WO2023201288A1 - Cd70 binding car-t cells comprising cd33 binding t-cell engaging antibody molecules - Google Patents

Cd70 binding car-t cells comprising cd33 binding t-cell engaging antibody molecules Download PDF

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WO2023201288A1
WO2023201288A1 PCT/US2023/065710 US2023065710W WO2023201288A1 WO 2023201288 A1 WO2023201288 A1 WO 2023201288A1 US 2023065710 W US2023065710 W US 2023065710W WO 2023201288 A1 WO2023201288 A1 WO 2023201288A1
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cell
car
cells
acid sequence
team
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WO2023201288A9 (en
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Marcela V. Maus
Mark LEICK
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General Hospital Corp
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General Hospital Corp
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Priority to AU2023254840A priority Critical patent/AU2023254840A1/en
Priority to JP2024560356A priority patent/JP2025515995A/ja
Priority to CA3248652A priority patent/CA3248652A1/en
Priority to CN202380047510.1A priority patent/CN119365202A/zh
Priority to US18/856,583 priority patent/US20250249037A1/en
Priority to EP23789144.5A priority patent/EP4507706A4/en
Publication of WO2023201288A1 publication Critical patent/WO2023201288A1/en
Anticipated expiration legal-status Critical
Publication of WO2023201288A9 publication Critical patent/WO2023201288A9/en
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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    • A61K40/10—Cellular immunotherapy characterised by the cell type used
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    • C07K16/2875—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Definitions

  • Chimeric Antigen Receptor T cells have been highly effective for certain hematologic malignancies.
  • the TNF-alpha family member CD70 has emerged as a promising surface target antigen in AML.
  • antigen escape can occur after antigen-specific cancer treatment, leading to antigen-negative cancer relapse.
  • the disclosure is directed to methods and compositions for treating cancers characterized by cells expressing CD70 and optionally cells expressing a lower level of CD70 or that do not express CD70 (e.g., such that the cancer is no longer characterized by cells expressing CD70 in response to an anti-CD70 treatment (e.g., CD70 antigen escape)).
  • the disclosure is directed, in part, to compositions and methods that overcome such antigen escape.
  • the disclosure provides a dual targeting strategy in which a CD70 CAR T cell is engineered to express and secrete a T-cell engaging antibody molecule (TEAM).
  • the TEAM comprises a cancer binding moiety (e.g., anti-CD33 antibody) and an immune cell binding moiety (e.g., an anti-CD3 antibody).
  • the TEAM may direct binding of the CAR-T cell or bystander cells to the target cancer cells (e.g., cancer cells expressing CD33) or myeloid derived suppressor cells (MDSCs) (e.g., MDSCs expressing CD33) independent of CD70 CAR expression.
  • the disclosure is directed to a cell comprising a chimeric antigen receptor (CAR) that binds CD70 and a T-cell engaging antibody molecule (TEAM) that binds to CD33.
  • the cell is an immune cell.
  • the immune cell is a T-cell, a NK cell, a dendritic cell, a macrophage, a B cell, a neutrophil, an eosinophil, a basophil, a mast cell, a myeloid derived suppressor cell, a mesenchymal stem cell, a precursor thereof, or a combination.
  • the immune cell is a T-cell.
  • the cell is collected from a subject, optionally a human subject.
  • the CAR comprises: (i) an extracellular target binding domain comprising a polypeptide that binds CD70; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.
  • the extracellular target binding domain comprises the CD70-binding domain of CD27.
  • the extracellular target binding domain comprises the extracellular domain of CD27.
  • the extracellular target binding domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 1, 8, or 9.
  • the extracellular target binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 1, 8, or 9.
  • the extracellular target binding domain comprises an anti-CD70 antibody, optionally an scFv.
  • the transmembrane domain is the transmembrane domain of CD27.
  • the intracellular signaling domain comprises (i) an ITAM-containing signaling domains and/or (ii) one or more signaling domains from one or more co-stimulatory proteins or cytokine receptors.
  • the intracellular signaling domain comprises a CD3y, CD3s, CD36, or CD3( ⁇ domain.
  • the intracellular signaling domain comprises a CD3( ⁇ domain.
  • the costimulatory domain comprises a CD28, 4- IBB, 2B4, KIR, 0X40, ICOS, MYD88, IL2 receptor, or SynNotch domain.
  • the costimulatory domain comprises a 4- IBB domain.
  • the extracellular target binding domain further comprises a signal peptide.
  • the signal peptide comprises a CD27 signal peptide.
  • the CAR comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 2-7. In some embodiments, the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 2-7.
  • the TEAM comprises an anti-CD33 antibody or a functional fragment thereof (e.g, a VH and/or VL domain of an anti-CD33 antibody).
  • the anti-CD33 antibody is selected from the group consisting of a fragment antigen-binding region (Fab region), a single-chain variable fragment (scFv), a diabody, a nanobody or a monoclonal antibody.
  • the anti-CD33 antibody is an scFv.
  • the anti-CD33 antibody comprises a VH domain having the amino acid sequence of SEQ ID NO: 20 and/or a VL domain having the amino acid sequence of SEQ ID NO: 19.
  • the VH domain is N-terminal of the VL domain. In some embodiments, the VL domain is N-terminal of the VH domain.
  • the TEAM comprises an immune cell binding moiety. In some embodiments, the immune cell binding moiety binds CD3, CD8, CD4, CXCR3, CCR4, GARP, LAP, CD25, CTLA-4, or CD 16. In some embodiments, the immune cell binding moiety is selected from the group consisting of a fragment antigen-binding region (Fab region), a single-chain variable fragment (scFv), a diabody, a nanobody or a monoclonal antibody. In some embodiments, the immune cell binding moiety is an anti-CD3 scFv.
  • Fab region fragment antigen-binding region
  • scFv single-chain variable fragment
  • the immune cell binding moiety is an anti-CD3 scFv.
  • the TEAM comprises a linker between the anti-CD33 antibody and the immune cell binding moiety.
  • the linker is a non-cleavable linker, optionally a (GGGGS)3 (SEQ ID NO: 27) linker.
  • the TEAM further comprises a secretion tag, optionally a IgK secretion tag.
  • the TEAM comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 17-18.
  • the cell comprises a polynucleotide molecule comprising a nucleic acid sequence encoding an amino acid sequence of any one of SEQ ID NOs: 17-18.
  • the nucleic acid sequence encoding the TEAM is codon-optimized.
  • the cell comprises a first polynucleotide molecule comprising a nucleic acid sequence encoding the CAR and a second polynucleotide molecule comprising a nucleic acid sequence encoding the TEAM.
  • the cell comprises a polynucleotide molecule comprising a nucleic acid sequence encoding the CAR and a nucleic acid sequence encoding the TEAM.
  • the polynucleotide molecule further comprises a nucleic acid sequence encoding a linker between the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the TEAM, optionally wherein the linker is a cleavable linker.
  • the cleavable linker is self- cleavable, optionally a P2A, E2A, F2A, or T2A self-cleavable linker.
  • the cleavable linker comprises a protease motif.
  • the linker comprises an internal ribosome entry site (IRES).
  • the polynucleotide molecule comprises a promoter operably linked to the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the TEAM.
  • the promoter is a constitutively active promoter.
  • the promoter is an EFl alpha promoter.
  • the sense strand of the polynucleotide molecule comprises, from 5’ to 3’, the nucleic acid sequence encoding the CAR, the linker, and the nucleic acid sequence encoding the TEAM. In some embodiments, the sense strand of the polynucleotide molecule comprises, from 5’ to 3’, the nucleic acid sequence encoding the TEAM, the linker, and the nucleic acid sequence encoding the CAR. In some embodiments, the polynucleotide molecule comprises a nucleic acid sequence encoding an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 22-25.
  • the disclosure is directed to a polynucleotide comprising a nucleic acid sequence encoding the CAR and the TEAM as described herein. In some aspects, the disclosure is directed to a cell comprising the CAR and the TEAM as described herein.
  • the disclosure is directed to a method comprising administering to a subject the cell as described herein. In some aspects, the disclosure is directed to a method of treating a cancer characterized by cells expressing CD70, the method comprising administering to a subject in need thereof an effective amount of the cell as described herein. In some aspects, the disclosure is directed to a method of treating a cancer characterized by cancer cells expressing CD70 and CD33, the method comprising administering to a subject in need thereof an effective amount of a cell as described herein.
  • the disclosure is directed to a method of treating a cancer characterized by cancer cells expressing CD33, the method comprising administering to a subject in need thereof an effective amount of a cell as described herein.
