CD70-binding CAR-T cells containing CD33-binding T cell-engaging antibody molecules

Abstract

The present disclosure relates to methods and compositions for treating cancer characterized by cells containing a chimeric antigen receptor (CAR) that binds CD70 and a T-cell engaging antibody molecule (TEAM) that binds CD33, nucleic acid molecules encoding a chimeric antigen receptor (CAR) that binds CD70 and / or a TEAM that binds CD33, and compositions and methods related thereto.

Description

[Technical field]

[0001] [Related Applications] This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 331,758, filed April 15, 2022, and U.S. Provisional Application No. 63 / 341,995, filed May 13, 2022, the entire contents of each of which are incorporated herein by reference.

[0002] [Government funding] This invention was made with Government support under Grant No. 5R01CA238268-03 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Chimeric antigen receptor T cells (CAR-T) have been highly effective in certain hematological malignancies. The TNF-α family member CD70 has emerged as a promising surface target antigen in AML. However, antigen escape can occur after antigen-specific cancer therapy, leading to relapse in antigen-negative cancers. Summary of the Invention

[0004] The present disclosure relates to methods and compositions for treating cancers characterized by cells expressing CD70 and optionally cells that express low levels of CD70 or that do not express CD70 (e.g., cancers that are no longer characterized by CD70-expressing cells in response to anti-CD70 treatment (e.g., CD70 antigen escape)). The present disclosure relates, in part, to compositions and methods that overcome such antigen escape. The present disclosure provides a dual targeting strategy in which CD70 CAR T cells are engineered to express and secrete a T-cell engaging antibody molecule (TEAM). In some embodiments, the TEAM comprises a cancer-binding moiety (e.g., an anti-CD33 antibody) and an immune cell-binding moiety (e.g., an anti-CD3 antibody). Without wishing to be bound by theory, the TEAM may direct the CAR-T cells or bystander cells to bind to 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.

[0005] Thus, in one aspect, the disclosure relates to a cell comprising a chimeric antigen receptor (CAR) that binds CD70 and a T-cell engaging antibody molecule (TEAM) that binds CD33. In some embodiments, the cell is an immune cell. In some embodiments, 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. In some embodiments, the immune cell is a T cell. In some embodiments, the cell is obtained from a subject, optionally a human subject.

[0006] In some embodiments, the CAR comprises (i) an extracellular target binding domain comprising a polypeptide that binds to CD70; (ii) a transmembrane domain; and (iii) an intracellular signaling domain. In some embodiments, the extracellular target binding domain comprises a CD70 binding domain of CD27. In some embodiments, the extracellular target binding domain comprises an extracellular domain of CD27. In some embodiments, 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. In some embodiments, the extracellular target binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 1, 8, or 9. In some embodiments, the extracellular target binding domain comprises an anti-CD70 antibody, optionally an scFv. In some embodiments, the transmembrane domain is the transmembrane domain of CD27. In some embodiments, the intracellular signaling domain comprises (i) an ITAM-containing signaling domain and / or (ii) one or more signaling domains from one or more costimulatory proteins or cytokine receptors. In some embodiments, the intracellular signaling domain comprises a CD3γ, CD3ε, CD3δ, or CD3ζ domain. In some embodiments, the intracellular signaling domain comprises a CD3ζ domain. In some embodiments, the costimulatory domain comprises a CD28, 4-1BB, 2B4, KIR, OX40, ICOS, MYD88, IL2 receptor, or SynNotch domain. In some embodiments, the costimulatory domain comprises a 4-1BB domain. In some embodiments, the extracellular target binding domain further comprises a signal peptide. In some embodiments, the signal peptide comprises a CD27 signal peptide. In some embodiments, 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.

[0007] In some embodiments, the TEAM comprises an anti-CD33 antibody or a functional fragment thereof (e.g., the VH and / or VL domains of an anti-CD33 antibody). In some embodiments, 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. In some embodiments, the anti-CD33 antibody is an scFv. In some embodiments, 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. In some embodiments, the VH domain is N-terminal to the VL domain. In some embodiments, the VL domain is N-terminal to the VH domain. In some embodiments, the TEAM comprises an immune cell binding moiety. In some embodiments, the immune cell binding moiety binds to CD3, CD8, CD4, CXCR3, CCR4, GARP, LAP, CD25, CTLA-4, or CD16. 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. In some embodiments, the TEAM comprises a linker between the anti-CD33 antibody and the immune cell binding moiety. In some embodiments, the linker is a non-cleavable linker, optionally a (GGGGS)3 (SEQ ID NO: 27) linker. In some embodiments, the TEAM further comprises a secretion tag, optionally an IgK secretion tag. In some embodiments, the TEAM comprises an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 17-18. In some embodiments, 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. In some embodiments, the nucleic acid sequence encoding the TEAM is codon optimized.

[0008] In some embodiments, the cell comprises a first polynucleotide molecule comprising a nucleic acid sequence encoding a CAR and a second polynucleotide molecule comprising a nucleic acid sequence encoding a TEAM. In some embodiments, the cell comprises a polynucleotide molecule comprising a nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding a TEAM. In some embodiments, 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 the linker being a cleavable linker. In some embodiments, the cleavable linker is self-cleavable, optionally a P2A, E2A, F2A, or T2A self-cleaving linker. In some embodiments, the cleavable linker comprises a protease motif. In some embodiments, the linker comprises an internal ribosome entry site (IRES). In some embodiments, the polynucleotide molecule comprises a promoter operably linked to the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the TEAM. In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the promoter is an EF1 alpha promoter.

[0009] In some embodiments, the sense strand of the polynucleotide molecule comprises, from 5' to 3', a nucleic acid sequence encoding a CAR, a linker, and a nucleic acid sequence encoding a TEAM. In some embodiments, the sense strand of the polynucleotide molecule comprises, from 5' to 3', a nucleic acid sequence encoding a TEAM, a linker, and a nucleic acid sequence encoding a CAR. In some embodiments, the polynucleotide molecule comprises a nucleic acid sequence encoding an amino acid sequence at least 85% identical to any one of SEQ ID NOs:22-25.

