and the ICD2 are connected with a peptide linker, e.g., a glycine-glycine (GG) linker.
  • GG glycine-glycine
  • the CAR comprises: a) a single chain fragment variable (scFv) domain; b) a hinge domain; c) a transmembrane domain; and d) the intracellular signaling region.
  • the scFv domain is a scFv domain set forth in Table 8. [0019] In some embodiments, the scFv domain comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 1, 2, 3, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 4, 5, 6, respectively. In some embodiments, the scFv domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 7; and/or a light chain variable domain having an amino acid sequence of SEQ ID NO: 8. In some embodiments, the scFv domain comprises an amino acid sequence of SEQ ID NO: 9.
  • the scFv domain comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 10, 11, 12, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NO: 13, SAS, and SEQ ID NO: 15, respectively.
  • the scFv domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 16; and/or a light chain variable domain having an amino acid sequence of SEQ ID NO: 17.
  • the scFv domain comprises an amino acid sequence of SEQ ID NO: 18.
  • the CAR comprises: a) an antigen-binding domain (e.g., a means for binding an antigen); b) a hinge domain; c) a transmembrane domain; and d) the intracellular signaling region.
  • the antigen-binding domain can be any protein domain that specifically binds to a cancer antigen (e.g., an antibody, antibody fragment, a nanobody, a ligand domain that binds to a receptor on a cancer cell, a receptor that binds to a ligand on a cancer cell).
  • the antigen-binding domain is a scFv domain.
  • the scFv domain is a scFv domain set forth in Table 8.
  • the antigen binding domain is a means for binding an antigen. Example structures corresponding to means for binding antigens are provided in Table 8.
  • the antigen-binding domain binds to a cancer antigen.
  • the cancer antigen is selected from the cancer antigens listed in Table 11.
  • the antigen-binding domain binds to Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1). FoleyHoagUS11580949.8 Attorney Docket No.
  • the antigen-binding domain comprises any one or more CDR (e.g., any one or more of CDR1, CDR2, and/or CDR3) described herein.
  • the antigen-binding domain comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 1, 2, 3, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 4, 5, 6, respectively.
  • the antigen-binding domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 7; and/or a light chain variable domain having an amino acid sequence of SEQ ID NO: 8.
  • the antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 9.
  • the antigen-binding domain comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 10, 11, 12, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NO: 13, SAS, and SEQ ID NO: 15, respectively.
  • the antigen-binding domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 16; and/or a light chain variable domain having an amino acid sequence of SEQ ID NO: 17.
  • the antigen-binding domain is or comprises an amino acid sequence of SEQ ID NO: 18.
  • the hinge domain is selected from the hinge domains of Table 12. In some embodiments, the hinge domain is an IgG4 hinge domain. In some embodiments, the hinge domain is or comprises an amino acid sequence of SEQ ID NO: 19. [0026] In some embodiments, the transmembrane domain is selected from the transmembrane domains of Table 13. [0027] In some embodiments, the intracellular signaling region is or comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 23.
  • the intracellular signaling region is or comprises an amino acid sequence of SEQ ID NO: 23.
  • a method of treating a tumor in a subject comprising administering a composition comprising cells expressing a CAR polypeptide described herein.
  • the tumor expresses the antigen recognized by an scFv and/or an antigen-binding domain expressed by the CAR.
  • FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025
  • the cells are induced pluripotent cells (iPSCs), hematopoietic stem cells (HSCs), or immune cells.
  • the immune cell is a leukocyte.
  • the immune cell is a lymphocyte, a monocyte, a macrophage, a dendritic cell, a mast cell, a neutrophil, a basophil, or an eosinophil.
  • the immune cell is a lymphocyte selected from a B cell, an ⁇ T cell, ⁇ T cell, a Natural Killer (NK) cell, a Natural Killer T (NKT) cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T cell, or any combination thereof.
  • the immune cell is a cytotoxic T lymphocyte (CTL). In some embodiments, the immune cell is a primary CD8 + T cell. In some embodiments, the immune cell is an autologous immune cell from the subject. In some embodiments, the immune cell is an allogeneic immune cell. [0030] In some embodiments, the immune cell proliferates in the subject. In some embodiments, the immune cell kills the tumor cell in the subject. In some embodiments, a reduced percentage of PD1 + TIM-3 + or PD1 + LAG-3 + population is observed for the immune cell after administration to the subject compared to the cells expressing a control CAR.
  • CTL cytotoxic T lymphocyte
  • the immune cell is a primary CD8 + T cell.
  • the immune cell is an autologous immune cell from the subject. In some embodiments, the immune cell is an allogeneic immune cell.
  • the immune cell proliferates in the subject. In some embodiments, the immune cell kills the tumor cell in the subject. In some embodiments, a
  • control CAR comprises the same antigen-binding domain and a 4-1BB-CD3z (BBZ) ICD.
  • BBZ 4-1BB-CD3z
  • a nucleic acid encoding a CAR polypeptide described herein is encoded by a nucleic acid sequence set forth in Table 15.
  • the ICD1 and/or the ICD2 is encoded by SEQ ID NO: 26.
  • the ICD1 and/or the ICD2 is encoded by SEQ ID NO: 27.
  • a vector comprising a nucleic acid described herein. In some embodiments, the vector is an expression vector.
  • the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector.
  • a cell comprising a nucleic acid described herein. In some aspects, provided herein is a cell expressing a CAR polypeptide described herein. In some embodiments, the cell is an iPSC, a HSC, or an immune cell. In some embodiments, the immune cell is a leukocyte. In some embodiments, the immune cell is a lymphocyte, a monocyte, a macrophage, a dendritic cell, a mast cell, a neutrophil, a basophil, or FoleyHoagUS11580949.8 Attorney Docket No.
  • the immune cell is a lymphocyte selected from a B cell, an ⁇ T cell, ⁇ T cell, a Natural Killer (NK) cell, a Natural Killer T (NKT) cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T cell, or any combination thereof.
  • the immune cell is a cytotoxic T lymphocyte (CTL).
  • the immune cell is a primary CD8 + T cell.
  • the immune cell proliferates in the presence of antigen-expressing target cells.
  • a reduced percentage of PD1 + TIM-3 + or PD1 + LAG-3 + population is observed for the immune cell after co-culturing with antigen-expressing target cells in a repeated manner compared to the same type of immune cell expressing a control CAR.
  • the control CAR comprises the same antigen- binding domain and a 4-1BB-CD3z (BBZ) ICD.
  • BBZ 4-1BB-CD3z
  • the CAR-expressing cell is generated using a genome-editing tool, e.g., a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), or a CRISPR/CAS system.
  • TALEN transcription activator-like effector nuclease
  • ZFN zinc-finger nuclease
  • the cell is an iPSC, a HSC, or an immune cell.
  • the immune cell is a leukocyte.
  • the immune cell is a lymphocyte, a monocyte, a macrophage, a dendritic cell, a mast cell, a neutrophil, a basophil, or an eosinophil.
  • the immune cell is a lymphocyte selected from a B cell, an ⁇ T cell, ⁇ T cell, a Natural Killer (NK) cell, a Natural Killer T (NKT) cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T cell, or any combination thereof.
  • the immune cell is a cytotoxic T lymphocyte (CTL).
  • the immune cell is a primary CD8 + T cell.
  • a cell bank comprising cells for adoptive immunotherapy, wherein the cells express the CAR herein.
  • FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 is a schematic illustration of an assay for CAR library pooled screening in CD8+ primary T cells.
  • Figure 2 shows flow cytometry plots demonstrating reduced levels of exhaustive phenotypes in the enriched CAR T cell libraries after completion of the tumor re-challenge screen.
  • FIG. 3 shows an overview of CAR library vector design and components.
  • Figure 4 shows schematics of CAR library pooled screening in CD8+ primary T cells (See also Figure 1).
  • Four different designs of CAR library distinguished by affinity of binder (R12: high affinity, 2A2: low affinity) and length of linker between the binder and transmembrane domain (CD8: long, IgG4: short) were used for pooled screening.
  • Individual libraries contain the same 10,000-member ICD combinations. Plasmids encoding these CAR molecules were packaged into lentiviral particles, which were treated onto primary CD8+ T cells for infection.
  • T cells were co-cultured with Jeko-1 Mantle Cell Lymphoma (MCL) target cells in a repeated manner (2-3 times of challenge every week for 3 weeks) to intentionally drive T cells into exhaustion and dysfunction.
  • MCL Mantle Cell Lymphoma
  • Figure 5 shows CAR-T cell counts throughout the assay.
  • Figure 6 quantifies the exhaustion phenotypes of CAR-T cells at endpoint using cell surface expression of canonical markers PD-1, TIM-3, and LAG-3(See also Figure 2). These plots show reduced frequencies of either PD-1+ TIM-3+ or PD-1+ LAG-3+ cells in CAR library expressing cells compared to those of BBZ CAR-T cells, indicating CAR-T cells with less exhaustive phenotypes relative to BBZ CAR-T cells.
  • Figure 7 shows the enrichment of individual CAR clones during the assay. Each dot represents a specific combination of CAR ICDs in either the R12-IgG4 design backbone (dark shade) or the 2A2-IgG4 design backbone (light shade). A threshold of 32-fold enrichment was set for selection of high-performing CAR designs, and is designated by the dashed line box. Fold-enrichment was calculated by the frequency of each barcode sequence at Day 20 normalized by the frequency at Day 0. [0045] Figure 8 shows enrichment of individual CAR clones across two biological replicates. Black dots are top hits that were defined in Figure 7.
  • FIG. 10 shows a heat map showing enrichment of ICD sequences in the ICD1 and ICD2 positions and segmented by immune function. A large proportion of CARs containing activating and co-stimulatory ICDs show enrichment, as expected.
  • ICDs from other immune cell types such as B cell, NK cell, and macrophage
  • Some ICDs also exhibit universal features, meaning that enrichment is FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 observed regardless of which ICD partner it is combined with.
  • FCRL5 in the ICD1 (membrane-proximal) position is shown in Figure 11.
  • Figure 11 shows volcano plots describing individual ICD enrichment at two different positions. Black dots indicate ICDs with statistically significant enrichment.
  • the size of dots represents the number of CAR clones individual ICD was incorporated at a specific position.
  • FCRL5 at ICD1 position was coupled with 75 different ICDs at ICD2 position and still shows significant enrichment in average across performance of 75 clones, implying that FCRL5 might be a universal ICD in the ICD1 position.
  • the FCRL5 sequence in the library contains the last 5 amino acids of the FCRL5 ICD sequence, and therefore lacks many functional motifs.
  • the linker between ICD1 and ICD2 in our CAR library encodes two glycines (GG) to be flexible and likely does not affect ICD functions. Alternatively, longer and flexible linker sequences, such as GGGGS (SEQ ID NO: 28), could be utilized.
  • FIG. 12 shows image-based cytotoxicity assay results with multiple tumor challenges. Dotted vertical lines on each graph indicate addition of fresh GFP+ A549 tumor cells to the co-culture. Normalized GFP+ A549 counts on the y-axis were calculated by normalizing the A549 tumor cell counts at each timepoint of challenge by counts at the beginning of challenge. CAR2 (28z), CAR4, and CAR10 (BBz) consistently removed tumor cells from the culture at every challenge up to 20 days. Each graph shows the mean of triplicates.
  • Figure 13 shows graphs describing results of a chronic stimulation assay with 10 different CAR constructs with three replicates for each design. Individually engineered CAR-T cells were co-cultured with A549 lung cancer cells over the course of >60+ days. Every 2-3 days, fresh tumor cells were introduced into the co-culture. All replicates of CAR4 dramatically outperformed other CAR designs including CAR10 (BBz) and CAR2 (28z) in terms of survival and proliferation. Fold expansion on the y-axis is calculated by normalizing T cell counts at each time-point by cell counts at Day 0 of the assay. [0051] Figure 14 shows graphs showing secreted cytokines from individual CAR design at different time-points throughout the chronic stimulation assay.
  • FIG. 15 depicts the differentiation state of CAR-T cells at Day 21 of the chronic stimulation assay.
  • CD45RA+CD62L+ stem-cell memory
  • CD45RA-CD62L+ central memory
  • CD45RA-CD62L- effector memory
  • CD45RA+CD62L- effector CD45RA+CD62L- effector.
  • CAR4 shows the reduced differentiation relative to control CARs CAR2 and CAR10.
  • Figure 16 shows images and a graph showing cytotoxicity of BBz and CAR4 T cells across multiple tumor targets.
  • BBz CAR-T cells clear out Jeko-1 and MDA-MB-468 tumor cells in a comparable amount to that of CAR4 cells.
  • CAR4 outperforms BBz against a challenging tumor cell line, A549.
  • Figure 17 shows the proliferation capacity of BBz and CAR4 across multiple tumor targets using a dye dilution assay. In all cases, CAR4 proliferates significantly better than BBz, as indicated by increased dilution of the CellTrace Violet dye across 6 days of assay.