  • the disclosure is directed to a method of treating a cancer characterized by cancer cells that have decreased CD70 expression, the method comprising administering to a subject in need thereof an effective amount of a cell as described herein.
  • the subject is human. In some embodiments, administering comprises infusion. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is a myeloid cancer. In some embodiments, the cancer is acute myeloid leukemia.
  • FIG. 1 shows a diagram of trCD27-CD8H/TM CAR secreting a CD33-CD3 bispecific T-cell engager (or T-cell engaging antibody molecule (TEAM)).
  • the CD27 CAR can bind to acute myeloid leukemia (AML) and the CD33 TEAM can engage a bystander T Cell to the AML cell.
  • AML acute myeloid leukemia
  • FIG. 2 shows graphs of flow cytometry data showing CD70 low relapse after CD70- targeted CAR treatment in an AML PDX.
  • NSG mice were engrafted with 5xl0 6 DFAM68555 AML patient derived xenograft cells on day -4.
  • mice received 2xl0 6 trCD27 CD8H/TM modified CD70-targeted CAR-T cells.
  • Most trCD8H/TM modified mice cleared tumor, however some mice relapsed with CD70 low tumor.
  • FIGs. 3A-3C show paired expression of CD33 and CD70 as combinatorial CAR targets in AML.
  • FIG. 3A shows co-expression of CD33 and CD70 in blood.
  • FIG. 8B shows expression of CD33 and CD70, alone and together, in primary AML samples.
  • FIG. 3C shows the co-expression of the CD33+CD70 antigen pair in AML cells compared with normal BM HSCs and T cells.
  • FIG. 4 is a schematic demonstrating a putative advantage of a CAR TEAM : CAR TEAM cells demonstrate rapid renal clearance, spare hepatocytes and have decreased hepatoxicity.
  • FIGs. 5A-5E show diagrams of exemplary CAR and TEAMs and graphs demonstrating that TEAMs are secreted by CAR-T cells and bind to appropriate target antigens.
  • FIG. 5A Structures of trCD27 CAR and/or CD33 TEAM constructs.
  • FIG. 5C supernatant from the indicated cells (rows) were added to the indicated cell types (column) and assessed for anti- His tag (TEAM) binding.
  • FIG. 5A-5E show diagrams of exemplary CAR and TEAMs and graphs demonstrating that TEAMs are secreted by CAR-T cells and bind to appropriate target antigens.
  • FIG. 5A Structures of trCD27 CAR and/or CD33 TEAM constructs.
  • FIG. 5B transduction efficiency of activated T-cells healthy donor T-cells (
  • FIG. 5D supernatant from Jurkat T cells transduced with the indicated constructs were incubated with plate bound Molml3 tumor cells as well as anti -His tag AF647 antibody (red). Fluorescent intensity was measured on an Incucyte with representative images as shown.
  • FIG. 5E supernatant from Jurkat T cells transduced with the indicated constructs were co-cultured with Molml3 tumor cells and untransduced (UTD) healthy primary human T cells and anti-CD69 antibody. Percent of cells expressing CD69 across time are shown.
  • PMA phorbol 12-myristate 13-acetate positive control.
  • FIG. 6 shows diagrams of CAR-T cell and TEAM action and graphs demonstrating that CD33 TEAMs mediate UTD-driven, target-dependent cytotoxicity.
  • Cells transduced with the trCD27 CAR-CD33-TEAM or trCD27-CD19-TEAM construct, or untransduced T- cells (UTD) were added to the top of a transwell insert.
  • Untransduced T-cells were added with Molml3WT or Molml3CD33- cells to the bottom of the transwell insert. Only the trCD27-CAR-CD33-TEAM construct was able to mediate Molml3WT cell clearance, which was target dependent and was abrogated with loss of CD33.
  • FIGs. 7A-7B show graphs and micrographs demonstrating simultaneous T-cell redirection through CARs and TEAMs is efficacious against heterogeneous tumors and outperform a mix of individual CARs.
  • FIG. 7A Individually targeted CD70 or CD33 CAR-T cells were compared to CD70CARCD33TEAM or CD70CARCD19TEAMs against AML targets with different levels of CD70 or CD33 (left).
  • FIGs. 8A-8E show CD70CAR CD33TEAM eradicates mixed tumor populations in vivo.
  • BBI bioluminescent
  • FIG. 8B-8C show serial BLI monitoring revealed tumor eradication in the CD70CAR CD33TEAM group of mice, but uncontrolled tumor growth in the CD70CAR CD19TEAM . P value as indicated by 2-way ANOVA.
  • FIG. 8D shows peripheral blood expansion determined by flow cytometry days 14-28 after CAR-T injection. P-values reported as the result of individual Mann-Whitney U tests.
  • FIG. 8E shows peripheral blood CAR-T phenotypic profiling via flow cytometry from the blood of mice at day +21 after CAR-T injection compared to unstimulated and nontransduced, donor matched T-cells. Representative examples shown.
  • CM central memory
  • EM effector memory
  • TDE terminal differentiated effectors
  • FIGs. 9A-9C shows CD70CAR CD33TEAM cells exhibit divergent expressional programs compared to CD70CAR CD19TEAM cells in vivo.
  • FIG. 9A shows a volcano plot highlighting differences in transcriptional programs between the constructs. Genes with adjusted p-value less than 0.05 (Benjamini -Hochberg) are colored red.
  • FIG. 9B shows pathway clustering was performed in an unbiased manner integrating all available pathways (top). Heatmap shown below for selected pathways.
  • FIG. 9C shows pathway score for TCR-signaling is shown for CD70CAR CD33TEAM (right) and CD70CAR CD19TEAM (left). P-value by unpaired t-test.
  • FIG. 10 shows administration of two different doses of the CD70CAR CD33TEAM CAR T cells to a patient derived xenograph (PDX) AML mouse model.
  • 5xl0 6 AML PDX cells were administered to the mouse at day -4.
  • 5xl0 5 or IxlO 6 CD70CAR CD33TEAM CAR T cells were administered to the mice.
  • Untreated and CD70CAR CD19TEAM were used as controls.
  • tumor cell (CD33+/CD45+) concentration was measured in blood from the mice. Results showed that administering 5xl0 5 and IxlO 6 CD70CAR CD33TEAM CAR T cells had similar effects on tumor cell concentration.
  • FIGs. 11 A-l IE show construction of a CD70-CAR-T cell secreting a CD33 T cell engaging antibody molecule.
  • FIG. 11 A shows NSG mice were engrafted with 5xl0 6 DFAM68555 AML patient derived xenograft cells on day -4. On day 0 mice received 2xl0 6 CD70-targeted CAR-T cells. Most CAR-T cell treated mice cleared tumor, however some mice relapsed with CD70 low tumor.
  • FIG. 1 IB shows CD70 CAR-T cell secreting a CD33- CD3 T cell engaging antibody molecule.
  • FIG. 1 ID shows construct diagrams of CARs used herein.
  • FIGs. 12A-12D show TEAMs are secreted by CAR-T cells and bind to appropriate target antigens.
  • FIG. 12A shows supernatant from the indicated conditions (rows) were added to the indicated cell types (columns) and assessed for anti-His tag (TEAM) binding by flow cytometry.
  • FIG. 12B shows supernatant from Jurkat T cells transduced with the indicated constructs were incubated with plate bound CD33 -expressing K562 tumor cells as well as anti-His tag AF647 antibody. Fluorescent intensity was measured on an Incucyte with representative images as shown.
  • FIG. 12A shows supernatant from the indicated conditions (rows) were added to the indicated cell types (columns) and assessed for anti-His tag (TEAM) binding by flow cytometry.
  • FIG. 12B shows supernatant from Jurkat T cells transduced with the indicated constructs were incubated with plate bound CD33 -expressing K562 tumor cells as well as anti-
  • FIG. 12C shows supernatant from Jurkat T cells transduced with the indicated constructs were co-cultured with Molml3 tumor cells, untransduced (UTD) healthy primary human T cells, and anti-CD69 antibody. Percent of cells expressing CD69 over time are shown.
  • PMA phorbol 12-myristate 13-acetate positive control.
  • FIGs. 13A-13C show simultaneous T-cell redirection through CARs and TEAMs is efficacious against heterogeneous tumors and outperform a mix of individual CARs.
  • FIG. 13A shows individually targeted CAR33 T cells were compared to 70 33 or 70 19 CAR-T cells against engineered Molml3wt, Molml3 CD70KO , Molml3 CD33KO AML targets at a 2: 1 effectortarget ratio.