[0010] In some aspects, the disclosure relates to polynucleotides comprising nucleic acid sequences encoding the CARs and TEAMs described herein. In some aspects, the disclosure relates to cells comprising the CARs and TEAMs described herein.

[0011] In some aspects, the disclosure relates to methods comprising administering to a subject the cells described herein. In some aspects, the disclosure relates to methods for treating a cancer characterized by cancer cells expressing CD70, comprising administering to a subject in need of treatment an effective amount of the cells described herein. In some aspects, the disclosure relates to methods for treating a cancer characterized by cancer cells expressing CD70 and CD33, comprising administering to a subject in need of treatment an effective amount of the cells described herein.

[0012] In some aspects, the disclosure relates to a method of treating a cancer characterized by cancer cells expressing CD33, comprising administering to a subject in need of treatment an effective amount of the cells described herein.

[0013] In some aspects, the disclosure relates to a method of treating a cancer characterized by cancer cells having reduced expression of CD70, comprising administering to a subject in need of treatment an effective amount of the cells described herein.

[0014] In some embodiments, the subject is a human. In some embodiments, the administering comprises infusion. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is a bone marrow cancer. In some embodiments, the cancer is acute myeloid leukemia. [Brief description of the drawings]

[0015] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the disclosure and may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0016] [Figure 1]Figure 1 shows a diagram of a trCD27-CD8H / TM CAR that secretes 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 bystander T cells to AML cells. [Diagram 2] Figure 2 shows a graph of flow cytometry data demonstrating CD70 low recurrence after CD70-targeted CAR therapy in AML PDX. NSG mice were transplanted with 5×106 DFAM68555 AML patient-derived xenograft cells on day -4. On day 0, the mice were administered 2×106 trCD27CD8H / TM-modified CD70-targeted CAR-T cells. Tumors were eliminated in most trCD8H / TM-modified mice, but CD70 low tumors recurred in some mice. [Figure 3A] Figures 3A - 3C show paired expression of CD33 and CD70 as combinatorial CAR targets in AML. Figure 3A shows co-expression of CD33 and CD70 in the blood. [Figure 3B] Figure 3B shows single and co-expression of CD33 and CD70 in primary AML samples. [Figure 3C] Figure 3C shows co-expression of the CD33+CD70 antigen pair in AML cells compared to normal BM HSC and T cells. [Figure 4] Figure 4 is a schematic diagram showing the putative advantages of CARTEAM. CARTEAM cells show rapid renal clearance, spare hepatocytes, and reduced hepatotoxicity. [Figure 5A] Figures 5A - 5E show diagrams of exemplary CARs and TEAMs and graphs demonstrating that the TEAM is secreted by CAR-T cells and binds to the appropriate target antigen. [Figure 5A] Structure of the trCD27 CAR and / or CD33 TEAM construct. [Figure 5B] Transduction efficiency of healthy donor T cells into activated T cells (n = 3 different healthy donors, representing at least 2 separate experiments). [Figure 5C]Supernatants from the indicated cells (rows) were added to the indicated cell types (columns) and assessed for anti-His tag (TEAM) binding. [Figure 5D] Supernatants from Jurkat T cells transduced with the indicated constructs were incubated with plate-bound Molm13 tumor cells as well as anti-His-tagged AF647 antibody (red). Fluorescence intensity was measured in an incucyte and representative images are shown. [Figure 5E] Supernatants from Jurkat T cells transduced with the indicated constructs were co-cultured with Molm13 tumor cells and untransduced (UTD) healthy primary human T cells and anti-CD69 antibody. Percentage of cells expressing CD69 over time is shown. PMA = phorbol 12-myristate 13-acetate positive control. [Figure 6] Figure 6 shows a diagram of the effects of CAR-T cells and TEAMs, and a graph showing that CD33 TEAM mediates UTD-driven target-dependent cytotoxicity. Cells transduced with trCD27 CAR-CD33-TEAM or trCD27-CD19-TEAM constructs, or untransduced T cells (UTD) were added to the top of the transwell insert. Untransduced T cells were added to the bottom of the transwell insert along with Molm13WT or Molm13CD33- cells. Only the trCD27-CAR-CD33-TEAM construct was able to mediate clearance of Molm13WT cells, which was target-dependent and abrogated by loss of CD33. [Figure 7A] Figures 7A-7B show graphs and micrographs demonstrating that simultaneous T cell redirection with CAR and TEAM is effective against heterogeneous tumors and is superior to a mixture of individual CARs. [Figure 7A] Individually targeted CD70 or CD33 CAR-T cells were compared with CD70CARCD33TEAM or CD70CARCD19TEAM against AML targets with different levels of CD70 or CD33 (left). CD70CARCD33TEAM cells showed superior cytotoxicity and CAR proliferation in real-time cytotoxicity assays in incubates. [Figure 7B]A representative image at the end of the experiment is shown in Figure 7B. Tumor cells are green. Data shown are representative of CAR-T cells produced in duplicate from T cells of three healthy donors. Experiments were repeated at least n=2 times. [Figure 8A] Figures 8A-8E show that CD70CARCD33TEAM eradicates mixed tumor populations in vivo. Figure 8A shows that NSG mice were injected with 5x105 mixed population Molm13 cells (90% Molm13CD70KO, 10% Molm13WT, n=10 mice / group) expressing the bioluminescence (BLI) reporter Click Beetle Green on day -7. On day 0, mice received 2x106 CD70CARCD19TEAM or CD70CARCD33TEAM cells (n=5 mice per individual T cell donor per group). [Figure 8B] Figures 8B-8C show that continuous BLI monitoring eradicated tumors in the CD70CARCD33TEAM mice, but did not control tumor growth in the CD70CARCD19TEAM mice. P values ​​are shown by two-way ANOVA. [Figure 8C] (the above) [Fig. 8D-E] Figure 8D shows peripheral blood proliferation quantified by flow cytometry on days 14-28 after CAR-T injection. P values ​​are reported as the result of individual Mann-Whitney U tests. Figure 8E shows peripheral blood CAR-T phenotypic profiling obtained by flow cytometry from mouse blood on day +21 after CAR-T injection compared to unstimulated and untransduced donor-matched T cells. Representative examples are shown; CM (central memory), EM (effector memory), and TDE (terminally differentiated effector). [Figure 9A]Figures 9A-9C show that CD70CARCD33TEAM cells display divergent expression programs compared to CD70CARCD19TEAM cells in vivo. Figure 9A shows a volcano plot highlighting the differences in transcriptional programs between the constructs. Genes with adjusted p-values ​​<0.05 (Benjamini-Hochberg) are colored red. [Figure 9B] Figure 9B shows that pathway clustering was performed in an unbiased manner by integrating all available pathways (top), and a heatmap of selected pathways is shown below. [Figure 9C] Figure 9C shows the pathway scores of TCR signaling for CD70CARCD33TEAM (right) and CD70CARCD19TEAM (left). P values ​​are from unpaired t-test. [Figure 10] FIG. 10 shows administration of two different doses of CD70CARCD33TEAMCAR T cells to a patient-derived xenograft (PDX) AML mouse model. 