  • Figure 18 shows heat maps showing enhanced survival and proliferation capacities of CAR4 compared to those of CAR10 (BBz) in the context of an A549 tumor challenge under immunosuppression.
  • CAR4 and CAR10 T cells were co-cultured with A549 tumor cells for 3 days in the presence of a combination of the immunosuppressive factors TGFb- 1 and adenosine.
  • CAR4 T cells show better resistance to immunosuppression as quantified by fold change in cell counts where the counts at endpoint were normalized by counts at Day 0 of the assay.
  • Figure 19 shows the cytotoxicity of CAR-T cells at different E:T ratios. The distinction in cytotoxicity across CAR designs becomes apparent from the E:T ratio of 1:4 where CAR4 seems to control tumor cells better than BBz and 28z. At the highest tumor burden condition of 1:8, CAR4 still clears out tumor cells, while BBz and 28z fail.
  • Figure 20 shows the differentiation and exhaustion phenotype of CAR-T in the high tumor burden culture condition (E:T of 1:8) for Figure 19.
  • the central memory T cell population (CD45RA-CD62L+) is noticeably higher in CAR4 compared to that of BBz and 28z.
  • CAR4 cells are also significantly less exhausted, denoted by smaller proportions of PD-1+, TIM3+, and/or LAG3+ populations compared to those of BBz and 28z.
  • Figure 21 is a graph showing tumor counts in a serial low IL-2 rechallenge assay with anti-GD2 CAR-T cells.
  • FIG. 22 is a graph showing tumor counts in a serial no IL-2 rechallenge assay with anti-GD2 CAR-T cells. Tumor counts are normalized by the number of tumor cells at the assay initiation for all challenges. Each data point represents a mean of duplicates. Error bars indicate standard deviations.
  • Figure 23 shows the level of cytokine secretion by each CAR-T group across multiple different conditions. Numbers indicate the level of each cytokine in pg/ml.
  • Figure 24 shows principle component analysis (PCA) clustering of CAR designs based on their metabolic profiles. Cells engineered with CARs with different signaling domains have distinct metabolites.
  • Figure 25 is a series of box plots showing concentrations of metabolites found from each CAR-T co-culture. Data sets are mean values of triplicate. Error bars represent standard deviation.
  • CAR Chimeric antigen receptors
  • First generation CAR-T designs had a single intracellular domain derived from the CD3 zeta chain.
  • Newer designs include multiple co-stimulatory or signaling domains in addition to CD3z, as well as other accessory sequences like cytokine expression.
  • All of the six currently approved CAR-T products are of the second generation design and all contain either CD28 or 4-1BB as the costimulatory domain.
  • FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 [0064]
  • the intracellular signaling domain can dramatically impact cell function and clinical outcomes (Kawalekar et al. (2016) Immunity 44(2):380-390).
  • the CD28 signaling domain leads to rapid, early cell proliferation in patients, while cells engineered with the 4-1BB domain tend to have more moderate but extended proliferation profiles.
  • the different signaling domains also lead to differences in T cell exhaustion, which is a state of dysfunction arising from excessive antigen stimulation and a barrier to increased therapeutic efficacy for T cell therapies.
  • CAR-T cells engage antigen-expressing tumor cells
  • T cell signaling is initiated through the CAR and can lead to T cell exhaustion, especially in the case of bulky solid tumors.
  • CAR-T cells engineered with CD28 and 4-1BB have different exhaustion profiles, with 4-1BB CAR-T cells generally exhibiting less exhaustion as compared to CD28 CAR-T cells (Majzner and Mackall (2019) Nature Medicine 25:1341-1355).
  • CAR-based cell therapies have not yet been successful in more challenging indications, such as bulky solid tumors, where T cell exhaustion is one of the limiting factors.
  • the present disclosure provides CAR polypeptides with new ICD combinations that, for example, can confer more favorable T cell phenotypes compared to the canonical CAR-T ICD combinations.
  • cytotoxic T cells expressing the CARs disclosed herein can, in some embodiments, exhibit superior T cell proliferation and survival, and less exhaustive phenotypes (e.g., lower populations of PD-1 + TIM3 + or PD1 + LAG-3 + ), compared to FDA-approved BBz control CARs.
  • the present disclosure demonstrates that provided CAR-based cell therapies are effective in treating solid tumors. For example, as shown in Example 3 and Figure 16, CAR4 has superior in killing of example solid tumors (MDA-MB-468 and A549) over BB Z control CAR.
  • a composition comprising cells expressing a CAR polypeptide as disclosed herein (e.g., a CAR4 polypeptide).
  • CAR polypeptides and polynucleotides encoding said CAR polypeptides wherein the CAR polypeptides comprise an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain FoleyHoagUS11580949.8 Attorney Docket No.
  • the intracellular domain pair comprises a first intracellular domain (e.g., ICD1) and a second intracellular domain (e.g., ICD2), and wherein the first intracellular domain and the second intracellular domain are individually any ICD disclosed herein, and/or the intracellular domain pair is any pair of ICD1 and ICD2 disclosed herein.
  • the intracellular signaling region further comprises an optional third intracellular domain (e.g., an ICD3, for example, as illustrated in Figure 3), wherein the optional third intracellular domain is distal to the membrane relative to ICD1 and ICD2.
  • the third intracellular domain comprises the sequence of any ICD disclosed herein.
  • CAR polypeptides and polynucleotides encoding said CAR polypeptides comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein the intracellular domain pair is selected from the intracellular domain pairs listed in Table 1, Table 2, or Table 3.
  • the ICD1 and/or ICD2 may comprise an ICD amino acid sequence set forth in Table 4.
  • the intracellular signaling region may comprise an amino acid sequence set forth in Table 5, Table 6, or Table 7.
  • the CAR polypeptide further comprises an antigen-binding domain, e.g., a scFv domain set forth in Table 8.
  • the antigen-binding domain binds to a cancer antigen such as ROR1.
  • the CAR polypeptide may also comprise a hinge domain (e.g., a hinge domain selected from Table 12) and a transmembrane domain (e.g., a transmembrane domain selected from Table 13).
  • the CAR polypeptide is a CAR4 polypeptide.
  • immune cells expressing a CAR polypeptide disclosed herein are also provided herein, as well as methods of treating a tumor in a subject by administering a composition comprising cells (e.g., immune cells) expressing a CAR polypeptide disclosed herein.
  • a composition comprising cells (e.g., immune cells) expressing a CAR polypeptide disclosed herein.
  • administering means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 professional and self-administering.
  • amino acid is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids.
  • Example amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing.
  • binding refers to an association, which may be a stable association, between two molecules, e.g., between a peptide and a binding partner or agent, e.g., small molecule, due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions.
  • cancer includes, but is not limited to, solid tumors and blood borne tumors.
  • the term cancer includes, but is not limited to, diseases of the skin, tissues, organs, bone, cartilage, blood, and vessels, including the cervix, anus, vagina, vulva, penis, tongue base, larynx, and tonsil.
  • CAR chimeric antigen receptor
  • a component present on the target cell for example an antibody-based specificity for a desired antigen (e.g., a tumor antigen) with an immune cell-activating intracellular domain to generate a chimeric protein.
  • CARs comprise an extracellular single chain antigen-binding domain (e.g., an scFv) fused to the intracellular signaling domain.
  • epipe means a protein determinant capable of specific binding to an antibody or immune cell (e.g., T cell).
  • Epitopes usually include chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which a CAR or antibody is capable of binding.
  • “Gene construct” refers to a nucleic acid, such as a vector, plasmid, viral genome or the like which includes a “coding sequence” for a polypeptide or which can otherwise transcribe to a biologically active RNA (e.g., antisense, decoy, ribozyme, etc.), may be transfected into cells, e.g., mammalian cells, and may cause expression of the coding sequence in cells transfected with the construct.
  • a biologically active RNA e.g., antisense, decoy, ribozyme, etc.
  • the gene construct may include one or more regulatory FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 elements operably linked to the coding sequence, as well as intronic sequences, polyadenylation sites, origins of replication, marker genes, etc.
  • ligand-binding domain and “antigen-binding domain” are used interchangeably herein, and refer to that portion of a chimeric antigen receptor that binds specifically to a predetermined antigen.
  • linker refers to a molecule or group of molecules connecting two compounds, such as two polypeptides.
  • the linker may be comprised of a single linking molecule or may comprise a linking molecule and a spacer molecule, intended to separate the linking molecule and a compound by a specific distance.
  • the term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences.
  • operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
  • the phrase “pharmaceutically acceptable” refers to those agents, compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; FoleyHoagUS11580949.8 Attorney Docket No.
  • GBB-74025 (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen- free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical
  • polynucleotide and “nucleic acid” are used interchangeably. They refer to a natural or synthetic molecule, or some combination thereof, comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide.
  • the polymeric form of nucleotides is not limited by length and can comprise either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • Polynucleotides may have any three-dimensional structure, and may perform any function.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • a polynucleotide may be further modified, such as by conjugation with a labeling component.
  • U nucleotides are interchangeable with T nucleotides.
  • the polynucleotide is not necessarily associated with the cell in which the nucleic acid is found in nature, and/or operably linked to a polynucleotide to which it is linked in nature.
  • precancerous lesions or “precancerous condition” refers to atypical cells and/or tissues that are associated with an increased risk of cancer.
  • a therapeutic that “prevents” a condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • an “intracellular signaling region” of a CAR is the part of the chimeric antigen receptor protein that is located within the cell and that is responsible for intracellular signaling following the binding of an extracellular antigen binding domain to the target.
  • the intracellular signal region can include multiple intracellular domains (ICDs) that may each convey a separate intracellular signal.
  • the intracellular signaling region comprises a pair of an intracellular domain 1 (ICD1) and an intracellular domain 2 (ICD2) listed in Tables 1-3, and/or the ICD1 and ICD2 are independently selected from any ICD described herein.
  • a polypeptide refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologics.
  • a specified ligand or antibody “specifically binds” to its particular “target” (e.g. an antibody specifically binds to an endothelial antigen) when it does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism.
  • a first molecule that “specifically binds” a second molecule has an affinity constant (Ka) greater than about 10 5 M –1 (e.g., 10 6 M –1 , 10 7 M –1 , 10 8 M –1 , 10 9 M –1 , 10 10 M –1 , 10 11 M –1 , and 10 12 M –1 or more) with that second molecule.
  • Ka affinity constant
  • a CAR specifically binds to its peptide/MHC with an affinity of at least a KD of about 10 -4 M or less, and binds to the predetermined antigen/binding partner with an affinity (as expressed by KD) that is at least 10 fold less, at least 100 fold less or at least 1000 fold less than its affinity for binding to a non-specific and unrelated peptide/MHC complex (e.g., one comprising a BSA peptide or a casein peptide).
  • a non-specific and unrelated peptide/MHC complex e.g., one comprising a BSA peptide or a casein peptide.
  • the term “subject” means a human or non-human animal selected for treatment or therapy.
  • FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025
  • the terms “transformation”, “transfection”, or “transduction” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell (e.g., a mammalian cell) including introduction of a nucleic acid to the chromosomal DNA of said cell.
  • treatment refers to clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology.
  • Desirable effects of treatment include decreasing the rate of progression, ameliorating or palliating the pathological state, and remission or improved prognosis of a particular disease, disorder, or condition.
  • An individual is successfully “treated,” for example, if one or more symptoms associated with a particular disease, disorder, or condition are mitigated or eliminated.
  • the term “vector” refers to the means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components.
  • Vectors include plasmids, viruses, bacteriophage, pro-viruses, phagemids, transposons, and artificial chromosomes, and the like, to which the nucleic acid has been linked, and may or may not be able to replicate autonomously or integrate into a chromosome of a host cell.
  • Such vectors may include any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element).
  • agents may be used alone or conjointly administered with another type of therapeutic agent.
  • the phrase “conjoint administration” or “administered conjointly” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the subject, which may include synergistic effects of the two agents).
  • the different therapeutic agents can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially.
  • the different therapeutic agents can be administered within about one hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about a week of one another.
  • a subject who receives such treatment can benefit from a combined effect of different therapeutic agents.
  • Chimeric Antigen Receptors are receptors comprising a targeting moiety that is associated with an intracellular signaling region comprising one or more intracellular 21 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 domains (ICDs) in a single fusion molecule.
  • the binding moiety of a CAR comprises an antigen-binding domain, e.g., a single-chain fragment variable (scFv) comprising the light and heavy chain variable fragments of a monoclonal antibody joined by a flexible linker.
  • the binding moiety further comprises transmembrane and hinge domains.
  • the CARs provided herein comprise an intracellular signaling region.
  • the intracellular signaling region comprising an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the intracellular domain pair is selected from the intracellular domain pairs listed in Table 1 below.