  • Flow cytometric assessed expression of indicated targets is shown (left) and results of real time cytotoxicity assay (FIG. 13B-top) and CAR-T expansion (FIG. 1 SB- bottom).
  • FIG. 14A-14D shows simultaneous T-cell redirection through CARs and TEAMs outperforms standard CAR-T cells in a patient derived xenograft model of AML.
  • FIG. 14B shows circulating CD33+ tumor cells are killed with higher efficacy by the CD70 CAR-T cell secreting a CD33 TEAM (70 33 ) than the CD70 CAR-T cell secreting a CD19 TEAM (70 19 ).
  • FIG. 14C shows the number of circulating 70 33 and 70 19 CAR-T cells at day +14.
  • FIG. 14D Survival of mice in the experiment. P-value represents log-rank mantel cox.
  • FIGs. 15A-15H show that 70 33 eradicates mixed tumor populations in vivo.
  • BLI bioluminescent
  • FIG. 15B shows peripheral blood expansion of CAR-T cells determined by flow cytometry days 14-28 after CAR-T injection.
  • FIGs. 15C-15D show serial BLI monitoring of tumor growth. P-value represents 2-way ANOVA.
  • FIG. 15B shows peripheral blood expansion of
  • FIG. 15E show peripheral blood CAR-T phenotypic profiling via flow cytometry from the blood of mice at day +21 after CAR-T injection compared to unstimulated and nontransduced, donor matched T-cells. Representative examples shown. CM (central memory), EM (effector memory), TDE (terminal differentiated effectors).
  • FIG. 15F shows FACS sorted CAR-T cells from the spleens of the mice at day +28 from the mixed tumor model underwent gene expression analysis via nanostring with the CAR-T characterization panel in addition to a custom ‘drop-in’ gene set. Volcano plot highlighting differences in transcriptional programs between the constructs.
  • FIG. 15G shows pathway hierarchical clustering as performed in an unbiased manner integrating all available pathways (top). Heatmap shown for selected pathways.
  • FIG. 15H shows pathway score for TCR- signaling is shown for 70 33T and 70CAR 19 . P-value by unpaired t-test.
  • FIGs. 16A-16G show TEAMs effectively redirect AML patients’ T cells to kill tumor cells.
  • FIG. 16A shows clinical characteristics of AML patients whose T cells were utilized.
  • FIG. 16B shows flow T cell phenotyping of AML patient T-cells relative to healthy donor T cells.
  • FIG. 16C shows representative phenotyping and PD-1 expression among AML patients’ T cells.
  • FIG. 16D shows co-culture of TEAM producer cells with isolated AML patients’ T cells and Molml3 tumor targets. Real time cytotoxicity measured on the incucyte.
  • FIG. 16E shows normalized cytokine measurement after 18 hours obtained from patients relative to healthy donor.
  • FIG. 16F shows transwell assay with TEAM producer cells on top and isolated AML patient T cells on bottom with tumor targets. Real time cyototoxicity is measured (top) and activation (CD69) bottom.
  • FIG. 16G shows representative images at 40 hours of activation.
  • FIG. 17 shows simultaneous T-cell redirection through CARs and TEAMs is efficacious against heterogeneous tumors with endogenous antigen expression levels and outperform a mix of individual CARs.
  • Individually targeted CAR33 T cells were compared to 70 33 or 70 19 CAR-T cells against the AML cell lines Kasumil, Monomacl, and OCI-AML3 at a 2: 1 effectortarget ratio.
  • P-values represent 2-way ANOVA of the indicated conditions.
  • FIG. 18 shows pathway hierarchical clustering was performed in an unbiased manner integrating all available pathways (top). Heatmap showing all nanostring available pathways.
  • CARs Chimeric Antigen Receptors
  • the present disclosure provides CAR-T cells targeting the tumor necrosis alpha family member, CD70, and the use of the CAR-T cells for the treatment of hematologic malignancies (e.g., acute myeloid leukemia (AML)).
  • CD70 is consistently expressed on myeloid blasts and leukemic stem cells but is highly restricted expression in healthy human tissues.
  • CD70-targeting CAR-T cells achieved antigen-specific activation, cytokine production, and cytotoxic activity in models of leukemia in vitro and in vivo, e.g., as described in PCT/US2020/051018, which is incorporated by reference in its entirety.
  • CARs chimeric antigen receptors
  • a “chimeric antigen receptor (CAR)” refers to a receptor protein that has been engineered to perform both antigen-binding and cell activating functions.
  • a CAR comprises a plurality of linked domains having distinct functions.
  • CAR domains include those with antigen-binding functions, those with structural functions, and those with signaling functions.
  • a CAR comprises at least an extracellular ligand domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule as defined below.
  • the CAR comprises an optional leader sequence (also referred to as “signal peptide”), an extracellular antigen binding domain, a hinge, a transmembrane domain, and an intracellular stimulatory domain.
  • the domains in the CAR are in the same polypeptide chain, e.g., comprise a chimeric fusion protein. In some embodiments, the domains in the CAR are not contiguous with each other.
  • the CAR described herein comprises an extracellular target binding domain comprising a polypeptide that binds Cluster of Differentiation 70 (CD70).
  • CD70 refers to a polypeptide that is encoded by the human CD70 gene (NCBI Gene ID: 970). As described herein, expression of CD70 is highly restricted in normal human (noncancer) tissues.
  • CD70 is expressed in numerous cancers, for example, bladder cancer, breast invasive carcinoma, cervical cancer, cholangiocarcinoma, colorectal cancer, diffuse large B-cell lymphoma (DLBC), Esophagus, glioblastoma (GBM), head and neck cancer, low-grade gliomas (LGG), liver cancer, lung adeno cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular germ cell cancer, thymoma, thyroid cancer, uterine cancer, uveal melanoma, clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma, papillary renal cell carcinoma (pRCC), acute myeloid leukemia, and adenoid cystic carcinoma (ACC) (Pan-Cancer Atlas 2018).
  • CD70 is a cytokine that contains a cytoplasmic, transmembrane, and extracellular domain
  • the polypeptide that binds CD70 comprises a CD70-binding domain of Cluster of Differentiation 27 (CD27) also called the CD27 antigen.
  • CD27 refers to a polypeptide that is encoded by the human CD27 gene (NCBI GENE ID: 939, Uniprot ID: P26842). An example of the CD27 amino acid sequence is provided below.
  • the CD27 protein has extracellular, transmembrane, and cytoplasmic domains.
  • the CD70 binding domain is located within the extracellular signaling domain of CD27.
  • the extracellular region contains multiple cysteine- rich domains (CRD): CDR1, CDR2, and CDR3.
  • CRD2 domain In some embodiments, the CD70 binding domain is located within the CRD2 domain.
  • the CD70-binding domain in CD27 comprises a peptide comprising the amino acid sequence of TRPHCESCRHCN (SEQ ID NO: 9) that is located in the extracellular domain of CD27.
  • the extracellular targeting binding domain of the CAR described herein comprises a polypeptide comprising an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical) to the amino acid sequence of SEQ ID NO: 9.
  • the extracellular targeting binding domain of the CAR described herein comprises the amino acid sequence of SEQ ID NO: 9.
  • the extracellular targeting binding domain of the CAR described herein comprises a polypeptide comprising the extracellular domain of CD27 or a functional fragment thereof (e.g., a fragment capable of binding CD70).
  • a functional fragment comprises SEQ ID NO: 9, or a variant thereof.
  • the extracellular targeting binding domain of the CAR described herein comprises a polypeptide comprising an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical) to the amino acid sequence of SEQ ID NO: 1, 8 or 9.
  • the extracellular targeting binding domain of the CAR described herein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
  • the polypeptide that binds CD70 in the extracellular targeting binding domain of the CAR described herein comprises an anti-CD70 antibody.
  • antibody used herein encompasses antibodies of different formats and antibody fragments.
  • antibody includes but is not limited to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single-chain variable fragment (scFV), a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen binding domain, such as a recombinant fibronectin domain, and the like.
  • scFV single-chain variable fragment
  • VHH variable domain
  • the antigen binding domain it is beneficial for the antigen binding domain to be derived from the same species in which the CAR will ultimately be used in. For example, for use in humans, it may be beneficial for the antigen binding domain of the CAR to comprise human or humanized residues for the antigen binding domain of an antibody or antibody fragment.
  • the polypeptide that binds CD70 in the extracellular targeting binding domain of the CAR described herein comprises a scFv that binds to CD70.