5×106 AML PDX cells were administered to mice on day −4. On day 0, mice were administered 5×105 or 1×106 CD70CARCD33TEAMCAR T cells. Untreated and CD70CARCD19TEAM were used as controls. On day 15, tumor cell (CD33+ / CD45+) concentrations in the blood of mice were measured. The results show that administration of 5×105 and 1×106 CD70CARCD33TEAMCAR T cells had similar effects on tumor cell concentrations. [Figure 11A-B] Figures 11A-11E show the construction of CD70-CAR-T cells secreting CD33 T cell-engaging antibody molecules. Figure 11A shows NSG mice were implanted with 5x106 DFAM68555 AML patient-derived xenograft cells on day -4. On day 0, mice were administered 2x106 CD70-targeted CAR-T cells. Most CAR-T cell-treated mice had tumor disappearance, but some mice had recurrence of CD70low tumors. Figure 11B shows CD70CAR-T cells secreting CD33-CD3 T cell-engaging antibody molecules. [Figure 11C] (the above) [Fig. 11D-E] Figure 11D shows a schematic of the CAR construct used here. Figure 11E shows the transduction efficiency of primary healthy donor T cells (n=3 different healthy donors, representative of at least 2 separate experiments). [Figure 12A] Figures 12A-12D show that TEAM is secreted by CAR-T cells and binds to the appropriate target antigen. Figure 12A shows that supernatants from the indicated conditions (rows) were added to the indicated cell types (columns) and assessed for anti-His tag (TEAM) binding by flow cytometry. [Figure 12B-C] FIG 12B shows that supernatants from Jurkat T cells transduced with the indicated constructs were incubated with plate-bound CD33-expressing K562 tumor cells and anti-His tagged AF647 antibody. Fluorescence intensity was measured in an incucyte and representative images are shown. FIG 12C shows that supernatants from Jurkat T cells transduced with the indicated constructs were co-cultured with Molm13 tumor cells, untransduced (UTD) healthy primary human T cells, and anti-CD69 antibody. Percentage of cells expressing CD69 over time is shown. PMA = phorbol 12-myristate 13-acetate positive control. [Figure 12D] Figure 12D shows that cells transduced with 7033 or 7019 constructs or UTD were added to the top of the transwell insert. UTD was added to the bottom of the transwell insert along with Molm13WT or Molm13CD33KO cells. Tumor growth was measured over time. P values ​​represent two-way ANOVA between the indicated conditions. Experiments were repeated at least n=2 times. [Figure 13A]Figures 13A-13C show that simultaneous T cell redirection with CAR and TEAM is effective against heterogeneous tumors and is superior to a mixture of individual CARs. Figure 13A shows that individually targeted CAR33 T cells were compared with 7033 or 7019 CAR-T cells against engineered Molm13wt, Molm13CD70KO, and Molm13CD33KOAML targets at an effector:target ratio of 2:1. [Figure 13B-1] Expression of the indicated targets assessed by flow cytometry (left) and results of real-time cytotoxicity assays (Figure 13B top) and CAR-T proliferation (Figure 13B bottom) are shown. [Figure 13B-2] (the above) [Figure 13C] Figure 13C shows representative images from incucytes after 40 hours in the indicated conditions. Data shown represents combined data from duplicate runs of CAR-T cells manufactured from T cells from three healthy donors. Experiments were repeated at least n=2 times. P values ​​represent two-way ANOVA for the indicated conditions. [Figure 14A-B] Figures 14A-14D show that simultaneous T cell redirection via CAR and TEAM outperforms standard CAR-T cells in a patient-derived xenograft model of AML. Figure 14A shows that 5x106 DFAM68555 AML PDX cells were injected via tail vein into NSG mice (n=5 / group) on day -4. Four days later, on day 0, 5x105 or 1x106 7033 or 7019 CAR-T cells were injected via tail vein. Figure 14B shows that CD70CAR-T cells secreting CD33 TEAM (7033) kill circulating CD33+ tumor cells more efficiently than CD70CAR-T cells secreting CD19 TEAM (7019). [Fig. 14C-D] Figure 14C shows the number of circulating 7033 and 7019 CAR-T cells on day +14. Figure 14D shows the survival rate of mice in the experiment. P values ​​represent log-rank Mantel-Cox. [Figure 15A-B]Figures 15A-15H show that 7033 eradicates mixed tumor populations in vivo. Figure 15A shows that NSG mice were injected with 5x105 mixed population Molm13 cells expressing the bioluminescence (BLI) reporter click beetle green (90% Molm13CD70KO, 10% Molm13WT, n=10 mice / group) on day -7. On day 0, mice received 2x106 of either 7019 or 70CAR33 cells (n=5 mice per individual T cell donor per group). Figure 15B shows peripheral blood proliferation of CAR-T cells quantified by flow cytometry on days 14-28 after CAR-T injection. [Figure 15C] Figures 15C-15D show serial BLI monitoring of tumor growth. P values ​​represent two-way ANOVA. [Fig. 15D-E] Figure 15D (above). Figure 15E shows peripheral blood CAR-T phenotypic profiling by flow cytometry from mouse blood at +21 days post-CAR-T injection compared to unstimulated and untransduced donor-matched T cells. Representative examples are shown. CM (central memory), EM (effector memory), TDE (terminally differentiated effector). [Figure 15F] Figure 15F shows that CAR-T cells FACS sorted at day +28 from the spleens of mice in the mixed tumor model underwent gene expression analysis in Nanostring using a custom "drop-in" gene set plus a CAR-T characterization panel. Volcano plots highlight differences in transcriptional programs between constructs. Genes with adjusted p-values ​​<0.05 (Benjamini-Hochberg) are colored dark grey. [Fig. 15G-H] Figure 15G shows pathway hierarchical clustering performed in an unbiased manner integrating all available pathways (top). Heatmaps of selected pathways are shown. Figure 15H shows pathway scores of TCR signaling for 7033T and 70CAR19. p-values ​​are from unpaired t-test. [Figure 16A-B]Figures 16A-16G show that TEAM effectively redirects T cells from AML patients to kill tumor cells. Figure 16A shows the clinical characteristics of the AML patients whose T cells were utilized. Figure 16B shows the flow T cell phenotype comparing T cells from AML patients with those from healthy donors. [Figure 16C] Figure 16C shows representative phenotypes and PD-1 expression in T cells from AML patients. [Figure 16D] Figure 16D shows co-culture of TEAM producing cells with isolated AML patient T cells and Molm13 tumor targets. Real-time cytotoxicity measured in incucyte. [Figure 16E] FIG. 16E shows normalized cytokine measurements obtained from patients compared to healthy donors after 18 hours. [Figure 16F] Figure 16F shows a transwell assay using TEAM producing cells on the top and isolated AML patient T cells on the bottom with tumor targets. Real-time cytotoxicity was measured (top) and activation (CD69) is shown on the bottom. [Figure 16G] FIG. 16G shows representative images at 40 hours of activation. [Figure 17] Figure 17 shows that simultaneous T cell redirection with CAR and TEAM is effective against heterogeneous tumors with endogenous antigen expression levels and is superior to a mixture of individual CARs. Individually targeted CAR33 T cells were compared with 7033 or 7019 CAR-T cells at a 2:1 effector:target ratio against AML cell lines Kasumi1, Monomac1, and OCI-AML3. Expression of the indicated targets assessed by flow cytometry (left) and results of real-time cytotoxicity assays (top) and CAR-T proliferation (bottom) are shown. Data shown represent combined data from CAR-T cells manufactured from T cells of three healthy donors performed in duplicate. Experiments were repeated at least n=2 times. P values ​​represent two-way ANOVA for the indicated conditions. [Figure 18]Figure 18 shows that pathway hierarchical clustering was performed in an unbiased manner by integrating all available pathways (top). The heatmap shows the available pathways for all nanostrings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] [CD-70-binding chimeric antigen receptor (CAR)] The present disclosure provides, in some aspects, CAR-T cells targeted to the tumor necrosis alpha family member CD70 and uses of the CAR-T cells for the treatment of hematological malignancies (e.g., acute myeloid leukemia (AML)). CD70 is consistently expressed on myeloblasts and leukemia stem cells, but has very limited expression in healthy human tissues. As previously demonstrated, CAR-T cells targeted to CD70 achieved antigen-specific activation, cytokine production, and cytotoxic activity in in vitro and in vivo leukemia models, for example, as described in PCT / US2020 / 051018, which is incorporated by reference in its entirety.