  • Table 1 Example intracellular domain pairs. 22 FoleyHoagUS11580949.8 Attorney Docket No.
  • the CAR polypeptides provided herein comprise an intracellular signaling region comprising an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the intracellular domain pair is selected from the intracellular domain pairs listed in Table 2 below.
  • IDC1 is CD3_X23mut.
  • ICD2 is CD40. Table 2: Example intracellular domain pairs. 23 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No.
  • the CAR polypeptides provided herein comprise an intracellular signaling region comprising an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the intracellular domain pair is selected from the intracellular domain pairs listed in Table 3 below.
  • IDC1 is CD3_X23mut.
  • ICD2 is CD40.
  • Table 3 Example intracellular domain pairs.
  • the ICD1 and/or the ICD2 comprises a full-length of the ICD listed in Tables 1-3 above.
  • the ICD1 and/or the ICD2 comprises a functionally active fragment of the ICD listed in Tables 1-3 above.
  • the ICD1 and/or the ICD2 may be an activating domain, a co-stimulatory domain, an inhibitory domain, or a domain from other immune cell types, such as B cell, NK cell, or macrophage.
  • the CAR disclosed herein may comprise a ICD1 and ICD2 pair listed in Tables 1-3 above, and the ICD1 and/or the ICD2 consists of or comprises an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an ICD amino acid sequence set forth in Table 4 below.
  • the CAR disclosed herein comprises a ICD1 and ICD2 pair listed in Tables 1-3 above, and the ICD1 and/or the ICD2 consists of or comprises an amino acid sequence set forth in Table 4 below.
  • the ICD1 is or comprises SEQ ID NO: 21 and FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 ICD2 is or comprises SEQ ID NO: 22.
  • the ICD1 is or comprises SEQ ID NO: 22 and ICD2 is or comprises SEQ ID NO: 21.
  • Table 4 Example ICD amino acid sequences 26 FoleyHoagUS11580949.8 Attorney Docket No.
  • the CAR described herein comprises an intracellular signaling region consisting of or comprising an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence set forth in Table 5 below.
  • the CAR comprises an intracellular signaling region that is or comprises an amino acid sequence that is identical to a sequence set forth in Table 5, but for 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 sequence differences (amino acid additions, deletions, and/or substitutions).
  • the sequence differences are amino acid substitutions (e.g., conservative amino acid substitutions).
  • the CAR disclosed herein comprises an intracellular signaling region consisting of or comprising an amino acid sequence as set forth in Table 5 below. Table 5: Example intracellular signaling regions.
  • the CAR described herein comprises an intracellular signaling region consisting of or comprising an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence set forth in Table 6 below.
  • the CAR comprises an intracellular signaling region consisting of or comprising an amino acid sequence that is identical to a sequence set forth in Table 6, but for 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 sequence differences (amino acid additions, deletions, and/or substitutions).
  • the sequence differences are amino acid substitutions (e.g., conservative amino acid substitutions).
  • the CAR disclosed herein comprises an intracellular signaling region comprising an amino acid sequence set forth in Table 6 below.
  • the CAR disclosed herein comprises an intracellular signaling region comprising SEQ ID NO: 23.
  • Table 6 Example intracellular signaling regions. FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No.
  • the CAR described herein comprises an intracellular signaling region comprising or consisting of an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence set forth in Table 7 below.
  • the CAR comprises an intracellular signaling region consisting of or comprising an amino acid sequence that is identical to a sequence set forth in Table 7, but for 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 sequence differences (amino acid additions, deletions, and/or substitutions).
  • the sequence differences are amino acid substitutions (e.g., conservative amino acid substitutions).
  • the CAR disclosed herein comprises an intracellular signaling region comprising or consisting of an amino acid sequence set forth in Table 7 below.
  • the CAR disclosed herein comprises an intracellular signaling region comprising SEQ ID NO: 23. Table 7: Example intracellular signaling regions.
  • the CAR described herein comprises an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 has a sequence of SAPHR (SEQ ID NO: 90).
  • the CAR described herein comprises comprising an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 has a sequence of SAPHR (SEQ ID NO: 90), and the ICD2 is or comprises an ICD amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence set forth in Table 4.
  • ICD1 has a sequence of SAPHR (SEQ ID NO: 90)
  • the ICD2 is or comprises an ICD amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%
  • the CAR described herein comprises comprising an intracellular domain pair comprising first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD2 has a sequence of SAPHR (SEQ ID NO: 90).
  • the CAR described herein comprises comprising an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD2 has a sequence of SAPHR (SEQ ID NO: 90), and the ICD1 is or comprises an ICD amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence set forth in Table 4. [0102] In some embodiments, the ICD1 and the ICD2 are connected with a peptide linker.
  • the peptide linker may be flexible and may not affect ICD functions.
  • the peptide linker is glycine-glycine (GG).
  • longer and flexible linker sequences such as GGGGS (SEQ ID NO: 28), can also be utilized.
  • linkers at the front of ICD1 and the back of ICD2 that bridge ICD combinations to the CAR backbone are single glycine residues to minimize any potential effect from linker sequences.
  • the binding domain and/or extracellular domain of a CAR provided herein provides the CAR with the ability to bind to the target antigen of interest.
  • a binding domain (e.g., a ligand-binding domain or antigen-binding domain) can be any protein, polypeptide, oligopeptide, or peptide that possesses the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, or a component thereof).
  • a biological molecule e.g., a cell surface receptor or tumor protein, or a component thereof.
  • a binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest.
  • a binding domain may be antibody light chain and heavy chain variable regions, or the light and heavy chain variable regions can be joined together in a single chain and in either orientation (e.g., V L -V H or V H -V L ).
  • assays are known for identifying binding domains of the present disclosure that specifically bind with a particular target, including Western blot, ELISA, flow cytometry, or surface plasmon resonance analysis (e.g., using BIACORE analysis).
  • the target may be an antigen of clinical interest against which it would be desirable to trigger an effector immune response that results in tumor killing.
  • the binding domain of the CAR is a single chain fragment variable (scFv) specific for a cancer antigen, and may be a murine, human or humanized scFv.
  • Single chain antibodies may be cloned from the V region genes of a hybridoma specific for a desired target.
  • VH variable region heavy chain
  • VL variable region light chain
  • a binding domain comprises an antibody- derived binding domain but can be a non-antibody derived binding domain.
  • an antibody-derived binding domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of the antibody, which fragment is involved in binding with the antigen.
  • the CAR further comprises a single chain fragment variable (scFv) domain.
  • the scFv domain may be a scFv domain set forth in Table 8 below, or a scFv that includes CDR and/or variable region sequences set forth in Table 8.
  • the CAR comprises an antigen binding domain (e.g., a scFv domain) comprising heavy chain and light chain CDR sequences of an antibody or scFv provided in Table 8.
  • the CAR comprises an antigen binding domain (e.g., a scFv domain) comprising a heavy chain variable region sequence and a light chain variable region sequence of an antibody or scFv provided in Table 8.
  • the CAR further comprises a scFv domain consisting of or comprising an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence set forth in Table 8.
  • FoleyHoagUS11580949.8 Attorney Docket No.
  • the CAR polypeptide comprises a means for binding a target antigen.
  • Example structures corresponding to means for binding antigens are provided in Table 8.
  • Table 8 Example binding domain sequences 40 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 [0107]
  • the scFv domain is a ROR1-specific scFv.
  • the scFv domain is a ROR1-specific R12 clone scFv.
  • the scFv domain comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 1, 2, 3, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 4, 5, 6, respectively.
  • the scFv domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 7; and/or a light chain variable domain having an amino acid sequence of SEQ ID NO: 8.
  • the heavy chain variable domain disclosed herein may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence set forth in SEQ ID NO: 7.
  • the light chain variable domain disclosed herein may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence set forth in SEQ ID NO: 8.
  • the scFv domain is or comprises an amino acid sequence of SEQ ID NO: 9. FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 Table 9: scFv R12 sequences [0108]
  • the scFv domain is a ROR1-specific scFv.
  • the scFv domain is a ROR1-specific 2A2 clone scFv. 2A2 scFv sequences are provided in Table 10.
  • the scFv domain comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 10, 11, 12, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NO: 13, SAS, and SEQ ID NO:15, respectively.
  • the scFv domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 16; and/or a light chain variable domain having an amino acid sequence of SEQ ID NO: 17.
  • the heavy chain variable domain disclosed herein may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence set forth in SEQ ID NO: 16.
  • the light chain variable domain disclosed herein may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence set forth in SEQ ID NO: 17.
  • the scFv domain is or comprises an amino acid sequence of SEQ ID NO: 18.
  • Table 10 scFv 2A2 sequences FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 [0109]
  • the CAR described herein further comprises an antigen- binding domain.
  • the term “antigen binding domain” or “antigen binding fragment” of an antibody as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., ROR1). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.
  • Such antibody embodiments may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens.
  • binding fragments encompassed within the term “antigen binding domain” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publication WO 90/05144 A1 herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR).
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigen binding domain” of an antibody.
  • scFv molecules may be incorporated into a fusion protein.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123).
  • the antigen-binding domain is a scFv domain described herein.
  • the antigen-binding domain binds to a cancer antigen.
  • the cancer antigen may be selected from the cancer antigens listed in Table 11 below. Table 11: Example cancer antigens FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 47 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No.
  • the antigen-binding domain binds to Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1).
  • ROR1 is highly expressed in a variety of cancers and its expression has been linked to poor clinical outcomes (Zhang et al. (2012) PloS One 7(3):e31127).
  • ROR1 is an oncofetal antigen, with expression primarily restricted to embryonic tissues and low expression in healthy adult tissue, and is generally considered to be a safe target for CAR-T therapy.
  • ROR1 targeted CAR-T cells have been demonstrated in second generation formats including both CD28-CD3z (Hudecek et al. (2010) Blood 116(22):4532-4541) and 4-1BB-CD3z (Lee et al. (2022) Cancers 14(15):3618) signaling domain combinations as well as in a third generation format (Cortes et al. (2021) Blood 138(1):4804).
  • the ROR1 targeted CARs described herein comprises new combinations of intracellular signaling domains with functionality superior to both CD28-Cd3z and 4-1BB-Cd3z FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 CAR designs.
  • the ROR1 targeted CAR-T cells described herein showed a higher proliferation/survival and less exhaustive phenotypes (e.g., lower populations of PD1 + TIM-3 + or PD1 + LAG-3 + cells after administration to the subject) compared to ROR1 targeted 4-1BB-CD3z (BBZ) CAR-T cells.
  • the antigen-binding domain binds to GD2.
  • the GD2 is a disialoganglioside glycosphingolipid expressed primarily on the cell surface. GD2 expression in normal tissues is rare and primarily restricted to the central nervous system (CNS), peripheral nerves and melanocytes. In cancerous cells, GD2 is uniformly expressed in neuroblastomas and most melanomas and to a variable degree in bone and soft-tissue sarcomas, small cell lung cancer, renal cell carcinoma, and brain tumors (Navid et al., Curr Cancer Drug Targets 2010; 10:200-209). [0114] Several anti-GD2 antibodies have been clinically validated.
  • Example clinically validated anti-GD2 antibodies include 3F8 (Cheung et al., 1985, Cancer Res 45, 2642-9), 14.18 (Mujoo et al., 1989, Cancer Res 49, 2857-61) and derivatives and variants thereof.
  • a CAR of the present disclosure comprises an anti-GD2 binding domain.
  • the CARs of the present disclosure may comprise a linker between the various domains, added for appropriate spacing and conformation of the molecule.
  • the linker between any of the domains of the chimeric antigen receptor may be between 1-20 or more than 20 amino acids long.
  • the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long.
  • the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long. Ranges including the numbers described herein are also included herein, e.g., a linker 10-30 amino acids long.
  • linkers suitable for use in the CAR described herein are flexible linkers.
  • Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.
  • 1 amino acid e.g., Gly
  • 2 amino acids to 15 amino acids from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.
  • FoleyHoagUS11580949.8 Attorney Docket No.
  • Example flexible linkers include glycine polymers (G)n, glycine-serine polymers, where n is an integer of at least one, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.
  • Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between domains of fusion proteins such as the CARs described herein. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains.
  • the binding domain of the CAR may be followed by a “spacer,” or, “hinge,” which refers to the region that moves the antigen-binding domain away from the effector cell surface to enable proper cell/cell contact, antigen-binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419).
  • the hinge region in a CAR is generally between the transmembrane (TM) and the binding domain.
  • a hinge region is an immunoglobulin hinge region and may be a wild type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region.
  • Other example hinge regions used in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 ⁇ , CD4, CD28 and CD7, which may be wild-type hinge regions from these molecules or may be altered.
  • the CAR described herein further comprises a hinge domain.
  • the hinge domain may be selected from the hinge domains of Table 12 below.