  • the CD70 antibody comprises an antibody or antigen binding domain (e.g., CDRs or VH and VL) as described in US11434298, US7491390, US8124738, US11377500, or US9701752, each of which is incorporated by reference in its entirety.
  • an antibody or antigen binding domain e.g., CDRs or VH and VL
  • the antibody is a human antibody or an antibody fragment. In some embodiments, the antibody a humanized antibody or an antibody fragment.
  • a humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g., European Patent Nos.
  • framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.)
  • a humanized antibody or antibody fragment has one or more amino acid residues remaining in it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • humanized antibodies or antibody fragments comprise one or more CDRs from non-human immunoglobulin molecules and framework regions wherein the amino acid residues comprising the framework are derived completely or mostly from human germline.
  • the antibody is derived from a display library.
  • a display library is a collection of entities; each entity includes an accessible polypeptide component and a recoverable component that encodes or identifies the polypeptide component.
  • the polypeptide component is varied so that different amino acid sequences are represented.
  • the polypeptide component can be of any length, e.g., from three amino acids to over 300 amino acids.
  • a display library entity can include more than one polypeptide component, for example, the two polypeptide chains of a Fab.
  • a display library can be used to identify an antigen binding domain. In a selection, the polypeptide component of each member of the library is probed with the antigen, or a fragment there, and if the polypeptide component binds to the antigen, the display library member is identified, typically by retention on a support.
  • Retained display library members are recovered from the support and analyzed.
  • the analysis can include amplification and a subsequent selection under similar or dissimilar conditions. For example, positive and negative selections can be alternated.
  • the analysis can also include determining the amino acid sequence of the polypeptide component and purification of the polypeptide component for detailed characterization.
  • a variety of formats can be used for display libraries. Examples include the phage display.
  • the protein component is typically covalently linked to a bacteriophage coat protein.
  • the linkage results from translation of a nucleic acid encoding the protein component fused to the coat protein.
  • the linkage can include a flexible peptide linker, a protease site, or an amino acid incorporated as a result of suppression of a stop codon.
  • Phage display is described, for example, in U.S. Pat. No.
  • Bacteriophage displaying the protein component can be grown and harvested using standard phage preparatory methods, e.g. PEG precipitation from growth media. After selection of individual display phages, the nucleic acid encoding the selected protein components can be isolated from cells infected with the selected phages or from the phage themselves, after amplification. Individual colonies or plaques can be picked, the nucleic acid isolated and sequenced.
  • display formats include cell based display (see, e.g., WO 03/029456), protein-nucleic acid fusions (see, e.g., U.S. Pat. No. 6,207,446), ribosome display, and E. coli periplasmic display.
  • the transmembrane domain of the CARs described herein may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to a target.
  • a transmembrane domain of particular use in this invention may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • CD8 e.g., CD8 alpha, CD8 beta
  • CD9 CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • a transmembrane domain may include at least the transmembrane region(s) of, e g., KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD 103, rfGAL, CD 11 a, LFA-1, ITGAM, CD 11b, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2,
  • the transmembrane domain is a CD28 transmembrane domain or CD8 transmembrane domain. In some embodiments, transmembrane domain is the transmembrane domain of CD27. In some embodiments, the transmembrane domain of CD27 comprises an amino acid sequence of ILVIFSGMFLVFTLAGALFL (SEQ ID NO: 10).
  • the transmembrane domain can be attached to the extracellular region of the CAR, e.g., the ligand domain of the CAR, via a hinge, e.g., a hinge from a human protein.
  • a hinge e.g., a hinge from a human protein.
  • the hinge can be a human Ig (immunoglobulin) hinge, e.g., an IgG4 hinge, or a CD8a hinge.
  • the cytoplasmic domain or region of the CAR described herein includes one or more intracellular signaling domains.
  • An intracellular signaling domain is capable of activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced.
  • Examples of intracellular signaling domains for use in the CAR described herein include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.
  • TCR T cell receptor
  • T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic domain, e.g., a costimulatory domain).
  • intracellular signaling domain refers to an intracellular portion of a molecule.
  • the intracellular signaling domain can generate a signal that promotes an immune effector function of the CAR containing cell, e.g., a CAR T cell or CAR-expressing NK cell.
  • immune effector function e.g., in a CAR T cell or CAR-expressing NK cell
  • examples of immune effector function include cytolytic activity and helper activity, including the secretion of cytokines.
  • the intracellular signal domain transduces the effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain.
  • intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • the one or more intracellular signaling domains comprise a primary intracellular signaling domain.
  • exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation.
  • a primary intracellular signaling domain comprises a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • IT AM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 theta, CD3 eta, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FceRI, CD66d, DAP10, and DAP12.
  • the intracellular signaling domain of the CAR comprises a CD3-zeta (CD3Q signaling domain.
  • the CD3-zeta (CD3Q signaling domain comprises the amino acid sequence of: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO: 11).
  • the CD3-zeta (CD3Q signaling domain of the CAR described herein comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical) to the amino acid sequence of SEQ ID NO: 11.
  • the one or more intracellular signaling domain comprise a costimulatory intracellular domain.
  • a costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule.
  • the intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.
  • Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals (e.g., antigen independent stimulation), and those derived from cytokine receptors.
  • the one or more intracellular signaling domains comprise a primary intracellular signaling domain, and a costimulatory intracellular signaling domain from one or more co-stimulatory proteins or cytokine receptors.
  • costimulatory molecule refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response.
  • Examples of such molecules include a MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD1 la/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, IT
  • the co-stimulatory domain of the CARs described herein comprises one or more signaling domains from one or more co-stimulatory protein or cytokine receptor selected from CD28, 4- IBB, 2B4, KIR, CD27, 0X40, ICOS, MYD88, IL2 receptor, and SynNotch.
  • the co- stimulatory domain of the CARs described herein comprises a 4-1BB costimulatory signaling domain.
  • the 4- IBB co-stimulatory signaling domain comprises the amino acid sequence of: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 12).
  • the 4-1BB co-stimulatory signaling domain of the CAR described herein comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical) to the amino acid sequence of SEQ ID NO: 12.
  • the intracellular signaling domain of the CAR described herein comprise the primary signaling domain, e.g., an ITAM containing domain such as a CD3-zeta signaling domain, by itself or combined with a costimulatory signaling domain (e.g., a costimulating domain from one or more co-stimulatory protein or cytokine receptor selected from CD28, 4- IBB, 2B4, KIR, CD27, 0X40, ICOS, MYD88, IL2 receptor, and SynNotch).
  • the intracellular signaling domain of the CAR described herein comprise a CD3-zeta (CD3Q signaling domain and a 4-1BB costimulatory signaling domain.
  • different linker sequences may be used between the different domains of the CAR, e.g., a (GGGS)n (SEQ ID NO: 35) linker, wherein n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 ,13, 14, 15, 16, 17, 18, 19, or 20).
  • the linker is (GGGS)? (SEQ ID NO: 36).
  • the CAR comprises additional sequences from CD27, e.g., the stalk and hinge region of CD27, between the extracellular target binding domain and the transmembrane region.
  • the stalk and hinge region of CD27 comprises the amino acid sequence of: PLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHW PPQRSLCSSDFIR (SEQ ID NO: 13).
  • the CAR does not comprise additional sequences from CD27, e.g., the stalk and hinge region of the between the extracellular target binding domain and the transmembrane region.
  • the CAR described herein comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical) to the amino acid sequence of any one of SEQ ID NOs: 2-7. In some embodiments, the CAR described herein comprises the amino acid sequence of any one of SEQ ID NOs: 2-7.
  • the CARs described herein further comprises a leader sequence (also referred herein to as a signal peptide) at the amino-terminus (N-terminus) of the antigen binding domain.
  • the CAR further comprises a leader sequence at the N-terminus of the antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.
  • the leader sequence is a CD27 signal peptide (e.g., a peptide having the amino acid sequence of: MARPHPWWLCVLGTLVGLS (SEQ ID NO: 14))
  • the leader sequence is an interleukin 2 signal peptide or a CD8 leader sequence.
  • the leader sequence comprises an amino acid sequence of: MALPVTALLLPLALLLHAARP (SEQ ID NO: 15).
  • the CARs described herein further comprises additional amino acid sequences (e.g., between the extracellular target binding domain and the leader sequence.
  • the additional sequence is an affinity tag (e.g., a Myc tag, EQKLISEEDL (SEQ ID NO: 16)).