[0018] Some aspects of the present disclosure provide a chimeric antigen receptor (CAR) comprising: (i) an extracellular target binding domain comprising a polypeptide that binds to CD70; (ii) a transmembrane domain; and (iii) an intracellular signaling domain.

[0019] "Chimeric antigen receptor (CAR)" refers to a receptor protein engineered to perform both antigen binding and cell activation functions. In some embodiments, a CAR comprises multiple linking domains with distinct functions. CAR domains include those with antigen binding function, those with structural function, and those with signaling function. In some embodiments, 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") that comprises a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, a CAR comprises an optional leader sequence (also referred to as a "signal peptide"), an extracellular antigen binding domain, a hinge, a transmembrane domain, and an intracellular stimulatory domain. In some embodiments, the domains in a CAR are in the same polypeptide chain, e.g., comprising a chimeric fusion protein. In some embodiments, the domains in a CAR are not contiguous to each other.

[0020] In some embodiments, the CAR described herein comprises an extracellular target binding domain that comprises a polypeptide that binds to Cluster of Differentiation 70 (CD70). "CD70" refers to the polypeptide encoded by the human CD70 gene (NCBI Gene ID: 970). As described herein, expression of CD70 is highly restricted in normal human (non-cancerous) tissues. However, CD70 is expressed in numerous cancers, including bladder cancer, invasive breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, diffuse large B-cell lymphoma (DLBC), esophageal cancer, glioblastoma (GBM), head and neck cancer, low-grade glioma (LGG), liver cancer, lung adenocarcinoma, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, gastric cancer, testicular germ cell carcinoma, 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 domain, a transmembrane domain, and an extracellular domain. The extracellular domain of CD70 is a ligand for CD27.