  • the hinge domain is an IgG4 hinge domain.
  • the CAR further comprises a hinge domain comprising an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence set forth in Table 12.
  • the hinge domain comprises an amino acid sequence of SEQ ID NO: 19.
  • Table 12 Example hinge domains FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 [0120]
  • the CAR described herein further comprises a transmembrane domain.
  • the “transmembrane” region or domain is the portion of the CAR that FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 anchors the extracellular binding portion to the plasma membrane of the immune effector cell, and facilitates binding of the binding domain to the target antigen.
  • the transmembrane domain may be a CD3 ⁇ transmembrane domain.
  • Other transmembrane domains that may be employed in some embodiments include those obtained from CD8, CD8 ⁇ , CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, and CD154.
  • the transmembrane domain is synthetic in which case it would comprise predominantly hydrophobic residues such as leucine and valine.
  • the transmembrane domain is selected from a transmembrane domain of CD28, CD8 ⁇ , ICOS, 4-1BB, CD4, Tim4, OX40, CD27, CD2, LFA-1, CD30, CD40, PD-1, CD7, LIGHT, NKG2C, B7-H3, NKG2D, NKp44, NKp46, DAP12, CD16, NKp30, FcR ⁇ , DAP10, 2B4, or DNAM-1.
  • the transmembrane domain may be selected from the transmembrane domains of Table 13 below.
  • the transmembrane domain is a CD28 transmembrane domain.
  • the CAR further comprises a transmembrane domain comprising an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence set forth in Table 13.
  • the transmembrane domain consists of or comprises an amino acid sequence of SEQ ID NO: 20.
  • Example transmembrane domains FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 [0122]
  • the CAR polypeptides encompassed by the present disclosure may comprise an amino acid sequence set forth in Table 14 below. Table 14.
  • Example CAR polypeptides FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 55 FoleyHoagUS11580949.8
  • Attorney Docket No. GBB-74025 56 FoleyHoagUS11580949.8
  • GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 60 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 61 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 62 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 63 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 64 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 65 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 66 FoleyHoagUS11580949.8 Attorney Docket No.
  • GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 68 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 69 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 72 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 73 FoleyHoagUS11580949.8 Attorney Docket No.
  • GBB-74025 Polypeptides having substantial sequence similarities can have the same or similar catalytic and/or functional activity. Accordingly, in some embodiments, a derivative, equivalent, variant, fragment, or mutant of a CAR described herein or fragment thereof may also suitable for the methods and compositions provided herein.
  • the CAR further comprises an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence set forth in Table 14.
  • the CAR is or comprises an amino acid sequence that is identical to a sequence set forth in Table 14, but for 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 sequence differences (amino acid additions, deletions, and/or substitutions).
  • the sequence differences are amino acid substitutions (e.g., conservative amino acid substitutions).
  • variations or derivatives of the CARs are provided herein.
  • the altered CAR polypeptide may have an altered amino acid sequence, for example by conservative substitution, yet still induces antigen-specific proliferation, cytotoxicity, cytokine secretion, and/or other immune cell phenotypes, and are considered functional equivalents.
  • the term “conservative substitution” denotes the replacement of an amino acid residue by another, biologically similar residue. It is well known in the art that the amino acids within the same conservative group may typically substitute for one another without substantially affecting the function of a protein. According to certain embodiments, the derivatives, FoleyHoagUS11580949.8 Attorney Docket No.
  • GBB-74025 equivalents, variants, or mutants of a CAR are polypeptides that are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to an amino acid sequence of a CAR described herein (e.g., the amino acid sequences listed in Table 14) or fragment thereof.
  • the identity is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more.
  • the CAR polypeptides encompassed by the present disclosure may consist of or comprise an amino acid sequence derived from a CAR polypeptide set forth in Table 14.
  • the CAR polypeptides may include conservative or non-conservative mutations.
  • a CAR polypeptide may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more mutations.
  • a CAR polypeptide may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more mutations.
  • nucleic acids and polynucleotide vectors encoding the CAR polypeptides disclosed herein comprise an ICD1-encoding nucleic acid sequence set forth in Table 15 below, and/or an ICD2-encoding nucleic acid sequence set forth in Table 15 below.
  • Table 15 Example ICD-encoding nucleic acid sequences FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 FoleyHoagUS11580949.8 Attorney Docket No.
  • Nucleic acid sequences encoding the disclosed CARs, and regions thereof, can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.
  • Expression of nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide to a promoter, and incorporating the construct into an expression vector.
  • Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 [0130]
  • the polynucleotide encoding the CAR described herein is inserted into a vector.
  • the vector is a vehicle into which a polynucleotide encoding a protein may be covalently inserted so as to bring about the expression of that protein and/or the cloning of the polynucleotide.
  • Such vectors may also be referred to as “expression vectors”.
  • the isolated polynucleotide may be inserted into a vector using any suitable methods known in the art, for example, without limitation, the vector may be digested using appropriate restriction enzymes and then may be ligated with the isolated polynucleotide having matching restriction ends.
  • Expression vectors have the ability to incorporate and express heterologous or modified nucleic acid sequences coding for at least part of a gene product capable of being transcribed in a cell. In most cases, RNA molecules are then translated into a protein.
  • Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism.
  • vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are discussed infra.
  • An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
  • the expression vector may have the necessary 5 ⁇ upstream and 3 ⁇ downstream regulatory elements such as promoter sequences such as CMV, PGK and EF1 ⁇ promoters, ribosome recognition and binding TATA box, and 3 ⁇ UTR AAUAAA transcription termination sequence for the efficient gene transcription and translation in its respective host cell.
  • Suitable promoters include the constitutive promoter of simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), HIV LTR promoter, MoMuLV promoter, avian leukemia virus promoter, EBV immediate early promoter, and Rous Sarcoma Virus promoter.
  • Human gene promoters may also be used, including, but not limited to the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • inducible promoters are also contemplated as part of the vectors expressing chimeric antigen receptor. This provides a molecular switch capable of turning on expression of the polynucleotide sequence of interest or turning off expression.
  • Examples of inducible FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, or a tetracycline promoter.
  • the expression vector may have additional sequence such as 6 ⁇ -histidine (SEQ ID NO: 458), c-Myc, and FLAG tags which are incorporated into the expressed CARs.
  • the expression vector may be engineered to contain 5 ⁇ and 3 ⁇ untranslated regulatory sequences that sometimes can function as enhancer sequences, promoter regions and/or terminator sequences that can facilitate or enhance efficient transcription of the nucleic acid(s) of interest carried on the expression vector.
  • An expression vector may also be engineered for replication and/or expression functionality (e.g., transcription and translation) in a particular cell type, cell location, or tissue type.
  • Expression vectors may include a selectable marker for maintenance of the vector in the host or recipient cell.
  • the vectors are plasmids, autonomously replicating sequences, and transposable elements.
  • Additional example vectors include, without limitation, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • animal viruses include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).
  • expression vectors are Lenti-XTM Bicistronic Expression System (Neo) vectors (Clontech), pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DESTTM, pLenti6/V5-DESTTM, and pLenti6.2N5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
  • the coding sequences of the CARs disclosed herein can be ligated into such expression vectors for the expression of the chimeric protein in mammalian cells.
  • the nucleic acids encoding the CAR are provided in a viral vector.
  • a viral vector can be that derived from, for example, a retrovirus (e.g., a foamy virus) or lentivirus.
  • a retrovirus e.g., a foamy virus
  • viral vector refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle.
  • the viral vector can contain the coding sequence for the various chimeric proteins described herein in place of nonessential viral genes.
  • the vector and/or particle FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 can be utilized for the purpose of transferring DNA, RNA or other nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
  • the viral vector containing the coding sequence for a CAR described herein is a retroviral vector or a lentiviral vector.
  • retroviral vector refers to a vector containing structural and functional genetic elements that are primarily derived from a retrovirus.
  • Retroviral vectors for use herein can be derived from any known retrovirus (e.g., type c retroviruses, such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)).
  • type c retroviruses such as Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)).
  • type c retroviruses such as Molone
  • Retroviruses also include human T cell leukemia viruses, HTLV-1 and HTLV-2, and the lentiviral family of retroviruses, such as Human Immunodeficiency Viruses, HIV-1, HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV), and other classes of retroviruses.
  • a lentiviral vector refers to a vector derived from a lentivirus, a group (or genus) of retroviruses that give rise to slowly developing disease.
  • HIV human immunodeficiency virus
  • HIV type 1 HIV type 2
  • visna- maedi a caprine arthritis-encephalitis virus
  • equine infectious anemia virus feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
  • FV feline immunodeficiency virus
  • BIV bovine immune deficiency virus
  • SIV simian immunodeficiency virus
  • Preparation of the recombinant lentivirus can be achieved using the methods according to Dull et al. and Zufferey et al. (Dull et al., J. Virol., 1998; 72: 8463- 8471 and Zufferey et al., J. Virol. 1998; 72:9873-9880).
  • Retroviral vectors i.e., both lentiviral and non-lentiviral
  • retroviral vectors can be formed using standard cloning techniques by combining the desired DNA sequences in the order and orientation described herein (Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals; Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci.
  • Suitable sources for obtaining retroviral (i.e., both lentiviral and non-lentiviral) sequences for use in forming the vectors include, for example, genomic RNA and cDNAs available from commercially available sources, including the Type Culture Collection (ATCC), Rockville, Md. The sequences also can be synthesized chemically.
  • the vector may be introduced into a host cell to allow expression of the polypeptide within the host cell.
  • the expression vectors may contain a variety of elements for controlling expression, including without limitation, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements may be selected as appropriate by a person of ordinary skill in the art, as described above.
  • the promoter sequences may be selected to promote the transcription of the polynucleotide in the vector.
  • Suitable promoter sequences include, without limitation, T7 promoter, T3 promoter, SP6 promoter, beta-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter.
  • Enhancer sequences may be selected to enhance the transcription of the polynucleotide.
  • Selectable markers may be selected to allow selection of the host cells inserted with the vector from those not, for example, the selectable markers may be genes that confer antibiotic resistance. Signal sequences may be selected to allow the expressed polypeptide to be transported outside of the host cell.
  • the vector may be introduced into a host cell (an isolated host cell) to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein.
  • the cloning vectors may contain sequence components generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art.
  • the origin of replication may be selected to promote autonomous replication of the vector in the host cell.
  • the present disclosure provides isolated host cells containing the vectors provided herein.
  • the host cells containing the vector may be useful in expression or cloning of the polynucleotide contained in the vector.
  • Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells.
  • Suitable prokaryotic cells for this purpose include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobactehaceae such as Escherichia, e.g., E.
  • the CARs are introduced into a host cell using transfection and/or transduction techniques known in the art.
  • transfection and, “transduction,” refer to the processes by which an exogenous nucleic acid sequence is introduced into a host cell.
  • the nucleic acid may be integrated into the host cell DNA or may be maintained extrachromosomally.
  • the nucleic acid may be maintained transiently or may be a stable introduction.
  • Transfection may be accomplished by a variety of means known in the art including but not limited to calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
  • Transduction refers to the delivery of a gene(s) using a viral or retroviral vector by means of viral infection rather than by transfection.
  • retroviral vectors are transduced by packaging the vectors into virions prior to contact with a cell.
  • a nucleic acid encoding a CAR carried by a retroviral vector can be transduced into a cell through infection and pro virus integration.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure.
  • Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic-resistance genes.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene.
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5 ⁇ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
  • an example delivery vehicle is a liposome.
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources.
  • DMPC dimyristyl phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • DMPG dimyristyl phosphatidylglycerol
  • the cells engineered to express the disclosed CAR polypeptides are immune effector cells.
  • other types of cells such as induced pluripotent stem cells (iPSC) or hematopoietic stem cells (HSC) are engineered and differentiated into immune effector cells that express the disclosed CAR polypeptides.
  • iPSC induced pluripotent stem cells
  • HSC hematopoietic stem cells
  • the iPSC, HSC, or immune effector cells are obtained from the subject to be treated (i.e., are autologous).
  • immune effector cell lines or donor effector cells allogeneic are used.
  • Immune effector cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as FicollTM separation. For example, cells from the circulating blood of an individual may be obtained by apheresis.
  • immune effector cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of immune effector cells can be further isolated by positive or negative selection techniques.
  • immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time period sufficient for positive selection of the desired immune effector cells.
  • enrichment of FoleyHoagUS11580949.8 Attorney Docket No.
  • the present disclosure provides methods for making the immune effector cells which express the CARs described herein.
  • the method comprises transfecting or transducing immune effector cells isolated from a subject, such as a subject having a cancer antigen (e.g., ROR1) expressing tumor cell, such that the immune effector cells express one or more CAR as described herein.
  • the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly re-administered into the individual.
  • the immune effector cells are first activated and stimulated to proliferate in vitro prior to being genetically modified to express a CAR.