  • TEAM T-cell engaging antibody molecule
  • the present application discloses cells (e.g., T cells) comprising a CAR that binds CD70 (CD70 CAR) (e.g., as described above) and TEAM that binds CD33 (CD33 TEAM).
  • a TEAM as described herein, is a molecule comprising a first binding moiety (e.g., an anti-CD33 antibody) and a second binding moiety (e.g., an immune cell binding moiety).
  • the CD33 TEAM helps overcome cancer antigen escape (e.g., the cancer cell no longer expresses the molecule targeted by the CAR) by providing a secondary binding site (i.e., CD33) for engaging immune cells to the cancer.
  • the CD33 TEAM engages T cells to the cancer.
  • the CD33 TEAM may engage both the CAR-T cell and native T cell to the cancer cells.
  • CAR-T cell expressing a CD70 CAR and a CD33 TEAM have increased efficacy in treating relapsed cancer cells that have CD70 loss.
  • the CD33 TEAM comprises an anti-CD33 antibody and an immune cell binding moiety.
  • the anti-CD33 antibody includes but is not limited to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single-chain variable fragment (scFv), a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen binding domain, such as a recombinant fibronectin domain, and the like.
  • scFv single-chain variable fragment
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VHH variable domain
  • the anti-CD33 antibody comprises from N-terminal to C-terminal a VH domain and then a VL domain. In some embodiments, the anti-CD33 antibody comprises from N-terminal to C-terminal a VL domain and then a VH domain. In some embodiments, the CD33 antibody is an scFv. In some embodiments, the CD33 antibody comprises a VL domain of SEQ ID NO: 19 or a variant thereof, and a VH domain of SEQ ID NO: 20 or a variant thereof.
  • the anti-CD33 antibody comprises any one of the antibodies or antigen binding domains (e.g., CDRs or VH and VL) as described in US 11136390, US10556951, US10787514, US8759494, US10000566, US11174313, US10711062, US11466082, and US20210317208, each of which is incorporated by reference in it’s entirety.
  • CDRs or VH and VL antibodies or antigen binding domains
  • the immune cell binding moiety is a molecule that binds to a protein expressed on the surface of a T-cell, an NK cell, a dendritic cell, a macrophage, a B cell, a neutrophil, an eosinophil, a basophil, a mast cell, a myeloid-derived suppressor cell, a mesenchymal stem cell, or combinations thereof, or any precursor, derivative, or progenitor cells thereof.
  • the immune cell binding moiety is a molecule that binds to a protein expressed on the surface of a T Cell (e.g., CD3).
  • the immune cell binding moiety binds to a cell surface marker of a T Cell.
  • the immune cell binding moiety is selected from the group consisting of CD3, CD8, CD4, CXCR3, CCR4, GARP, LAP, CD25, CTLA-4, or CD16.
  • the immune cell binding moiety is an antibody (e.g., an antibody that binds to a protein expressed on the surface of a T-Cell) as described herein.
  • immune cell binding moiety includes but is not limited to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single-chain variable fragment (scFv), a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen binding domain, such as a recombinant fibronectin domain, and the like.
  • the immune cell binding moiety is an scFv.
  • the immune cell binding moiety is an anti- CD3 antibody.
  • the anti-CD3 antibody comprises from N-terminal to C-terminal a VH domain and then a VL domain.
  • the anti-CD33 antibody comprises from N-terminal to C-terminal a VL domain and then a VH domain.
  • the immune cell binding moiety comprises an amino acid sequence of SEQ ID NO: 21 (anti-CD3 scFv) or a variant thereof.
  • the anti-CD3 antibody comprises any one of the antibodies or antigen binding domains (e.g., CDRs or VH and VL) as described in US9657102, US20210244815, US11530275, US10759858, US20220380464, US20210253701, and US11505606.
  • CDRs or VH and VL antibodies or antigen binding domains as described in US9657102, US20210244815, US11530275, US10759858, US20220380464, US20210253701, and US11505606.
  • the CD33 TEAM comprises a linker between the CD33 antibody and the immune cell binding moiety.
  • the linker is a peptide linker.
  • the linker is non-cleavable.
  • the linker is a glycine linker.
  • the linker is a GlySer linker.
  • the GlySer linker comprises the amino acid sequence GGGS (SEQ ID NO: 34).
  • the GlySer linker comprises the amino acid sequence (GGGS)2 (SEQ ID NO: 37), (GGGS)3 (SEQ ID NO: 26), (GGGS)4 (SEQ ID NO: 38), (GGGS)5 (SEQ ID NO: 39), (GGGS)6 (SEQ ID NO: 40).
  • the GlySer linker comprises the amino acid sequence (GGGGS)2 (SEQ ID NO: 41), (GGGGS)3 (SEQ ID NO: 27), (GGGGS)4 (SEQ ID NO: 28), (GGGGS)5 (SEQ ID NO: 42), (GGGGS)6 (SEQ ID NO: 43).
  • the linker comprises an amino acid sequence of any one of SEQ ID NOs: 26- 30. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NOs: 26. In some embodiments, the linker is any linker described herein.
  • the CD33 TEAM comprises a signal peptide.
  • the signal peptide is an IgK signal peptide, a CD8 signal peptide, or a CD27 signal peptide.
  • the signal peptide comprises the amino acid sequence of any one of SEQ ID NOs: 14, 15 or 33, or including the amino acid sequence of any one of SEQ ID NOs: 14, 15 or 33, or including an amino acid sequence having at least 75%, at least 80%, at least 85%, 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 at least 100% sequence identity to the sequence of any one of SEQ ID NOs: 14, 15 or 33.
  • the CD33 TEAM comprises a CD33 scFv, a non-cleavable linker, and a CD3 scFv. In some embodiments, the CD33 TEAM comprises a CD33 scFv, a non-cleavable linker, a CD3 scFv, a His6 tag, a T2A self-cleavable sequence, and a fluorescent protein (e.g., mCherry).
  • a fluorescent protein e.g., mCherry
  • the CD33 TEAM is codon-optimized.
  • Codon-optimization is a process of introducing silent mutations into a nucleic acid sequence encoding a protein (e.g., a nucleic acid sequence encoding a CD33 TEAM) that improve the expression of the protein (e.g., the CD33 TEAM). Codon-optimization does not alter the amino acid sequence of the protein, Methods of codon optimization are well known in the art, e.g., as described in Mauro et al., BioDrugs 32.1 (2016): 69-81 and .
  • the CD33 TEAM comprises an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to any one of SEQ ID NOs: 17-18.
  • the CD33 TEAM comprises an amino acid sequence of any one of SEQ ID NOs: 17-18.
  • the CD33 TEAM consists of an amino acid sequence of any one of SEQ ID NOs: 17-18.
  • the CD70 CAR CD33 TEAM construct comprises a CD70 CAR as described herein and a CD33 TEAM as described herein.
  • a construct, as used herein, refers to a nucleic acid sequence or an amino acid sequence that comprises one or more components (e.g. comprises a CD70 CAR and a CD33 TEAM).
  • the CD70 CAR - CD33 TEAM construct comprises a CD70 CAR comprising CD27 or a fragment thereof that is capable of binding to CD70 as described herein.
  • the CD70 CAR - CD33 TEAM construct comprises a linker between the CD70 CAR and the CD33 team. In some embodiments, the linker is a cleavable linker.
  • the cleavable linker is a protease cleavable linker.
  • the linker is a self-cleavable linker (e.g., P2A (SEQ ID NO: 32), E2A, F2A, or T2A (SEQ ID NO: 31)).
  • the linker comprises an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • the CD33 TEAM may be secreted from the cell and may bind to cancer cells expressing CD33 and to immune cells (e.g.,CAR-T cells, untransduced T cells, endogenous T cells, and/or bystander T Cells expressing CD3).
  • the CD33 TEAM may activate CAR-T cells, untransduced T cells, endogenous T cells, and/or bystander T Cells expressing CD3.
  • the CD70 CAR and the CD33 TEAM are each operably linked to a promoter, e.g., a promoter as described herein. In some embodiments, the CD70 CAR and the CD33 TEAM are operably linked to the same promoter. In some embodiments, the CD70 CAR and the CD33 TEAM are operably linked to different promotors. In some embodiments, the promoters are constitutive promoters and described herein. In some embodiments, the promoters and inducible promoters and describe herein. In some embodiments, the CD70 CAR CD33 TEAM is operably linked to an EFlalpha promoter.
  • the CD70 CAR CD33 TEAM construct comprises from N- terminal to C-terminal a CD70 CAR (e.g. CD27 CAR), a cleavable linker (e.g. P2A), and a CD33 TEAM.