[0021] In some embodiments, the polypeptide that binds to CD70 comprises the CD70-binding domain of cluster of differentiation 27 (CD27), also known as the CD27 antigen. "CD27" refers to the polypeptide encoded by the human CD27 gene (NCBI gene ID: 939, Uniprot ID: P26842). An example of a CD27 amino acid sequence is provided below. MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 8)

[0022] The CD27 protein has an extracellular domain, a transmembrane domain, and a cytoplasmic domain. In some embodiments, the CD70 binding domain is located within the extracellular signaling domain of CD27. In some embodiments, the extracellular region includes multiple cysteine-rich domains (CRDs), namely CDR1, CDR2, and CDR3. In some embodiments, the CD70 binding domain is located within the CRD2 domain.

[0023] In some embodiments, the CD70 binding domain of CD27 comprises a peptide comprising the amino acid sequence of TRPHCESCRHCN (SEQ ID NO:9), which is located in the extracellular domain of CD27. In some embodiments, the extracellular target binding domain of a 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. In some embodiments, the extracellular target binding domain of a CAR described herein comprises the amino acid sequence of SEQ ID NO:9.

[0024] In some embodiments, the extracellular target binding domain of a CAR described herein comprises a polypeptide comprising the extracellular domain of CD27 or a functional fragment thereof (e.g., a fragment capable of binding to CD70). In some embodiments, the functional fragment comprises SEQ ID NO:9 or a variant thereof. In some embodiments, the extracellular target binding domain of a 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. In some embodiments, the extracellular target binding domain of a CAR described herein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:1.

[0025] In some embodiments, the polypeptide that binds to CD70 in the extracellular target binding domain of the CAR described herein comprises an anti-CD70 antibody. The term "antibody" as used herein encompasses different types of antibodies and antibody fragments. In some embodiments, antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including, but not limited to, single chain variable fragments (scFV), single domain antibodies, such as the heavy chain variable domain (VH), light chain variable domain (VL) and variable domain (VHH) of camelid-derived nanobodies, and alternative scaffolds known in the art to function as antigen binding domains, such as recombinant fibronectin domains. In some embodiments, it is beneficial for the antigen binding domain to be derived from the same species in which the CAR will ultimately be used. For example, when used in humans, it may be beneficial for the antigen binding domain of the CAR to comprise human or humanized residues of the antigen binding domain of an antibody or antibody fragment. In some embodiments, the polypeptide that binds to CD70 within the extracellular target binding domain of a CAR described herein comprises an scFv that binds to CD70.

[0026] In some embodiments, the CD70 antibody comprises an antibody or antigen binding domain (e.g., CDRs or VH and VL) described in U.S. Pat. No. 1,434,298, U.S. Pat. No. 7,491,390, U.S. Pat. No. 8,124,738, U.S. Pat. No. 1,377,500, or U.S. Pat. No. 9,701,752, each of which is incorporated by reference in its entirety.

[0027] In some embodiments, the antibody is a human antibody or antibody fragment. In some embodiments, the antibody is a humanized antibody or antibody fragment. Humanized antibodies can be modified by techniques such as CDR grafting (see, e.g., European Patent No. EP 239400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein by reference in its entirety), veneering, or resurfacing (see, e.g., European Patent Nos. EP 592106 and EP 519596, each of which is incorporated herein by reference in its entirety; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Science, 1999, 1999, 1999, 1999, 1999, 1990, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 19 ... Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein by reference in its entirety), and methods such as those described in, e.g., U.S. Patent Application Publication No. 2005 / 0042664, U.S. Patent Application Publication No. 2005 / 0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein ... and Caldas et al., Protein Immunol., 169:1119-25 (2002), each of which is incorporated herein by reference in its entirety. Eng., 13(5):353-60(2000), Morea et al., Methods, 20(3):267-79(2000), Baca et al., J. Biol. Chem., 272(16):10678-84(1997), Roguska et al., Protein Eng.,9(10):895-904(1996), Couto et al., Cancer Res.,55(23 Supp):5973s-5977s(1995), Couto et al., Cancer Res.,55(8):1717-22(1995), Sandhu They can be produced using a variety of techniques known in the art, including those disclosed in JS, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994).Often, framework residues in the framework regions are replaced with corresponding residues from the CDR donor antibody to alter (e.g., improve) antigen binding. These framework replacements are identified by methods well known in the art, such as modeling the interactions of 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.)

[0028] A humanized antibody or antibody fragment contains one or more amino acid residues that are derived from a non-human source. These non-human amino acid residues are often referred to as "import" residues, and are typically taken from an "import" variable domain. As provided herein, a humanized antibody or antibody fragment comprises one or more CDRs and framework regions derived from a non-human immunoglobulin molecule, wherein the amino acid residues comprising the framework are derived entirely or predominantly from human germline. Several techniques for humanization of antibodies or antibody fragments are well known in the art and may be performed essentially by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody, i.e., CDR grafting, according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)) (EP 239400; PCT Publication No. WO 91 / 09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference). In such humanized antibodies and antibody fragments, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. Humanized antibodies are often human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing (EP 592106; EP 519596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the contents of which are incorporated herein by reference in their entireties.

[0029] In some embodiments, the antibodies are obtained 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 components vary such that different amino acid sequences are represented. The polypeptide components can be of any length, for example, from 3 amino acids to 300 amino acids or more. A display library entity can include more than one polypeptide component, for example, two polypeptide chains of a Fab. In one exemplary embodiment, a display library can be used to identify antigen binding domains. In selection, the polypeptide component of each member of the library is probed with an antigen or a fragment thereof, and if the polypeptide component binds to the antigen, the display library member is identified, typically by retention on a support.

[0030] The retained display library members are recovered from the support and analyzed. Analysis can include amplification and subsequent selection under similar or different conditions. For example, alternating positive and negative selections can be performed. Analysis can also include determining the amino acid sequence of the polypeptide components and purifying the polypeptide components for detailed characterization.