  • the immune effector cells may be cultured before or after being genetically modified (i.e., transduced or transfected to express a CAR as described herein).
  • the source of cells Prior to in vitro manipulation or genetic modification of the immune effector cells described herein, the source of cells may be obtained from a subject.
  • the immune effector cells for use with the CARs as described herein comprise T cells.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cell can be obtained from a unit of blood collected from the subject using any number of techniques known to the skilled person, such as FICOLL separation.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocyte, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing.
  • the cells are washed with PBS.
  • the washed solution lacks calcium, and may lack magnesium or may lack many, if not all, divalent cations.
  • a washing step may be accomplished by methods known to those in the art, such as by using a semiautomated flowthrough centrifuge. After washing, the cells may be resuspended in a variety of biocompatible buffers or other saline FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 solution with or without buffer.
  • T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient.
  • PBMCs peripheral blood mononuclear cells
  • a specific subpopulation of T cells, such as CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO + T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • a method for use herein is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 b, CD16, HLA-DR, and CD8.
  • Flow cytometry and cell sorting may also be used to isolate cell populations of interest.
  • PBMCs may be used directly for genetic modification with the CARs using methods as described herein.
  • T lymphocytes after isolation of PBMC, T lymphocytes are further isolated and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and/or expansion.
  • CD8 + cells can be obtained by using standard methods.
  • CD8 + cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of those types of CD8 + cells.
  • memory T cells are present in both CD62L + and CD62L- subsets of CD8 + peripheral blood lymphocytes.
  • PBMC are sorted into CD62L-CD8 + and CD62L + CD8 + fractions after staining with anti-CD8 and anti-CD62L antibodies.
  • the expression of phenotypic markers of central memory TCM include CD45RO, CD62L, CCR7, CD28, CD3, and CD127 and are negative for granzyme B.
  • central memory T cells are CD45RO + , CD62L + , CD8 + T cells.
  • effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin.
  • naive CD8+T lymphocytes are characterized by the expression of phenotypic markers of naive T cells including CD62L, CCR7, CD28, CD3, CD 127, and CD45RA.
  • phenotypic markers of naive T cells including CD62L, CCR7, CD28, CD3, CD 127, and CD45RA.
  • FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025
  • CD4 + T cells are further sorted into subpopulations.
  • CD4 + T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.
  • CD4 + lymphocytes can be obtained by standard methods.
  • naive CD4 + T lymphocytes are CD45RO ⁇ , CD45RA + , CD62L + CD4 + T cell.
  • central memory CD4 + cells are CD62L positive and CD45RO positive.
  • effector CD4 + cells are CD62L and CD45RO negative.
  • the immune effector cells such as T cells, can be genetically modified following isolation using known methods, or the immune effector cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified.
  • the immune effector cells such as T cells
  • Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Pat. Nos.6,905,874; 6,867,041; 6,797,514; WO2012079000.
  • such methods include contacting PBMC or isolated T cells with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a bead or other surface, in a culture medium with appropriate cytokines, such as IL-2 (e.g., recombinant human IL-2).
  • a stimulatory agent and costimulatory agent such as anti-CD3 and anti-CD28 antibodies
  • cytokines such as IL-2 (e.g., recombinant human IL-2).
  • Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a “surrogate” antigen presenting cell (APC).
  • the T cells may be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Pat. Nos.6,040,177; 5,827,642; and WO2012129514.
  • the immune effector cells comprise any leukocyte involved in defending the body against infectious disease and foreign materials.
  • the immune effector cells can comprise lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combinations thereof.
  • the immune effector cells can comprise T lymphocytes, preferably cytotoxic T lymphocytes (CTLs).
  • CTLs cytotoxic T lymphocytes
  • the immune effector cell is an NK cell, a Treg, a gamma delta T cell, a macrophage, a B cell, a, NKT cell, or a plasmacytoid dendritic cell.
  • T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4 + T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen- presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.
  • APCs antigen- presenting cells
  • T C cells kill virally infected cells and tumor cells, and are also implicated in transplant rejection.
  • CD8 + T cells since they express the CD8 glycoprotein at their surface.
  • CD8 + T cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells.
  • MHC class I molecules which are present on the surface of all nucleated cells.
  • IL-10 adenosine and other molecules secreted by regulatory T cells, the CD8 + cells can be inactivated to an anergic state, which prevents autoimmune diseases.
  • Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re- exposure to their cognate antigen, thus providing the immune system with “memory” against past infections. Memory cells may be either CD4 + or CD8 + . Memory T cells typically express the cell surface protein CD45RO.
  • Regulatory T cells T reg cells, formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell- mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus.
  • T reg cells Two major classes of CD4 + T reg cells have been described: naturally occurring T reg cells and adaptive T reg cells.
  • Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive immune system with the innate immune system.
  • NKT cells recognize glycolipid antigen presented by a molecule called CD1d.
  • MHC major histocompatibility complex
  • CD1d glycolipid antigen presented by a molecule called CD1d.
  • the T cells comprise a mixture of CD4 + cells.
  • the T cells are enriched for one or more subsets based on cell surface expression.
  • the T cells are cytotoxic CD8 + T lymphocytes.
  • Natural-killer (NK) cells are CD56 + CD3 – large granular lymphocytes that can kill virally infected and transformed cells, and constitute a critical cellular subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 201253:1666–1676). Unlike cytotoxic CD8 + T lymphocytes, NK cells launch cytotoxicity against tumor cells without the requirement for prior sensitization, and can eradicate MHC-I-negative cells (Narni-Mancinelli E, et al. Int Immunol 201123:427–431).
  • NK cells are safer effector cells, as they may avoid the potentially lethal complications of cytokine storms (Morgan RA, et al. Mol Ther 201018:843–851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011365:725–733), and on-target, off-tumor effects.
  • Binding Properties of Chimeric Antigen Receptors [0165] As used herein, the term “binding” in the context of the binding of a chimeric antigen receptor to, e.g., a predetermined antigen, such as a cell surface protein or fragment thereof (or to an antigen bound to a cell surface protein such as an HLA molecule).
  • Binding typically refers to an interaction or association between a minimum of two entities or molecular structures, such as an antigen-binding domain:antigen interaction.
  • binding affinity typically corresponds to a K D value of about 10 -7 M or less, such as about 10 -8 M or less, such as about 10 -9 M or less when determined by, for instance, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody or chimeric antigen receptor as the analyte (or antiligand).
  • SPR surface plasmon resonance
  • K D M refers to the dissociation equilibrium constant of a particular antigen-binding domain:antigen interaction.
  • antigen X compared to the binding affinity of the molecule (e.g., chimeric antigen receptor) to another interactive partner molecule (e.g. antigen Y) may be expressed as a binding ratio determined by dividing the larger K D value (lower, or weaker, affinity) by the smaller K D (higher, or stronger, affinity), for example, expressed as 5-fold or 10-fold greater binding affinity, as the case may be.
  • Cell-based binding strategies such as fluorescent-activated cell sorting (FACS) binding assays, are also routinely used, and FACS data correlates well with other methods such as radioligand competition binding and SPR (Benedict, CA, J Immunol Methods.
  • a chimeric antigen receptor of the present disclosure binds to the predetermined antigen or cell surface molecule (receptor) having an affinity corresponding to a K D value that is at least ten-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein).
  • a chimeric antigen receptor of the present disclosure can bind to an HLA-presented antigen described herein.
  • the affinity of a chimeric antigen receptor with a K D value that is equal to or less than ten-fold lower than a non-specific antigen may be considered non-detectable binding.
  • k d (sec -1 or 1/s) refers to the dissociation rate constant of a particular antigen-binding domain:antigen interaction, or the dissociation rate constant of a chimeric antigen receptor. Said value is also referred to as the k off value.
  • k a (M -1 x sec -1 or 1/M x 1/s) refers to the association rate constant of a particular antigen-binding domain:antigen interaction, or the association rate constant of a chimeric antigen receptor.
  • K A (M -1 or 1/M) refers to the association equilibrium constant of a particular antigen-binding domain:antigen interaction, or the association equilibrium constant of a chimeric antigen receptor. The association equilibrium constant is obtained by dividing the k a by the k d .
  • EC50 refers to the half-maximal effective concentration, which includes the concentration of a chimeric antigen receptor that induces a response halfway between the baseline and maximum after a specified exposure time.
  • the EC 50 essentially represents the concentration of a chimeric antigen receptor where 50% of its maximal effect is FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 observed.
  • the EC 50 value equals the concentration of a chimeric antigen receptor of the present disclosure that gives half-maximal binding to cells expressing an antigen (e.g., a tumor-associated antigen), as determined by e.g., a FACS binding assay.
  • an antigen e.g., a tumor-associated antigen
  • decreased binding can be defined as an increased EC 50 chimeric antigen receptor concentration that enables binding to the half-maximal amount of target cells.
  • the present disclosure provides chimeric antigen receptors with antigen-binding domains derived from antibodies that bind an antigen (e.g., a human antigen, a cancer antigen) with high affinity (e.g., nanomolar or sub-nanomolar K D values).
  • the present disclosure provides chimeric antigen receptors with antigen-binding domains derived from corresponding antibodies that bind human antigen (e.g., at 25oC) with a K D of less than about 5 nM as measured by surface plasmon resonance.
  • the corresponding antibodies bind an antigenic protein with a K D of less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 700 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, or less than about 25 pM as measured by surface plasmon resonance.
  • the present disclosure also provides chimeric antigen receptors with antigen-binding domains derived from corresponding antibodies that bind the antigenic protein with a dissociative half-life (t1 ⁇ 2) of greater than about 10 minutes or greater than about 125 minutes as measured by surface plasmon resonance at 25oC.
  • the corresponding antibodies bind the antigenic protein with a t1 ⁇ 2 of greater than about 3 minutes, greater than about 4 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 FoleyHoagUS11580949.8 Attorney Docket No.
  • the present disclosure also provides chimeric antigen receptors with antigen-binding domains derived from corresponding antibodies that bind specifically to human cell lines which express endogenous ROR1, as determined by a FACS binding assay.
  • Therapeutic Methods [0176] Immune effector cells expressing the CARs disclosed herein may in some embodiments elicit a therapeutically beneficial immune response against antigen-expressing (e.g., ROR1-expressing) cancer cells.
  • an anti-tumor immune response elicited by the disclosed CAR-modified immune effector cells may be an active or a passive immune response.
  • the CAR-mediated immune response may be part of an adoptive immunotherapy approach in which CAR-modified immune effector cells induce an immune response specific to a cancer antigen (e.g., ROR1).
  • CAR-expressing immune effector cells prepared as described in some embodiments herein can be utilized in methods and compositions for adoptive immunotherapy in accordance with known techniques, or variations thereof that will be apparent to those skilled in the art based on the instant disclosure. See, e.g., US Patent Application Publication No. 2003/0170238 to Gruenberg et al; see also U.S. Pat.
  • a solid tumor e.g., a solid tumor
  • methods of treating cancer e.g., a solid tumor
  • a composition comprising cells expressing a CAR polypeptide disclosed herein.
  • CAR- based cell therapies are effective in treating both blood cancer and solid tumors.
  • CAR4 can effectively kill an example blood tumor (Jeko-1), and CAR4 has superior in killing of example solid tumors (MDA-MB-468 and A549) over BB Z control.
  • a composition comprising cells expressing a CAR polypeptide as disclosed herein (e.g., a CAR4 polypeptide).
  • FoleyHoagUS11580949.8 Attorney Docket No.
  • GBB-74025 Provided herein are methods of treating a tumor in a subject, the method comprising administering a composition comprising cells expressing a CAR polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein: (i) ICD1 comprises an amino acid sequence of SEQ ID NO: 22 and ICD2 comprises an amino acid sequence of SEQ ID NO: 21, or (ii) ICD1 comprises an amino acid sequence of SEQ ID NO: 21 and ICD2 comprises an amino acid sequence of SEQ ID NO: 22 [0180] Provided herein are methods of treating a tumor in a subject, the method comprising administering a composition comprising cells expressing a CAR polypeptide comprising an intracellular signaling region comprising an amino acid sequence of SEQ ID NO: 23.
  • CAR4 refers to the CAR polypeptide comprising an intracellular signaling region comprising amino acid sequences listed as CD3z_X23mut (SEQ ID NO: 22) as ICD1and CD40 (SEQ ID NO: 21) as ICD2, herein. Therefore, the CAR4 intracellular signaling region comprises SEQ ID NO: 23 (i.e., CD3z_X23mut (SEQ ID NO: 22) and CD40 (SEQ ID NO: 21), combined).
  • the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a “pharmaceutically acceptable” carrier) in a treatment-effective amount.
  • Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized.
  • the infusion medium can be supplemented with human serum albumin.