  • the CD70 CAR CD33 TEAM construct comprises from N-terminal to C-terminal a CD33 TEAM, a cleavable linker (e.g. P2A), and a CD70 CAR (e.g. CD27 CAR).
  • the CD70 CAR CD33 TEAM comprises a truncated CD27 CAR and a CD33 TEAM.
  • the CD70 CAR CD33 TEAM comprises from N-terminal to C-Terminal a truncated CD27 (trCD27), a CD8 Hinge, a CD8 transmembrane, a 4-1BB co-stimulatory signaling domain, a CD3-zeta domain, a P2A cleavable peptide, an IgK leader, a CD33 scFv and a CD3 scFv.
  • the CD70 CAR CD33 TEAM comprises from N-terminal to C-Terminal a truncated CD27 (trCD27), a CD8 Hinge, a CD8 transmembrane, a 4-1BB intracellular signaling domain, a CD3-zeta signaling domain, a P2A cleavable peptide, an IgK leader, a CD33 scFv, a non- cleavable linker, and a CD3 scFv.
  • the CD70 CAR CD33 TEAM comprises from N-terminal to C-Terminal a truncated CD27 (trCD27), a CD8 Hinge, a CD8 transmembrane, a 4- IBB intracellular signaling domain, a CD3-zeta domain, a P2A cleavable peptide, an IgK leader, a CD33 scFv, a non-cleavable linker, a CD3 scFv, a His6 tag, a T2A cleavable peptide, and a fluorescent protein (e.g., mCherry).
  • trCD27 truncated CD27
  • CD8 Hinge CD8 Hinge
  • CD8 transmembrane a 4- IBB intracellular signaling domain
  • CD3-zeta domain a P2A cleavable peptide
  • an IgK leader a CD33 scFv
  • the CD70 CAR CD33 TEAM construct comprises an amino acid sequence that is at least at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 22-25. In some embodiments, the CD70 CAR CD33 TEAM construct comprises an amino acid sequence of any one of SEQ ID NOs: 22-25. In some embodiments, the CD70 CAR CD33 TEAM construct comprises an amino acid sequence of any one of SEQ ID NOs: 22-23. In some embodiments, the CD70 CAR CD33 TEAM construct consists of an amino acid sequence of any one of SEQ ID NOs: 22-25.
  • the disclosure provides nucleic acid molecules (e.g., vectors) for expressing CD70 CAR CD33 TEAM constructs in cells, e.g., T cells.
  • the nucleic acid molecule comprises a nucleotide sequence encoding the CD70 CAR CD33 TEAM constructs described herein.
  • the nucleic acid sequences coding for the desired molecules 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.
  • the gene of interest can be produced synthetically, rather than cloned.
  • the desired CD70 CAR CD33 TEAM constructs can be expressed in the cells by way of transposons.
  • expression of natural or synthetic nucleic acids CARs is typically achieved by operably linking a nucleic acid encoding the CAR to a promoter, and incorporating the construct into an expression vector.
  • the vectors can be suitable for replication and integration into eukaryotes.
  • Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the expression constructs of the disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties.
  • promoter elements e.g., enhancers
  • promoters regulate the frequency of transcriptional initiation.
  • these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • Another example of a suitable promoter is Elongation Factor-la (EF-la).
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the disclosure is not limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the disclosure.
  • an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • the promoter is an EF-la promoter.
  • the nucleic acid comprising a nucleotide sequence encoding the CD70 CAR CD33 TEAM construct described herein is a vector.
  • the nucleic acid can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retrovirus vectors are used.
  • lentivirus vectors are used.
  • adeno- associated virus (AAV) vectors can also be used.
  • Retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • a "lentivirus” as used herein refers to a genus of the Retroviridae family.
  • Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.
  • transfection or transformed or transduced
  • transfection or transformation
  • transduction or electroporation
  • transfected or transformed or transduced
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • this application discloses cells comprising a CD70 CAR and a CD33 TEAM as described herein.
  • the cell comprises a CD70 CAR - CD33 TEAM construct, as described herein.
  • the cells are immune cells.
  • the immune cell is a mammalian immune cell. In some embodiments, the immune cell is a human immune cell.
  • an “immune cell” can be a T-cell, an NK cell, a dendritic cell, a macrophage, a B cell, a neutrophil, an eosinophil, a basophil, a mast cell, a myeloid-derived suppressor cell, a mesenchymal stem cell, or combinations thereof, or any precursor, derivative, or progenitor cells thereof.
  • the immune cell is a T cell. In some embodiments, the immune cell is a human T cell.
  • Immune cells e.g., T cells
  • T 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.
  • the immune cells e.g., T cells
  • any number of immune cell lines including but not limited to T cell lines, including, for example, Hep-2, Jurkat, and Raji cell lines, available in the art, may be used.
  • immune cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FicollTM separation.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, NK 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 steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations.
  • initial activation steps in the absence of calcium lead to magnified activation.
  • a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated "flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions.
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca 2+ -free, Mg 2+ -free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • buffers such as, for example, Ca 2+ -free, Mg 2+ -free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • immune 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 T cells such as CD3 + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO + T cells, can be further isolated by positive or negative selection techniques.
  • Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method 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 CD 14, CD20, CD1 lb, CD 16, HLA-DR, and CD8.
  • it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4 + , CD25 + , CD62L hi , GITR + , and FoxP3 + .
  • T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.
  • the engineered immune cells may be autologous. Being “autologous” means the immune cells are obtained from a subject, engineered to express a CAR described herein, and administered to the same subject. Administration of autologous cells to a subject may result in reduced rejection of the immune cells as compared to administration of non- autologous cells.
  • the engineered immune cells e.g., T cells
  • allogeneic immune cells may be derived from a human donor and administered to a human recipient who is different from the donor.
  • this application discloses a CAR-T Cell comprising a CD70 CAR (e.g. CD27 extracellular binding domain) and a CD33 TEAM (e.g. comprising an anti- CD33 antibody and an anti-CD3 antibody) as described herein.
  • the CAR-T cell comprises a CD70 CAR - CD33 TEAM construct encoding an amino acid sequence of any one of SEQ ID NOs: 22-25.
  • this application discloses methods of treating cancer (e.g. cancer characterized by cells expressing CD70 and CD33) comprising administering an effective amount of a cell (e.g. a CAR-T cell comprising a CD70 CAR and a CD33 TEAM) to a subject.
  • a cell e.g. a CAR-T cell comprising a CD70 CAR and a CD33 TEAM
  • the method is for treating a cancer characterized by cells expressing CD70, and the method comprises administering to a subject in need thereof an effective amount of the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) or the compositions comprising the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) described herein.
  • the method is for treating a cancer characterized by cells expressing CD70 and cells that express a lower level of CD70 (i.e., lower than the level of the aforementioned cancer cells expressing CD70) or that do not express CD70, and the method comprises administering to a subject in need thereof an effective amount of the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) or the compositions comprising the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) described herein.
  • the method comprises administering CD70 CAR CD33 TEAM CAR-T cells to a subject having or diagnosed as having a cancer that express CD70.
  • the method comprises administering CD70 CAR CD33 TEAM CAR-T cells to a subject having cancer cells that express CD70. In some embodiments, the method comprises administering CD70 CAR CD33 TEAM CAR-T cells to a subject having cancer cells that express CD70 and CD33. In some embodiments, the method comprises administering CD70 CAR CD33 TEAM CAR-T cells to a subject having cancer cells that express CD33. In some embodiments, the method comprises administering CD70 CAR CD33 TEAM CAR-T cells to a subject having cancer cells that are resistant to CD70 CAR-based treatments (e.g., CD70 expression is decreased or a mutation decreases CD70 CAR binding to CD70 of the cancer cell).
  • the method comprises administering CD70 CAR CD33 TEAM CAR-T cells to a subject having cancer cells that have decreased CD70 expression.
  • Decreased CD70 expression is decreased relative to a cancer cell that express CD70 at an amount that is sufficient for CD70 CAR T Cell directed killing of the cancer cell (e.g., a CD70 expressing cancer cell that has not yet been treated with an anti-CD70 therapeutic).
  • decreased CD70 expression is at least 0.1% (e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%) decreased compared to a CD70 expressing cancer cell that has not yet been treated with an anti-CD70 therapeutic (e.g., a CD70 CAR) and/or a CD70 expressing cancer cell that is expected to be killed by an anti-CD70 CAR T Cell.