[0031] Various formats can be used for display libraries. Examples include phage display. In phage display, the protein components are typically covalently linked to a bacteriophage coat protein. Linkage occurs by translation of a nucleic acid encoding the protein components fused to the coat protein. Linkage can include flexible peptide linkers, protease sites, or amino acids incorporated as a result of suppression of a stop codon. Phage display is described, for example, in U.S. Pat. No. 5,223,409; WO 92 / 18619; WO 91 / 17271; WO 92 / 20791; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; WO 90 / 02809. Bacteriophage displaying the protein components can be grown and harvested using standard phage preparation methods, such as PEG precipitation from growth medium. After selection of individual display phages, the nucleic acid encoding the selected protein components can be isolated from cells infected with the selected phage or from the phage itself after amplification. Individual colonies or plaques can be picked and the nucleic acid isolated and sequenced. Other 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.

[0032] The transmembrane domain of the CAR described herein can be derived from either natural or recombinant sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In one embodiment, the transmembrane domain can transmit a signal to the intracellular domain whenever the CAR binds to the target. Transmembrane domains of particular use in this invention can include at least the transmembrane regions of, for example, the alpha, beta or zeta chains 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. In some embodiments, the transmembrane domain includes at least one of the following: e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, rfGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT Transmembrane domains may include those of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMFl, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, NKG2C, and CD19. In some embodiments, the transmembrane domain is a CD28 transmembrane domain or a CD8 transmembrane domain. In some embodiments, the transmembrane domain is a CD27 transmembrane domain.In some embodiments, the transmembrane domain of CD27 comprises the amino acid sequence of ILVIFSGMFLVFTLAGALFL (SEQ ID NO: 10).

[0033] In some embodiments, 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 derived from a human protein. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge, e.g., an IgG4 hinge, or a CD8a hinge.

[0034] In some embodiments, the cytoplasmic domain or region of the CARs described herein includes one or more intracellular signaling domains. The intracellular signaling domain can activate at least one of the normal effector functions of an immune cell into which the CAR is introduced. Examples of intracellular signaling domains for use in the CARs described herein include the cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act in concert to initiate signaling following antigen receptor engagement, as well as any derivatives or variants of these sequences, and any recombinant sequences that have the same functional capabilities.

[0035] T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains).

[0036] As the term is used herein, "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain can generate a signal that promotes immune effector function of a CAR-containing cell, such as a CAR T cell or a CAR-expressing NK cell. Examples of immune effector function, for example in a CAR T cell or a CAR-expressing NK cell, include cytolytic activity and helper activity, including secretion of cytokines. In embodiments, the intracellular signaling domain transmits an effector function signal, directing the cell to perform a specialized function. The entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits an effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0037] In some embodiments, the one or more intracellular signaling domains comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary or antigen-dependent stimulation. In some embodiments, the primary intracellular signaling domain comprises a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences that contain ITAMs 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. In some embodiments, the intracellular signaling domain of the CAR comprises a CD3 zeta (CD3ζ) signaling domain. In some embodiments, the CD3 zeta (CD3ζ) signaling domain comprises the amino acid sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 11). In some embodiments, the CD3 zeta (CD3ζ) signaling domain of a CAR described herein comprises an amino acid sequence 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.

[0038] In some embodiments, the one or more intracellular signaling domains comprise a costimulatory intracellular signaling domain. A costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule. An intracellular signaling domain may comprise the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain, or a functional fragment thereof. Exemplary costimulatory intracellular signaling domains include those derived from molecules that carry costimulatory signals (e.g., antigen-independent stimulation) and those derived from cytokine receptors. In some embodiments, the one or more intracellular signaling domains comprise a primary intracellular signaling domain and a costimulatory intracellular signaling domain from one or more costimulatory proteins or cytokine receptors.

[0039] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or its ligand, that is required for an efficient immune response. Examples of such molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFl), NKp44, NKp3 0, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT These include ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGLl, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.For example, CD27 co-stimulation has been demonstrated to promote the proliferation, effector function, and survival of human CART cells in vitro and enhance the persistence and anti-tumor activity of human T cells in vivo (Song et al. Blood. 2012;119(3):696-706). In some embodiments, the co-stimulatory domain of the CAR described herein comprises one or more co-stimulatory proteins selected from CD28, 4-1BB, 2B4, KIR, CD27, OX40, ICOS, MYD88, the IL2 receptor, and SynNotch, or one or more signaling domains from a cytokine receptor. In some embodiments, the co-stimulatory domain of the CAR described herein comprises a 4-1BB co-stimulatory signaling domain. In some embodiments, the 4-1BB co-stimulatory signaling domain comprises the amino acid sequence of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 12). In some embodiments, 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.

[0040] In some embodiments, the intracellular signaling domain of the CAR described herein comprises an ITAM-containing domain, such as a primary signaling domain, e.g., the CD3 zeta signaling domain, alone or in combination with a co-stimulatory signaling domain (e.g., a co-stimulatory domain from one or more co-stimulatory proteins selected from CD28, 4-1BB, 2B4, KIR, CD27, OX40, ICOS, MYD88, the IL2 receptor, and SynNotch, or a cytokine receptor). In some embodiments, the intracellular signaling domain of the CAR described herein comprises a CD3 zeta (CD3ζ) signaling domain and a 4-1BB co-stimulatory signaling domain.

[0041] In some embodiments, different linker sequences, e.g., (GGGS), are used between different domains of the CAR n(SEQ ID NO:35) linkers may be used, where n is 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the linker is (GGGS) 7 (SEQ ID NO: 36). In some embodiments, the CAR comprises additional sequences from CD27 between the extracellular target binding domain and the transmembrane region, such as the stalk and hinge region of CD27. In some embodiments, the stalk and hinge region of CD27 comprises the amino acid sequence of PLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR (SEQ ID NO: 13). In some embodiments, the CAR does not comprise additional sequences of CD27, such as the stalk and hinge region between the extracellular target binding domain and the transmembrane region.

[0042] In some embodiments, a 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, a CAR described herein comprises the amino acid sequence of any one of SEQ ID NOs: 2-7.