  • a treatment-effective amount of cells in the composition is at least 2 cells (for example, at least 1 CD8 + central memory T cell and at least 1 CD4 + helper T cell subset), or is more typically greater than 10 2 cells and up to 10 6 or up to and including 10 8 or 10 9 cells, and can be more than 10 10 cells.
  • the number of cells will depend upon the ultimate use for which the composition is intended as will the type of cells included therein.
  • the cells may be autologous or heterologous to the patient undergoing therapy.
  • the cells may be allogeneic.
  • the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and/or chemokines (e.g., IFN- ⁇ , IL-2, IL-12, TNF- ⁇ , IL-18, and TNF- ⁇ , GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1 ⁇ , etc.) as described herein to enhance induction of the immune response.
  • mitogens e.g., PHA
  • lymphokines e.g., lymphokines, cytokines, and/or chemokines (e.g., IFN- ⁇ , IL-2, IL-12, TNF- ⁇ , IL-18, and TNF- ⁇ , GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1 ⁇ , etc.) as described
  • the CAR expressing immune effector cell populations may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.
  • Pharmaceutical compositions disclosed herein may comprise a CAR-expressing immune effector cell population, such as T cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • the anti-tumor immune response induced in a subject by administering CAR expressing T cells described herein using the methods described herein, or other methods known in the art may include cellular immune responses mediated by cytotoxic T cells capable of killing infected cells, by regulatory T cells, and/or by helper T cells.
  • Humoral immune responses mediated primarily by helper T cells capable of activating B cells thus leading to antibody production, may also be induced.
  • a variety of techniques may be used for analyzing the type of immune responses induced by the compositions disclosed herein, which are well described in the art; e.g., Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.
  • compositions may be carried out in any convenient manner, including by injection, transfusion, or implantation.
  • compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the disclosed compositions are administered to a patient by intradermal or subcutaneous injection.
  • the disclosed compositions are administered by i.v. injection.
  • the compositions may also be injected directly into a tumor, lymph node, or site of infection.
  • the disclosed CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to additional cancer treatments.
  • the CAR-modified immune effector cells may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.
  • immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies
  • immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies
  • cytoxin fludaribine
  • cyclosporin FK506, rapamycin
  • mycophenolic acid steroids
  • steroids FR901228
  • cytokines irradiation
  • the CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T-cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
  • the cell compositions are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.
  • subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • subjects receive an infusion of the expanded immune cells.
  • expanded cells are administered before or following surgery to treat cancer or pre-cancerous lesions in the subject.
  • FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 Administration Regimens [0190]
  • multiple doses of the engineered cells may be administered to a subject over a defined time course.
  • the methods according to this aspect comprise sequentially administering to a subject multiple doses of the cells.
  • “sequentially administering” means that each dose is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months).
  • the present disclosure provides methods which comprise sequentially administering to the patient a single initial dose, followed by one or more secondary doses, and optionally followed by one or more tertiary doses.
  • the terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the engineered cells of the present disclosure.
  • the “initial dose” is the dose which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”);
  • the “secondary doses” are the doses which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses.
  • the initial, secondary, and tertiary doses may all contain the same amount of engineered cells, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of engineered cells contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).
  • each secondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 11 ⁇ 2, 2, 21 ⁇ 2, 3, 31 ⁇ 2, 4, 41 ⁇ 2, 5, 51 ⁇ 2, 6, 61 ⁇ 2, 7, 71 ⁇ 2, 8, 81 ⁇ 2, 9, 91 ⁇ 2, 10, 101 ⁇ 2, 11, 111 ⁇ 2, 12, 121 ⁇ 2, 13, 131 ⁇ 2, 14, 141 ⁇ 2, 15, 151 ⁇ 2, 16, 161 ⁇ 2, 17, 171 ⁇ 2, 18, 181 ⁇ 2, 19, 191 ⁇ 2, 20, 201 ⁇ 2, 21, 211 ⁇ 2, 22, 221 ⁇ 2, 23, 231 ⁇ 2, 24, 241 ⁇ 2, 25, 251 ⁇ 2, 26, 261 ⁇ 2, or more) weeks after the immediately preceding dose.
  • 1 to 26 e.g., 1, 11 ⁇ 2, 2, 21 ⁇ 2, 3, 31 ⁇ 2, 4, 41 ⁇ 2, 5, 51 ⁇ 2, 6, 61 ⁇ 2, 7, 71 ⁇ 2, 8, 81 ⁇ 2, 9, 91 ⁇ 2, 10, 101 ⁇ 2, 11, 111 ⁇ 2, 12, 121 ⁇ 2, 13, 131 ⁇ 2, 14, 141 ⁇ 2, 15, 151 ⁇ 2, 16, 161 ⁇ 2, 17, 171 ⁇ 2, 18, 181 ⁇ 2, 19, 19
  • the immediately preceding dose means, in a sequence of multiple administrations, the dose which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.
  • the methods according to this aspect of the present disclosure may comprise administering to a patient any number of secondary and/or tertiary doses.
  • any number of secondary and/or tertiary doses For example, in certain FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient.
  • each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose.
  • each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose.
  • the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen.
  • the frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
  • Indications [0195]
  • the cancer to be treated expresses a cancer antigen (e.g., ROR1) to which the CAR expressed by the immune effector cells (e.g., T cells) specifically binds.
  • a cancer antigen e.g., ROR1
  • the immune effector cells e.g., T cells
  • the cells are expressing ROR1 targeting CARs described herein, and cancers that may be treated by methods and compositions comprising such cells include, but are not limited to, blood cancers (e.g., B cell chronic lymphoblastic leukemia, mantle cell lymphoma and acute lymphoblastic leukemia) and epithelial cancers (e.g., non-small cell lung cancer, triple negative breast cancer, and ovarian cancer).
  • blood cancers e.g., B cell chronic lymphoblastic leukemia, mantle cell lymphoma and acute lymphoblastic leukemia
  • epithelial cancers e.g., non-small cell lung cancer, triple negative breast cancer, and ovarian cancer.
  • the methods and compositions provided herein relate to the treatment of solid tumors.
  • the methods and compositions provided herein relate to the treatment of a carcinoma.
  • carcinoma refers to a malignant growth made up of epithelial cells tending to infiltrate the surrounding tissues, and/or resist physiological and non- FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 physiological cell death signals and gives rise to metastases.
  • Non-limiting examples of carcinomas include, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet- ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma,
  • the methods and compositions provided herein relate to the treatment of a sarcoma.
  • sarcoma generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 embedded in a fibrillar, heterogeneous, or homogeneous substance.
  • Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing' s sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic s
  • Additional example neoplasias that can be treated using the methods and compositions described herein include Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenal cortical cancer.
  • the cancer treated is a melanoma.
  • melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • melanomas are Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungual melanoma, and superficial spreading melanoma.
  • the methods and compositions provided herein relate to the treatment of a leukemia.
  • leukemia is meant broadly progressive, malignant diseases of the hematopoietic organs/systems and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow.
  • Non-limiting examples of leukemia diseases include, acute nonlymphocytic leukemia, chronic lymphocytic FoleyHoagUS11580949.8 Attorney Docket No.
  • GBB-74025 leukemia acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leuk
  • tumors that can be treated using methods and compositions described herein include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, colorectal cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, as well as metastases of all the above.
  • tumors include hepatocellular carcinoma, hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, pulmonary squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well differentiated, moderately differentiated, poorly differentiated or undifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma, hypernephroma, hypernephroid adenocarcinoma, bile duct carcinoma,
  • GBB-74025 including: acute myelogenous leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma, Hodgkin' s lymphoma, non-Hodgkin' s lymphoma.
  • EMBODIMENTS [0203] Provided below are certain example embodiments related to the CAR polypeptides provided herein. [0204] Example embodiment 1.
  • a chimeric antigen receptor (CAR) polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein the intracellular domain pair is selected from the intracellular domain pairs listed in Table 1.
  • Example embodiment 2 The CAR of embodiment 1, wherein the ICD1 comprises an ICD amino acid sequence set forth in Table 4.
  • Example embodiment 3 The CAR of embodiment 1 or 2, wherein the ICD2 comprises an ICD amino acid sequence set forth in Table 4.
  • Example embodiment 4 The CAR of embodiment 1, wherein the intracellular signaling region comprises an amino acid sequence set forth in Table 5.
  • a chimeric antigen receptor (CAR) polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein the intracellular domain pair is selected from the intracellular domain pairs listed in Table 2.
  • Example embodiment 6 The CAR of embodiment 5, wherein the ICD1 comprises an ICD amino acid sequence set forth in Table 4.
  • Example embodiment 7 The CAR of embodiment 5 or 6, wherein the ICD2 comprises an ICD amino acid sequence set forth in Table 4.
  • Example embodiment 8 The CAR of embodiment 5, wherein the intracellular signaling region comprises an amino acid sequence set forth in Table 6.
  • Example embodiment 9 A chimeric antigen receptor (CAR) polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein the intracellular domain pair is selected from the intracellular domain pairs listed in Table 3.
  • Example embodiment 10 The CAR of embodiment 9, wherein the ICD1 comprises an ICD amino acid sequence set forth in Table 4.
  • Example embodiment 11 The CAR of embodiment 9 or 10, wherein the ICD2 comprises an ICD amino acid sequence of in Table 4.
  • Example embodiment 13 A chimeric antigen receptor (CAR) polypeptide comprising an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 has a sequence of SAPHR.
  • Example embodiment 14 The CAR of embodiment 13, wherein the ICD2 comprises an ICD amino acid sequence set forth in Table 4.
  • Example embodiment 15 A chimeric antigen receptor (CAR) polypeptide comprising an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD2 has a sequence of SAPHR.
  • Example embodiment 16 The CAR of embodiment 15, wherein the ICD1 comprises an ICD amino acid sequence set forth in Table 4.
  • Example embodiment 17 A chimeric antigen receptor (CAR) polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein ICD1 is CD3z_X23mut and ICD2 is CD40.
  • Example embodiment 18 The CAR of embodiment 17, wherein the ICD1 comprises an amino acid sequence of SEQ ID NO: 22.
  • Example embodiment 19 Example embodiment 19.
  • Example embodiment 20 A chimeric antigen receptor (CAR) polypeptide comprising an intracellular signaling region comprising an amino acid sequence of SEQ ID NO: 23.
  • Example embodiment 21 A chimeric antigen receptor (CAR) polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein ICD1 is CD40 and ICD2 is CD3z_X23mut.
  • Example embodiment 22 The CAR of embodiment 21, wherein the ICD1 comprises an amino acid sequence of SEQ ID NO: 21.
  • Example embodiment 23 The CAR of embodiment 21 or 22, wherein the ICD2 comprises an amino acid sequence of SEQ ID NO: 22.
  • Example embodiment 24 The CAR polypeptide of any one of embodiments 1-23, wherein the ICD1 and the ICD2 are connected with a peptide linker.
  • Example embodiment 25 The CAR polypeptide of embodiment 24, wherein the peptide linker is glycine-glycine (GG). [0229] Example embodiment 26.
  • Example embodiment 27 The CAR polypeptide of embodiment 26, where in the scFv domain is a scFv domain set forth in Table 8 [0231] Example embodiment 28.
  • Example embodiment 29 The CAR polypeptide of embodiment 28, wherein the scFv domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 7; and/or a light chain variable domain having an amino acid sequence of SEQ ID NO: 8.
  • FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 [0233] Example embodiment 30.
  • Example embodiment 31 The CAR polypeptide of embodiment 26, wherein the scFv domain comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 10, 11, 12, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 13, 14, 15, respectively.
  • Example embodiment 32 The CAR polypeptide of embodiment 31, wherein the scFv domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 16; and/or a light chain variable domain having an amino acid sequence of SEQ ID NO: 17.
  • Example embodiment 33 Example embodiment 33.
  • Example embodiment 34 The CAR polypeptide of any one of embodiments 1-25, wherein the CAR comprises: a) an antigen-binding domain; b) a hinge domain; c) a transmembrane domain; and d) the intracellular signaling region.
  • Example embodiment 35 The CAR polypeptide of embodiment 34, wherein the antigen-binding domain is a scFv domain.
  • Example embodiment 36 The CAR polypeptide of embodiment 35, where in the scFv domain is a scFv domain set forth in Table 8. [0240] Example embodiment 37.
  • Example embodiment 38 The CAR polypeptide of embodiment 37, wherein the cancer antigen is selected from the cancer antigens listed in Table 11.
  • Example embodiment 39 The CAR polypeptide of embodiment 38, wherein the antigen-binding domain binds to Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1).
  • Example embodiment 40 The CAR polypeptide of embodiment 40, wherein the antigen-binding domain binds to Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1).
  • the CAR polypeptide of embodiment 39 wherein the antigen-binding domain comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 1, 2, 3, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 4, 5, 6, respectively.
  • FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 [0244]
  • Example embodiment 41 The CAR polypeptide of embodiment 40, wherein the antigen-binding domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 7; and/or a light chain variable domain having an amino acid sequence of SEQ ID NO: 8. [0245]
  • Example embodiment 42 Example embodiment 42.
  • Example embodiment 43 The CAR polypeptide of embodiment 39, wherein the antigen-binding domain comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 10, 11, 12, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 13, 14, 15, respectively.
  • Example embodiment 44 The CAR polypeptide of embodiment 43, wherein the antigen-binding domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 16; and/or a light chain variable domain having an amino acid sequence of SEQ ID NO: 17.
  • Example embodiment 45 The CAR polypeptide of embodiment 44, wherein the antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 18.
  • Example embodiment 46 The CAR polypeptide of any one of embodiments 1-45, wherein the hinge domain is selected from the hinge domains of Table 12.
  • Example embodiment 47 The CAR polypeptide of embodiment 46, wherein the hinge domain is an IgG4 hinge domain.
  • Example embodiment 48 The CAR polypeptide of embodiment 47, wherein the hinge domain comprises an amino acid sequence of SEQ ID NO: 19.
  • Example embodiment 49 The CAR polypeptide of any one of embodiments 1-48, wherein the transmembrane domain is selected from the transmembrane domains of Table 13.
  • Example embodiment 50 A nucleic acid encoding the CAR polypeptide of any one of embodiments 1-49.
  • Example embodiment 51 The nucleic acid of embodiment 50, wherein the ICD1 is encoded by a nucleic acid sequence set forth in Table 15.
  • Example embodiment 52 The nucleic acid of embodiment 51, wherein the ICD1 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 26. 111 FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 [0256]
  • Example embodiment 53 The nucleic acid of embodiment 51, wherein the ICD1 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 27. [0257] Example embodiment 54.
  • Example embodiment 50 The nucleic acid of embodiment 50 or 51, wherein the ICD2 is encoded by a nucleic acid sequence set forth in Table 15.
  • Example embodiment 55 The nucleic acid of embodiment 54, wherein the ICD2 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 26.
  • Example embodiment 56 The nucleic acid of embodiment 54, wherein the ICD2 is encoded by a nucleic acid sequence set forth in SEQ ID NO: 27.
  • Example embodiment 57 A vector comprising the nucleic acid of any one of embodiments 50-56.
  • Example embodiment 58 The vector of embodiment 57, wherein the vector is an expression vector.
  • Example embodiment 59 Example embodiment 59.
  • Example embodiment 60 The vector of any one of embodiments 57 to 59, wherein the viral vector is lentiviral vector.
  • Example embodiment 61 A cell comprising the nucleic acid of any one of embodiments 50 to 56.
  • Example embodiment 62 A cell expressing the CAR polypeptide of any one of embodiments 1 to 49.
  • Example embodiment 63 The cell of embodiment 61 or 62, wherein the cell is an induced pluripotent stem cell (iPSC), a hematopoietic cell (HSC), or an immune cell.
  • iPSC induced pluripotent stem cell
  • HSC hematopoietic cell
  • Example embodiment 64 Example embodiment 64.
  • Example embodiment 65 The cell of any one of embodiments 63, wherein the cell is a leukocyte, optionally wherein the leukocyte is a lymphocyte, a monocyte, a macrophage, a dendritic cell, a mast cell, a neutrophil, a basophil, or an eosinophil.
  • the leukocyte is a lymphocyte, a monocyte, a macrophage, a dendritic cell, a mast cell, a neutrophil, a basophil, or an eosinophil.
  • NK Natural Killer
  • NKT Natural Killer T
  • ILC innate lymphoid cell
  • CIK cytokine induced killer
  • CTL cytotoxic T lymphocyte
  • LAK lymphokine activated killer
  • Example embodiment 65 wherein the cell is a primary CD8 + T cell.
  • Example embodiment 68 The cell of any one of embodiments 61 to 67, wherein the cell proliferates in the presence of antigen-expressing target cells.
  • Example embodiment 69 The cell of any one of embodiments 61 to 68, wherein the cell is resistant to exhaustion.
  • Example embodiment 70 The cell of embodiment 69, wherein the control CAR comprises the same antigen-binding domain and a 4-1BB-CD3z (BBZ) ICD.
  • Example embodiment 71 Example embodiment 71.
  • a method of generating a CAR-expressing cell comprising contacting the cell with a nucleic acid of any one of embodiments 50 to 56, or a vector of any one of embodiments 57 to 60.
  • Example embodiment 72 The method of embodiment 71, wherein the CAR- expressing cell is generated using a genome-editing tool.
  • Example embodiment 73 The method of embodiment 71 or 72, wherein the genome-editing tool is a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), or a CRISPR/CAS system.
  • TALEN transcription activator-like effector nuclease
  • ZFN zinc-finger nuclease
  • CRISPR/CAS system CRISPR/CAS system
  • Example embodiment 75 The method of any one of embodiments 71 to 74, wherein the cell is an induced pluripotent stem cell (iPSC), a hematopoietic cell (HSC), or an immune cell.
  • iPSC induced pluripotent stem cell
  • HSC hematopoietic cell
  • Example embodiment 75 The method of any one of embodiments 71 to 74, wherein the immune cell is a leukocyte.
  • Example embodiment 76 The method of any one of embodiments 71 to 75, wherein the immune cell is a lymphocyte, a monocyte, a macrophage, a dendritic cell, a mast cell, a neutrophil, a basophil, or an eosinophil.
  • Example embodiment 77 Example embodiment 77.
  • the immune cell is a lymphocyte selected from a B cell, an ⁇ T cell, ⁇ T cell, a Natural Killer (NK) cell, a Natural Killer T (NKT) cell, an innate lymphoid cell (ILC), a cytokine FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T cell, or any combination thereof.
  • CTL cytotoxic T lymphocyte
  • LAK lymphokine activated killer
  • Example embodiment 79 The method of embodiment 78, wherein the immune cell is a primary CD8 + T cell.
  • Example embodiment 80 A composition comprising cells of any one of embodiments 61 to 70.
  • Example embodiment 81 A method of treating a tumor in a subject, the method comprising administering a composition comprising cells expressing a CAR polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein the intracellular domain pair is selected from the intracellular domain pairs listed in Table 1.
  • Example embodiment 82 A method of treating a tumor in a subject, the method comprising administering a composition comprising cells expressing a CAR polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and
  • Example embodiment 81 wherein the ICD1 comprises an ICD amino acid sequence set forth in Table 4.
  • Example embodiment 83 The method of embodiment 81 or 72, wherein the ICD2 comprises an ICD amino acid sequence of in Table 4.
  • Example embodiment 84 The method of embodiment 81, wherein the intracellular signaling region comprises an amino acid sequence set forth in Table 5. [0288] Example embodiment 85.
  • a method of treating a tumor in a subject comprising administering a composition comprising cells expressing a CAR polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein the intracellular domain pair is selected from the intracellular domain pairs listed in Table 2.
  • Example embodiment 86 The method of embodiment 85, wherein the ICD1 comprises an ICD amino acid sequence set forth in Table 4.
  • the method of embodiment 85 or 86, wherein the ICD2 comprises an ICD amino acid sequence of in Table 4.
  • FoleyHoagUS11580949.8 Attorney Docket No.
  • Example embodiment 88 The method of embodiment 85, wherein the intracellular signaling region comprises an amino acid sequence set forth in Table 6.
  • Example embodiment 89 A method of treating a tumor in a subject, the method comprising administering a composition comprising cells expressing a CAR polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2), and wherein the intracellular domain pair is selected from the intracellular domain pairs listed in Table 3.
  • Example embodiment 90 The method of embodiment 89, wherein the ICD1 comprises an ICD amino acid sequence set forth in Table 4.
  • Example embodiment 91 The method of embodiment 89 or 90, wherein the ICD2 comprises an ICD amino acid sequence of in Table 4.
  • Example embodiment 92 The method of embodiment 91, wherein the intracellular signaling region comprises an amino acid sequence set forth in Table 7.
  • Example embodiment 93 A method of treating a tumor in a subject, the method comprising administering a composition comprising cells expressing a CAR polypeptide comprising an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD1 has a sequence of SAPHR.
  • Example embodiment 94 The method of embodiment 93, wherein the ICD2 comprises an ICD amino acid sequence set forth in Table 4.
  • Example embodiment 95 A method of treating a tumor in a subject, the method comprising administering a composition comprising cells expressing a CAR polypeptide comprising an intracellular domain pair comprising a first intracellular domain (ICD1) and a second intracellular domain (ICD2), wherein the ICD2 has a sequence of SAPHR.
  • Example embodiment 96 The method of embodiment 95, wherein the ICD1 comprises an ICD amino acid sequence set forth in Table 4.
  • Example embodiment 97 Example embodiment 97.
  • a method of treating a tumor in a subject comprising administering a composition comprising cells expressing a CAR polypeptide comprising an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair, wherein the intracellular domain pair comprises a first intracellular FoleyHoagUS11580949.8 Attorney Docket No. GBB-74025 domain (ICD1) and a second intracellular domain (ICD2), and wherein: (i) the ICD1 comprises an amino acid sequence of SEQ ID NO: 22 and ICD2 comprises an amino acid sequence of SEQ ID NO: 21, or (ii) ICD1 comprises an amino acid sequence of SEQ ID NO: 21 and the ICD2 comprises an amino acid sequence of SEQ ID NO: 22.
  • Example embodiment 98 A method of treating a tumor in a subject, the method comprising administering a composition comprising cells expressing a CAR polypeptide comprising an intracellular signaling region comprising an amino acid sequence of SEQ ID NO: 23.
  • Example embodiment 99 The method of any one of embodiments 81 to 98, wherein the ICD1 and the ICD2 are connected with a peptide linker.
  • Example embodiment 100 The method of embodiment 99, wherein the peptide linker is glycine-glycine (GG).
  • Example embodiment 101 Example embodiment 101.
  • Example embodiment 102 The method of embodiment 101, where in the scFv domain is a scFv domain set forth in Table 8.
  • Example embodiment 103 The method of embodiment 101, wherein the scFv domain comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 1, 2, 3, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 4, 5, 6, respectively.
  • Example embodiment 104 The method of embodiment 103, wherein the scFv domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 7; and/or a light chain variable domain having an amino acid sequence of SEQ ID NO: 8.
  • Example embodiment 105 The method of embodiment 104, wherein the scFv domain comprises an amino acid sequence of SEQ ID NO: 9.
  • Example embodiment 106 The method of embodiment 101, wherein the scFv domain comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 10, 11, 12, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 13, 14, 15, respectively.
  • Example embodiment 107 The method of embodiment 102, wherein the scFv domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 16; and/or a light chain variable domain having an amino acid sequence of SEQ ID NO: 17. [0311]
  • Example embodiment 110 The method of any one of embodiments 81 to 100, wherein the CAR comprises: a) an antigen-binding domain; b) a hinge domain; c) a transmembrane domain; and d) the intracellular signaling region.
  • Example embodiment 110 The method of embodiment 109, wherein the antigen- binding domain is a scFv domain.
  • Example embodiment 111 The method of embodiment 110, where in the scFv domain is a scFv domain set forth in Table 8.
  • Example embodiment 112. The method of any one of embodiments 109 to 111, wherein the antigen-binding domain binds to a cancer antigen.
  • Example embodiment 113 Example embodiment 113.
  • Example embodiment 112 wherein the cancer antigen is selected from the cancer antigens listed in Table 11.
  • Example embodiment 114 The method of embodiment 113, wherein the antigen- binding domain binds to Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1).
  • Example embodiment 115 The method of embodiment 114, wherein the antigen- binding domain comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 1, 2, 3, respectively; and light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ ID NOs: 4, 5, 6, respectively.
  • Example embodiment 116 Example embodiment 116.
  • Example embodiment 115 wherein the antigen- binding domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 7; and/or a light chain variable domain having an amino acid sequence of SEQ ID NO: 8.
  • Example embodiment 117 The method of embodiment 116, wherein the antigen- binding domain comprises an amino acid sequence of SEQ ID NO: 9.
  • Example embodiment 118 The method of embodiment 114, wherein the antigen- binding domain comprises heavy chain CDR1, CDR2, and CDR3 having an amino acid FoleyHoagUS11580949.8 Attorney Docket No.
  • Example embodiment 119 The method of embodiment 118, wherein the antigen- binding domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 16; and/or a light chain variable domain having an amino acid sequence of SEQ ID NO: 17.
  • Example embodiment 120 The method of embodiment 119, wherein the antigen- binding domain comprises an amino acid sequence of SEQ ID NO: 18.
  • Example embodiment 121 The method of any one of embodiments 81 to 120, wherein the hinge domain is selected from the hinge domains of Table 12.