  • an anti-CD70 therapeutic e.g., a CD70 CAR
  • decreased CD70 expression is at least 0.1% (e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%) decreased compared to CD70 expression of a Molml3 WT cell.
  • decreased CD70 expression refers to no detectable CD70 expression (e.g., no detectable CD70 cell surface expression).
  • the method comprises administering the CD70 CAR CD33 TEAM CAR-T Cells described herein to a subject having a cancer that expresses CD70.
  • the method of administering the CD70 CAR CD33 TEAM CAR-T Cells described herein to a subject having a cancer that expresses CD70 includes administering one or more rounds of CD70 CAR CD33 TEAM CAR-T Cells to cancers cells that have decreased CD70 expression.
  • the method comprises administering a CD70 CAR CAR-T Cell to a subject having a cancer that expresses CD70, and, if the cancer decreases CD70 expression or otherwise becomes resistant to the CD70 CAR-T cells, the method further comprises administering CD70 CAR CD33 TEAM CAR-T Cells.
  • treating refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease, a symptom of the target disease, or a predisposition toward the target disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of the disease, or the predisposition toward the disease.
  • treatment of cancer in a subject using CD70 CAR CD33 TEAM CAR-T Cells does not result systemic toxicity. In some embodiments, treatment of cancer in a subject using CD70 CAR CD33 TEAM CAR-T Cells does not result in CD33 TEAM systemic toxicity. In some embodiments, treatment of cancer in a subject using CD70 CAR CD33 TEAM CAR-T Cells does not result hepatotoxicity. In some embodiments, treatment of cancer in a subject using CD70 CAR CD33 TEAM CAR-T Cells does not result in CD33 TEAM hepatotoxicity.
  • the method is for treating a cancer characterized by cells expressing CD70, and the method comprises administering to a subject in need thereof an effective amount of the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) or the compositions comprising the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) described herein, and an effective amount of an agent that enhances expression of CD70 in the cancer (e.g., azacitidine or decitabine).
  • the engineered immune cells e.g., CD70 CAR CD33 TEAM CAR-T cells
  • an agent that enhances expression of CD70 in the cancer e.g., azacitidine or decitabine.
  • cancers characterized by cells that express CD70 include, without limitation, bladder cancer, breast invasive carcinoma, cervical cancer, cholangiocarcinoma, colorectal cancer, diffuse large B-cell lymphoma (DLBC), Esophagus, glioblastoma (GBM), head and neck cancer, low-grade gliomas (LGG), liver cancer, lung adeno cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular germ cell cancer, thymoma, thyroid cancer, uterine cancer, uveal melanoma, clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma, papillary renal cell carcinoma (pRCC), acute myeloid leukemia, and adenoid cystic carcinoma (ACC).
  • bladder cancer breast invasive carcinoma, cervical cancer, cholangiocarcinoma, colorectal cancer, diffuse large B-cell lymphoma
  • the cancer is a lymphoma.
  • the lymphoma is a B-cell Non-Hodgkin Lymphoma (NHL), mantle cell lymphoma, Burkitt’s lymphoma, B-cell lymphoblastic lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma, or T-cell lymphoma.
  • the cancer is a leukemia.
  • the leukemia is acute myeloid leukemia (AML), small lymphocytic lymphoma (SLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), B-cell lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), or T- cell leukemia.
  • AML acute myeloid leukemia
  • SLL small lymphocytic lymphoma
  • CML chronic myeloid leukemia
  • ALL acute lymphocytic leukemia
  • B-cell lymphoblastic leukemia B-cell lymphoblastic leukemia
  • chronic lymphocytic leukemia CLL
  • T- cell leukemia T- cell leukemia.
  • the cancer is a myeloid cancer. In some embodiments, the cancer is acute myeloid leukemia.
  • compositions comprising any one of the immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) described herein.
  • the composition comprising the engineered immune cells e.g., CD70 CAR CD33 TEAM CAR-T cells
  • the agent results in hypomethylation of CD-70 encoding gene in the cancer.
  • the agent is azacitidine or decitabine.
  • the composition comprises the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) and azacitidine.
  • the composition comprises the engineered immune cells (e.g., CD70-targeting CAR-T cells) and azacitidine, wherein azacitidine has a concentration of 100 pM or less (e.g., 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, 10 pM or less, 5 pM or less,l pM or less) in the composition.
  • azacitidine has a concentration of 100 pM or less (e.g., 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, 10 pM or less, 5 pM or less,l pM or less) in the
  • the composition comprises the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) and azacitidine, wherein azacitidine has a concentration of 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM in the composition.
  • engineered immune cells e.g., CD70 CAR CD33 TEAM CAR-T cells
  • azacitidine has a concentration of 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM in the composition.
  • the composition is a pharmaceutical composition.
  • the composition further comprises a pharmaceutically acceptable carrier, excipients or stabilizers typically employed in the art (all of which are termed “excipients”), for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and/or other miscellaneous additives.
  • excipients typically employed in the art (all of which are termed “excipients”), for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and/or other miscellaneous additives.
  • any one of the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) described herein or any one of the compositions comprising the engineered immune cells described herein is administered to a subject. Accordingly, some aspects of the present disclosure provide methods of administering to a subject any one of the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) or the compositions comprising the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) described herein.
  • the engineered immune cells e.g., CD70 CAR CD33 TEAM CAR-T cells
  • the agent e.g., azacitidine or decitabine
  • the engineered immune cell and the agent are formulated in a composition for administration.
  • the composition comprises the engineered immune cells (e.g., CD70-targeting CAR-T cells) and azacitidine, wherein azacitidine has a concentration of 100 pM or less (e.g., 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, 10 pM or less, 5 pM or less, 1 pM or less) in the composition.
  • azacitidine has a concentration of 100 pM or less (e.g., 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, 10 pM or less, 5 pM or less, 1 pM or less) in the
  • the composition comprises the engineered immune cells (e.g., CD70-targeting CAR-T cells) and azacitidine, wherein azacitidine has a concentration of 100 pM, 90 pM, 80 pM, 70 pM, 60 pM s, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM in the composition.
  • engineered immune cells e.g., CD70-targeting CAR-T cells
  • azacitidine has a concentration of 100 pM, 90 pM, 80 pM, 70 pM, 60 pM s, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM in the composition.
  • the engineered immune cells e.g., CD70 CAR CD33 TEAM CAR-T cells
  • the agent e.g., azacitidine or decitabine
  • the engineered immune cells e.g., CD70-targeting CAR-T cells
  • the waiting period is for the agent (e.g., azacitidine or decitabine) to enhance CD70 expression in the cancer and to clear out of the subject before the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) are administered.
  • the waiting period is 3 hours or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 12, 24 hours or more).
  • the agent e.g., azacitidine or decitabine
  • the agent enhances CD70 expression in the cancer by at least 10% (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, or more), compared to the same cancer without exposure to the agent (e.g., azacitidine or decitabine).
  • administering both the engineered immune cells (e.g., CD70- targeting CAR-T cells) and the agent (e.g., azacitidine or decitabine) to the subject enhances the therapeutic efficacy by at least at least 10% (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, or more), compared to when the engineered immune cells (e.g., CD70-targeting CAR-T cells) or the agent (e.g., azacitidine or decitabine) is administered alone.
  • Therapeutic efficacy may be measured by methods known in the art, e.g., clearance of cancer cells, prolonged survival of the subject.
  • an effective amount of the engineered immune cells e.g., CD70 CAR CD33 TEAM CAR-T cells
  • the agent that enhances CD70 expression in the cancer e.g., azacitidine or decitabine
  • a suitable route e.g., intravenous infusion
  • the immune cell population may be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition prior to administration, which is also within the scope of the present disclosure.
  • the subject to be treated may be a mammal (e.g., human, mouse, pig, cow, rat, dog, guinea pig, rabbit, hamster, cat, goat, sheep or monkey).
  • the subject may be suffering from cancer or an immune disorder (e.g., an autoimmune disease).
  • an effective amount refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more active agents.
  • Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, gender and weight, the duration of treatment, route of administration, excipient usage, co-usage (if any) with other active agents and like factors within the knowledge and expertise of the health practitioner.
  • the quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to produce a cell-mediated immune response. Precise mounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art.
  • the therapeutic methods described herein may be utilized in conjunction with other types of therapy for cancer, such as chemotherapy, surgery, radiation, gene therapy, and so forth.
  • Such therapies can be administered simultaneously or sequentially (in any order) with the immunotherapy described herein.
  • suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.