[0043] In some embodiments, the CAR described herein further comprises a leader sequence (also referred to herein as a signal peptide) at the amino-terminus (N-terminus) of the antigen binding domain. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the antigen binding domain, which is optionally cleaved from the antigen binding domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane. In some embodiments, the leader sequence is a CD27 signal peptide (e.g., a peptide having the amino acid sequence of MARPHPWWLCVLGTLVGLS (SEQ ID NO: 14)). In some embodiments, the leader sequence is an interleukin 2 signal peptide or a CD8 leader sequence. In some embodiments, the leader sequence comprises the amino acid sequence of MALPVTALLLPLALLLHAARP (SEQ ID NO: 15).

[0044] In some embodiments, the CAR described herein further comprises an additional amino acid sequence (e.g., between the extracellular target binding domain and the leader sequence). In some embodiments, the additional sequence is an affinity tag (e.g., Myc tag, EQKLISEEDL (SEQ ID NO: 16)).

[0045] [T cell engaging antibody molecule (TEAM)] In some aspects, the present application discloses a cell (e.g., a T cell) comprising a CAR that binds to CD70 (CD70 CAR) (e.g., as described above) and a TEAM that binds to CD33 (CD33 TEAM). The TEAM described herein is a molecule that comprises a first binding moiety (e.g., an anti-CD33 antibody) and a second binding moiety (e.g., an immune cell binding moiety). Without wishing to be bound by theory, the CD33 TEAM helps overcome cancer antigen escape (e.g., cancer cells no longer express the molecule targeted by the CAR) by providing a second binding site (i.e., CD33) for immune cells to engage the cancer. In some embodiments, the CD33 TEAM engages T cells to the cancer. The CD33 TEAM can engage both CAR-T cells and natural T cells to the cancer cells. As disclosed herein, CAR-T cells expressing a CD70 CAR and a CD33 TEAM have improved efficacy in treating recurrent cancer cells with CD70 loss.

[0046] In some embodiments, the CD33 TEAM comprises an anti-CD33 antibody and an immune cell binding portion. In some embodiments, 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 functional fragments thereof including, but not limited to, single chain variable fragments (scFv), single domain antibodies such as heavy chain variable domain (VH), light chain variable domain (VL) and variable domain (VHH) of camelid-derived nanobody, and alternative scaffolds known in the art to function as antigen binding domains such as recombinant fibronectin domain. In some embodiments, the anti-CD33 antibody comprises, from N-terminus to C-terminus, a VH domain followed by a VL domain. In some embodiments, the anti-CD33 antibody comprises, from N-terminus to C-terminus, a VL domain followed by 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.

[0047] In some embodiments, the anti-CD33 antibody comprises any one of the antibodies or antigen binding domains (e.g., CDRs or VH and VL) described in U.S. Patent No. 11136390, U.S. Patent No. 10556951, U.S. Patent No. 10787514, U.S. Patent No. 8759494, U.S. Patent No. 10000566, U.S. Patent No. 11174313, U.S. Patent No. 10711062, U.S. Patent No. 11466082, and U.S. Patent No. 20210317208, each of which is incorporated by reference in its entirety.

[0048] In some embodiments, the immune cell binding moiety is a molecule that binds to a protein expressed on the surface of T cells, NK cells, dendritic cells, macrophages, B cells, neutrophils, eosinophils, basophils, mast cells, bone marrow-derived suppressor cells, mesenchymal stem cells, or combinations thereof, or any precursor, derivative, or progenitor cell thereof. In some embodiments, the immune cell binding moiety is a molecule that binds to a protein (e.g., CD3) expressed on the surface of T cells. In some embodiments, the immune cell binding moiety binds to a cell surface marker of T cells. In some embodiments, the immune cell binding moiety is selected from the group consisting of CD3, CD8, CD4, CXCR3, CCR4, GARP, LAP, CD25, CTLA-4, or CD16. In some embodiments, the immune cell binding moiety is an antibody described herein (e.g., an antibody that binds to a protein expressed on the surface of T cells). In some embodiments, the immune cell binding moiety includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and functional fragments thereof including, but not limited to, single-chain variable fragments (scFv), heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VHH) of camelid-derived nanobodies, and alternative scaffolds known in the art that function as antigen-binding domains such as recombinant fibronectin domains. In some embodiments, the immune cell binding moiety is an scFv. In some embodiments, the immune cell binding moiety is an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody includes a VH domain followed by a VL domain from N-terminus to C-terminus. In some embodiments, the anti-CD33 antibody includes a VL domain followed by a VH domain from N-terminus to C-terminus. In some embodiments, the immune cell binding moiety includes the amino acid sequence of SEQ ID NO: 21 (anti-CD3 scFv) or a variant thereof.

[0049] In some embodiments, the anti-CD3 antibody comprises any one of the antibodies or antigen-binding domains (e.g., CDRs or VH and VL) described in U.S. Patent No. 9,657,102, U.S. Patent Application Publication No. 2021 / 0244815, U.S. Patent No. 11,530,275, U.S. Patent No. 10,759,858, U.S. Patent Application Publication No. 2022 / 0380464, U.S. Patent Application Publication No. 2021 / 0253701, and U.S. Patent No. 11,505,606.

[0050] In some embodiments, the CD33 TEAM comprises a linker between the CD33 antibody and the immune cell-binding portion. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is non-cleavable. In some embodiments, the linker is a glycine linker. In some embodiments, the linker is a GlySer linker. In some embodiments, the GlySer linker comprises the amino acid sequence GGGS (SEQ ID NO: 34). In some embodiments, the GlySer linker comprises the amino acid sequences (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). In some embodiments, the GlySer linker comprises the amino acid sequences (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). In some embodiments, the linker comprises any one of the amino acid sequences of SEQ ID NOs: 26 - 30. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the linker is any linker described herein.