  • Example embodiment 122 The method of embodiment 121, wherein the hinge domain is an IgG4 hinge domain.
  • Example embodiment 123 The method of embodiment 122, wherein the hinge domain comprises an amino acid sequence of SEQ ID NO: 19.
  • Example embodiment 124 The method of any one of embodiments 81 to 123, wherein the transmembrane domain is selected from the transmembrane domains of Table 13.
  • Example embodiment 125 The method of any one of embodiments 81 to 124, wherein the tumor expresses the antigen recognized by the antigen-binding domain of the CAR.
  • Example embodiment 126 Example embodiment 126.
  • Example embodiment 127 The method of any one of embodiments 81 to 126, wherein the cells are iPSCs, HSCs, or immune cells.
  • Example embodiment 127 The method of any one of embodiments 81 to 126, wherein the immune cell is a leukocyte.
  • Example embodiment 128 The method of any one of embodiments 81 to 127, wherein the immune cell is a lymphocyte, a monocyte, a macrophage, a dendritic cell, a mast cell, a neutrophil, a basophil, or an eosinophil.
  • Example embodiment 129 Example embodiment 129.
  • the immune cell is a lymphocyte selected from a B cell, an ⁇ T cell, ⁇ T cell, a Natural Killer (NK) cell, a Natural Killer T (NKT) cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, a regulatory T cell, or any combination thereof.
  • NK Natural Killer
  • NKT Natural Killer T
  • ILC innate lymphoid cell
  • CIK cytokine induced killer
  • CTL cytotoxic T lymphocyte
  • LAK lymphokine activated killer
  • Example embodiment 131 The method of any one of embodiments 81 to 130, wherein the immune cell is a primary CD8+ T cell.
  • Example embodiment 132 The method of any one of embodiments 81 to 131, wherein the immune cell is an autologous immune cell from the subject.
  • Example embodiment 133 The method of any one of embodiments 81 to 132, wherein the immune cell is an allogeneic immune cell.
  • Example embodiment 134 The method of any one of embodiments 81 to 133, wherein the immune cell proliferates in the subject.
  • Example embodiment 135. The method of any one of embodiments 81 to 134, wherein the immune cell kills the tumor cell in the subject.
  • Example embodiment 136 The method of any one of embodiments 81 to 135, wherein a reduced percentage of PD1 + TIM-3 + or PD1 + LAG-3 + population is observed for the immune cell after administration to the subject compared to the cells expressing a control CAR.
  • Example embodiment 137 The method of any one of embodiments 81 to 136, wherein the control CAR comprises the same antigen-binding domain and a 4-1BB-CD3z (BBZ) ICD.
  • Example embodiment 138 A cell bank comprising cells for adoptive immunotherapy, wherein the cells express the CAR of any one of embodiments 1 to 49.
  • Exemplification Example 1 Identification of Novel CAR Intracellular Signaling Regions [0342]
  • a platform that allows parallel comparison of 10,000 different 2nd generation CAR ICD combinations in primary CD8+ T cells was used to identify novel combination of intracellular signaling domains (ICDs) that conveyed favorable T cell phenotypes, including, for example, improved T cell persistence and fitness following activation.
  • Figure 1 provides a schematic illustration of the screen.
  • the CAR polypeptide comprises: an antigen- binding domain; a hinge domain; a transmembrane domain; and an intracellular signaling region, wherein the intracellular signaling region comprises an intracellular domain pair.
  • the FoleyHoagUS11580949.8 Attorney Docket No.
  • GBB-74025 intracellular domain pair comprises a first intracellular domain (ICD1) and a second intracellular domain (ICD2).
  • ICD1 is proximal to the membrane relative to ICD2, and ICD2 is distal to the membrane relative to ICD1.
  • R12 ScFv high affinity for ROR1, 2A2 ScFv: low affinity for ROR1
  • CD8 linker long, IgG4 linker: short
  • Individual libraries contained the same set of 10,000 ICD combinations.
  • Plasmids encoding these CAR molecules were packaged into lentiviral particles, which were treated onto primary CD8+ T cells for infection. These transduced T cells were co-cultured with Jeko-1 Mantle Cell Lymphoma (MCL) target cells in a repeated manner (2-3 times of challenge every week for 3 weeks) to drive T cells into exhaustion and dysfunction. Identification of CAR molecules having ICD combinations that prevent T cells from acquiring an exhaustion phenotype was attained by assessing proliferative capacity of these T cells at the end of the assay based on barcode enrichment, i.e., how many times the unique barcode sequences associated with individual ICD combinations were identified at the end of the assay compared to how much they were present at the beginning of the assay.
  • barcode enrichment i.e., how many times the unique barcode sequences associated with individual ICD combinations were identified at the end of the assay compared to how much they were present at the beginning of the assay.
  • next generation sequencing was used to identify the ICD combinations that were most enriched by the assay in the R12-IgG4 and 2A2-IgG4 CAR T cell pools.
  • the ICD combinations enriched by at least 30- fold in the R12-IgG4 library are provided in Table 1.
  • the ICD combinations enriched by at least 30-fold in the 2A2-IgG4 library are provided in Table 2.
  • the ICD combinations enriched by at least 10-fold in both libraries are provided in Table 3.
  • CARs having intracellular signaling regions containing the novel ICD combinations provided herein were FoleyHoagUS11580949.8 Attorney Docket No.
  • Lentivirus production and T cell transduction [0346] Lenti-X 293T cells (Takara) were seeded in collagen I (Corning) pre-coated T- 175 flask and expanded to achieve 90% confluency on the day of transfection.
  • Transfer plasmid encoding CAR library and packaging plasmid were mixed at 1:1 ratio and transfected into pre-seeded Lenti-X 293T cells with FugeneHD (Promega) under the manufacturer’s protocol. Lentiviral supernatant was collected at 48h and 72h, pooled, and concentrated with Lenti-X Concentrator (Takara) under the manufacturer’s protocol. On Day 0, primary CD8+ T cells isolated from leukopak of healthy donor were stimulated with CD3/CD28 antibody coated beads (Gibco) for 24h prior to transduction.
  • Pre-stimulated CD8+ T cells were mixed with CAR library lentivirus at MOI of 0.5 in the presence of polybrene (Cellecta) and spinfected at 800g for 1h at 32C. Cells were washed 1 day post-transduction (Day 2), then de-beaded and seeded in a 24-well G-REX plate (Wilson Wolf) the next day (Day 3), and expanded for 4 more days. Serial rechallenge assay [0347] 1E5 CAR-T cells and Jeko-1 target cells were seeded at 1:1 ratio in 96-well plate.
  • the sequencing reads were processed with following steps: First, the constant sequences at the 5’ and 3’ of the DNA barcode region were identified with a maximum error of 1. Next, barcode regions with wrong length (more than 2 bp deletion or insertion), or with frameshift (more than 4 bp location change) were filtered out. Then, putative barcodes were error-corrected to input barcodes with maximum hamming distance of 6. After obtaining the error-corrected barcodes, normalized barcode abundance was calculated by dividing the abundance of a given barcode with the total number of reads from a given sample. This normalized abundance was used for subsequent analyses.
  • Example 3 Assays of CAR4 and BBZ T Cells Chronic stimulation assay for arrayed screen [0350] Every time a rechallenge was performed, 1E5 A549 cells engineered to express GFP were seeded 2h prior to 1E5 CAR-T cells in 24-well plates. Every 3 days, 5% of culture volume was flow analyzed to assess CAR-T cell counts. The assay ran on the image-based equipment, IncuCyte, to assess target cell counts and T cell cytotoxicity. See Figure 12, Figure 13, and Figure 15.
  • GBB-74025 On day 4, 11, and 24 of the chronic stimulation assay, 100 ul of co-culture supernatant was collected for each CAR design. Multiplexed cytokine levels were measured by using the MSD U-plex platform (Meso Scale Diagnostics). See Figure 14. Multiple tumor target challenge [0353] Prior to co-culture of CAR-T and tumor cells, BBz and CAR4 T cells were stained with 5 uM of CellTrace Violet dye under manufacturer’s protocol. CAR-T cells were then mixed with A549, MDA-MB-468, or Jeko-1 target cell lines at 1:1 E:T ratio in the presence of low IL-2 cytokine support (5 IU/ml).
  • Example 4 Characterization of CAR4 T cell function Assessment of Persistent Cytotoxicity [0355] Cytotoxicity of Anti-GD2 CAR-T cells using CAR4 against MG-63 osteosarcoma tumor cells was assessed in comparison to cytotoxicity against CAR-T cells with BBz and 28OX40z. Anti-GD2 CAR-T cells with different signaling domain combinations were co- cultured with MG-63 osteosarcoma tumor cells at E:T ratio of 1:1 in a serial rechallenge assay in the presence and absence of IL-2 cytokine support. For every challenge, CAR-T cells were counted and the co-culture was normalized at E:T ratio of 1:1.
  • Cytokine secretion was characterized for each CAR-T group across multiple different conditions (as shown in Figure 23). Cytokine profiling was conducted with supernatant collected at the end of each rechallenge.
  • Anti-GD2 CAR-T cells using CAR4 generally secreted a larger amount of cytokines known to support anti-tumor function, such as IFN-g, TNF-a, GM- CSF, and IL-12p70, and lower amount of inhibitory cytokine (IL-4) and a cytokine known to be involved in CAR-T cell therapy toxicity (IL-6).
  • cytokines known to support anti-tumor function such as IFN-g, TNF-a, GM- CSF, and IL-12p70
  • IL-6 inhibitory cytokine
  • supernatant from the co-culture was collected, and multiplexed cytokine levels were measured by using the MSD U-plex platform (Meso Scale Diagnostics).
  • Samples were prepared using 500 ⁇ l of sample mixed with 500 ⁇ l of a NMR buffer containing a chemical shift reference and analytical standard (TMSP-2,2,3,3- D4 (D,98%) SODIUM-3-TRIMETHYLSILYL PROPIONATE purchased from Cambridge Isotope Laboratories, Inc.).
  • TMSP-2,2,3,3- D4 D,98% SODIUM-3-TRIMETHYLSILYL PROPIONATE purchased from Cambridge Isotope Laboratories, Inc.
  • the NMR buffer was prepared with Mono- and Dibasic Potassium Phosphate at 0.5M concentration and a pH of 7.1 M solutions of both Mono- and Dibasic Potassium Phosphate were prepared and a mixture of 61.5mL dibasic and 38.5mL monobasic potassium phosphate was prepared and the pH was measured at pH 7 as the Henderson– Hasselbalch equation evaluates to for a pK of 6.86 at 25C.
  • TMSP-d4 was added with a concentration of 30mM. Samples were filled into standard 5mM NMR tubes (Wilmad) and automated shimming routine was performed for each sample before measurement. FoleyHoagUS11580949.8 Attorney Docket No.
  • GBB-74025 For NMR data acquisition NMR samples were measured using a Spinsolve 80 MHz NMR (Magritek, Malvern, PA, USA) using 128 scans per sample, 15s relaxation delay and water presaturation. All samples were acquired with an acquisition time of 6.4s and a saturation period of 2s. Free induction decays were multiplied by an exponential weighting function equivalent to 1 Hz line broadening after which they were Fourier transformed from the time to the frequency domain and referenced to the TSP single peak at 0.0 ppm. All spectra were then phased and baseline corrected automatically. The spectra were converted into numerical vectors, representing the individual metabolites, by integrating across the spectrum using 0.04 ppm integral regions by using Decimator.
  • each CAR design was distinct from each other in terms of capacity of secretion and absorption of different metabolites.
  • Anti-ROR1 CAR-T cells with CAR4 have different metabolic characteristics than those of BBz (CAR10) and 28z (CAR2). These data suggest that CAR4 has distinct signaling pathways.
  • Energy Source of CAR4 Cells [0361] Metabolite concentrations from supernatants from each CAR-T cell group co- culture were measured (as shown in Figure 25). CAR4 showed relatively higher levels of lactate and pyruvate, which are products of glucose break-down, indicating that CAR4 may use glycolysis as a source of energy while maintaining long-term persistence in the tumor environment.
  • butyrate and acetate are short-chain fatty acids that are not produced by immune cells, therefore the measured levels of these metabolites show that CAR4 cells consumed less butyrate and consumed more acetate provided to the cells in the cell medium. It has been previously shown that butyrate consumption can expedite T cell apoptosis, and acetate consumption may lead T cells to enhance effector function in a nutrient-restricted environment.

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Abstract

La présente invention concerne des récepteurs antigéniques chimériques, des cellules exprimant de tels récepteurs antigéniques chimériques, des compositions de cellules exprimant de tels récepteurs antigéniques chimériques, ainsi que des procédés de fabrication et d'utilisation de ceux-ci.
PCT/US2023/036126 2022-10-28 2023-10-27 Récepteurs antigéniques chimériques comprenant une paire de domaines intracellulaires WO2024091669A1 (fr)

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