  • Non-limiting examples of other anti-cancer therapeutic agents useful for combination with the modified immune cells described herein include, but are not limited to, immune checkpoint inhibitors (e.g., PDL1, PD1, and CTLA4 inhibitors), anti -angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin- 1, tissue inhibitors of metalloproteases, prolactin, angiostatin, endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, and placental proliferin-related protein); a VEGF antagonist (e.g., anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments); chemotherapeutic compounds.
  • immune checkpoint inhibitors e.g., PDL1, PD1, and CTLA4 inhibitors
  • anti -angiogenic agents e.g., TNP-470, platelet factor 4, thrombospondin- 1, tissue inhibitors of
  • chemotherapeutic compounds include pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine); purine analogs (e.g., fludarabine); folate antagonists (e.g., mercaptopurine and thioguanine); antiproliferative or antimitotic agents, for example, vinca alkaloids; microtubule disruptors such as taxane (e.g., paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, and epidipodophyllotoxins; DNA damaging agents (e.g., actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin,
  • radiation, or radiation and chemotherapy are used in combination with the cell populations comprising modified immune cells described herein. Additional useful agents and therapies can be found in Physician's Desk Reference, 59.sup.th edition, (2005), Thomson P D R, Montvale N.J.; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20.sup.th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15. sup. th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.
  • AML Acute myeloid leukemia
  • intensive chemotherapy is now curative in forty percent of cases of adults 1 . While this represents a substantial improvement, there remains a significant unmet clinical need for older and relap sed/refractory patients where cure rates rapidly fall below ten percent 1 .
  • Treatment of AML had changed little over fifty years since the advent of intensive “induction” cytotoxic chemotherapy, however, since 2017 there have been eight FDA drug approvals including for inhibitors of hedgehog, BCL-2, FLT3, IDH1/2, a CD33 antibody drug conjugate, as well as a more potent liposomal formulation of induction chemotherapy 2 . While these interventions represent substantial progress, the majority of AML patients still fail to respond or relapse and die from their disease.
  • CAR-T cell therapy has rapidly revolutionized the treatment of lymphoid malignancies with two FDA approvals in 2017 for aggressive B-cell malignancies 10 11 and now four total. These therapies result in destruction of malignant clones, however, there is also an on target, off tumor effect resulting in the elimination of normal B-cells, causing hypogammaglobulinemia which, notably, is manageable via administration of intravenous immunoglobulin.
  • CD70 is a tumor necrosis alpha family member that serves as the ligand for CD27 which is involved in T-cell signaling.
  • CD70 has an extremely restricted expression on normal tissues but marked overexpression in a number of cancer types, including AML 19,20 .
  • AML drug targeting CD70, ARGX-110, an antibody drug conjugate (ADC) has shown impressive response rates in a phase I trial of newly diagnosed AML patients not fit for traditional therapy 21 .
  • ADC antibody drug conjugate
  • TEAM bispecific T-cell engaging antibody molecule
  • CARTEAM CAR-T cells
  • This technique has the added advantage of leveraging the non-transduced — but infused— T-cells representing typically >70% of all infused cells for clinical grade CAR-T cell products (due to the need for low MOIs to preclude multiple viral integration events) via the TEAM secreted from the CAR.
  • TEAM bispecific T-cell engaging antibody molecule
  • CARTEAM CAR-T cells
  • AML antigen is CD33 which is expressed in up to 90% of leukemic blasts, (also on normal myeloid cells and some progenitors but not CD34 + stem cells 31 ) 32 .
  • Comprehensive AML surfaceome analysis of AML cell lines and primary patient samples projected that simultaneous targeting of CD33 and CD70 in AML would be feasible in over 97% of patients with non-overlapping bystander tissue toxicides 19 (FIGs. 3A-3C).
  • FILLs. 3A-3C To simultaneously address antigenic heterogeneity, promote local anti-tumor activity through the recruitment of Tconventionai, Treg and exhausted Teff, and eliminate immunosuppressive MDSCs, a CD3/CD33 TEAM secreting CD70 targeted CAR (CARTEAM) was generated (FIG. 1 and FIG. 5A).
  • CD70 targeted CAR-T cells traffic to the bone marrow where they lyse AML blasts and simultaneously locally deliver extremely small quantities of a CD33 TEAM to engage Tconventionai cells and further potentiate the action of CD70 targeted CARs against AML blasts and MDSCs, but not cause systemic toxicity due to extremely rapid clearance of TEAMs in humans 22 (FIG. 4).
  • the trCD27-CAR-CD33-TEAM had comparable transduction efficiency to monotargeted constructs with greater than 60% across three healthy donors’ T-cells (FIG. 5B).
  • Results show that TEAMs are secreted by CAR-T cells (FIG. 5C).
  • Results also show that TEAMs from the supernatant from the trCD27-CAR- CD33-TEAM bind specifically to target-expressing cells (FIG. 5D).
  • Results also showed that the CD33 TEAM decrease the percent of cells expression CD69 (FIG. 5E).
  • CD70CAR CD33TEAM cells exhibited superior cytotoxicity and CAR expansion in this real-time cytotoxicity assay on the incucyte at a 2: 1 effector to target ratio.
  • CAR-T cells were manufactured from 3 healthy donors’ T-cells performed in duplicate.
  • BBI bioluminescent
  • mice received 2xl0 6 of either CD70CAR CD19TEAM or CD70CAR CD33TEAM cells (n 5 mice per individual T-cell donor per group).
  • Peripheral blood CAR-T phenotypic profiling was also performed via flow cytometry from the blood of mice at day +21 after CAR-T injection compared to unstimulated and nontransduced, donor matched T-cells (FIG. 8E).
  • CD70CAR CD33TEAM cells were also found to exhibit divergent expressional programs compared to CD70CAR CD19TEAM cells in vivo.
  • CAR-T cells were separated via fluorescence- activated cell sorting (FACS) from the spleens of the mice in the mixed tumor model.
  • FACS fluorescence- activated cell sorting
  • CAR-T cells were then lysed and underwent gene expression analysis via nanostring with the CAR-T characterization panel in addition to a custom ‘drop-in’ gene set (FIGs. 9A-9C).
  • CD33 which is expressed in up to 90% of leukemic blasts 31 , (also on normal myeloid cells and some progenitors but not CD34+ stem cells 32 ). Importantly, elimination of CD33+ cells via treatment of bone marrow autografts with a monoclonal antibody eliminates committed myeloid progenitors, however, while delayed, normal trilinear hematopoiesis occurs. 33 Unexpectedly, CD33 is also found on hepatocytes and treatment with the CD33 ADC, gemtuzumab ozogamicin (GO), has led to fatal liver toxicity in the form of veno-occlusive disease (VOD).
  • GO gemtuzumab ozogamicin
  • CD33 can also be found on immunosuppressive MDSCs in the bone marrows of patients with AML. Recently, successful synergistic targeting of these MDSCs and AML using a CD3/CD33 bispecific T cell engager has been demonstrated in vitro. 35 Comprehensive AML surfaceome analysis of AML cell lines and primary patient samples projected that simultaneous targeting of CD33 and CD70 in AML would be feasible in over 97% of patients with non-overlapping bystander tissue toxi cities. 19
  • CD3/CD33 TEAM secreting CD70 targeted CAR was generated and tested, based on a previously optimized CD70-targeted CAR-T platform.
  • mice were injected with 5xl0 6 fresh PDX cells on day -4 followed by 5xl0 5 or IxlO 6 70 33 or 70 19 CAR-T cells (U and 14 the previous treatment dose respectively) on day 0 (FIG. 14A).
  • T cells T cells used as manufacturing substrate due to immunosuppressive effects of the cancer itself, or T cell damage due to intensive chemoimmunotherapy.
  • PBMC peripheral blood mononuclear cell
  • FIG. 16A T cells were isolated and flow cytometric phenotyping was performed. It was found that AML patients’ T cells had a more differentiated and exhausted phenotype compared to healthy donor T cells (FIG. 16B-16C).
  • T cells acting as redirected bystanders via the TEAM their isolated T cells were cocultured with TEAM producer cells and AML targets.
  • T cells from AML patients produced inflammatory cytokines, though at lower numbers than healthy donor T cells, intriguingly except for IFNg (FIG.
  • Ochsenbein A Argx-110 Targeting CD70, in Combination with Azacitidine, Shows Favorable Safety Profile and Promising Anti-Leukemia Activity in Newly Diagnosed AML Patients in an Ongoing Phase 1/2 Clinical Trial. Abstract 2680. American Society of Hematology Annual Meeting. San Diego, CA2018.

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