[0051] In some embodiments, the CD33 TEAM comprises a signal peptide. In some embodiments, the signal peptide is an IgK signal peptide, a CD8 signal peptide, or a CD27 signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of any one of SEQ ID NOs: 14, 15, or 33, or comprises the amino acid sequence of any one of SEQ ID NOs: 14, 15, or 33, or comprises 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 any one of SEQ ID NOs: 14, 15, or 33.

[0052] In some embodiments, 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-cleaving sequence, and a fluorescent protein (e.g., mCherry).

[0053] In some embodiments, the CD33 TEAM is codon-optimized. Codon optimization is the process of introducing silent mutations into a nucleic acid sequence encoding a protein (e.g., a nucleic acid sequence encoding CD33 TEAM) that improves expression of the protein (e.g., CD33 TEAM). Codon optimization does not change the amino acid sequence of the protein. Methods of codon optimization are well known in the art, for example, as described in Mauro et al., BioDrugs 32.1 (2018):69-81.

[0054] In some embodiments, 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. In some embodiments, CD33 TEAM comprises an amino acid sequence of any one of SEQ ID NOs: 17-18. In some embodiments, CD33 TEAM consists of an amino acid sequence of any one of SEQ ID NOs: 17-18.

[0055] [CD70 CAR-CD33 TEAM construct] In some embodiments, the CD70 CAR CD33 TEAM construct comprises a CD70 CAR described herein and a CD33 TEAM described herein. Construct, as used herein, refers to a nucleic acid sequence or an amino acid sequence comprising one or more components (e.g., comprising a CD70 CAR and a CD33 TEAM). In some embodiments, the CD70 CAR-CD33 TEAM construct comprises a CD70 CAR comprising CD27 or a fragment thereof capable of binding to CD70 as described herein. In some embodiments, 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. In some embodiments, the cleavable linker is a protease cleavable linker. In some embodiments, the linker is a self-cleaving linker (e.g., P2A (SEQ ID NO: 32), E2A, F2A, or T2A (SEQ ID NO: 31)). In some embodiments, the linker comprises an internal ribosome entry site (IRES). Without being bound by theory, the CD70 CAR CD33 TEAM construct, when expressed as a fusion protein, may undergo cleavage at the cleavable linker between the CD70 CAR and the CD33 TEAM. This cleavage releases the CD33 TEAM. The CD33 TEAM may be secreted from cells and may bind to cancer cells and immune cells expressing CD33 (e.g., CAR-T cells, non-transduced T cells, endogenous T cells, and / or bystander T cells expressing CD3). In some embodiments, the CD33 TEAM may activate CAR-T cells, non-transduced T cells, endogenous T cells, and / or bystander T cells expressing CD3.

[0056] In some embodiments, the CD70 CAR and CD33 TEAM are each operably linked to a promoter, such as a promoter described herein. In some embodiments, the CD70 CAR and CD33 TEAM are operably linked to the same promoter. In some embodiments, the CD70 CAR and CD33 TEAM are operably linked to different promoters. In some embodiments, the promoter is a constitutive promoter and is described herein. In some embodiments, the promoter is an inducible promoter and is described herein. In some embodiments, the CD70 CAR and CD33 TEAM are operably linked to an EF1 alpha promoter.

[0057] In some embodiments, the CD70 CAR CD33 TEAM construct comprises, from the N-terminus to the C-terminus, a CD70 CAR (e.g., a CD27 CAR), a cleavable linker (e.g., P2A), and a CD33 TEAM. In some embodiments, the CD70 CAR CD33 TEAM construct comprises, from the N-terminus to the C-terminus, a CD33 TEAM, a cleavable linker (e.g., P2A), and a CD70 CAR (e.g., a CD27 CAR). In some embodiments, the CD70 CAR CD33 TEAM comprises a truncated CD27 CAR and a CD33 TEAM. In some embodiments, the CD70 CAR CD33 TEAM comprises, from the N-terminus to the C-terminus, a truncated CD27 (trCD27), a CD8 hinge, a CD8 transmembrane, a 4-1BB co-stimulatory signaling domain, a CD3 zeta domain, a P2A non-transducible cleavable peptide, an IgK leader, a CD33 scFv, and a CD3 scFv. In some embodiments, the CD70 CAR CD33 TEAM comprises, from the N-terminus to the C-terminus, 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. In some embodiments, the CD70 CAR CD33 TEAM comprises, from the N-terminus to the C-terminus, a truncated CD27 (trCD27), a CD8 hinge, a CD8 transmembrane, a 4-1BB 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).

[0058] In some embodiments, the CD70 CAR CD33 TEAM construct 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: 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.

[0059] In some aspects, the disclosure provides nucleic acid molecules (e.g., vectors) for expressing a CD70 CAR CD33 TEAM construct in a cell, e.g., a T cell. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a CD70 CAR CD33 TEAM construct described herein. The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, for example, by screening libraries from cells expressing the gene, or by inducing the gene from a vector known to contain the same, or by direct isolation from cells and tissues containing the same, using standard techniques. Recombinant DNA and molecular cloning techniques used herein are well known in the art and are described, for example, in Sambrook, J., Fritsch, EF and Maniatis, T. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989; and Silhavy, TJ, Bennan, ML and Enquist, LW EXPERIMENTS WITH GENE FUSIONS; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1984; and Ausubel, FM et al., IN CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing and Wiley-Interscience Publishing, 1987; each of which is incorporated herein by reference in its entirety. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0060] In some embodiments, the desired CD70 CAR CD33 TEAM construct may be expressed in cells by transposons. In some embodiments, expression of natural or synthetic nucleic acid CAR is typically achieved by operably linking the nucleic acid encoding the CAR to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration into eukaryotes. Typical cloning vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence. The expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety.

[0061] Additional promoter elements, e.g. enhancers, control the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, but recently, many promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is preserved even when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, activity begins to decrease when the spacing between promoter elements is increased to 50 bp. In some promoters, individual elements appear to be able to function cooperatively or independently to activate transcription.

[0062] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation factor 1a (EF-1a). However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present disclosure is not limited to the use of constitutive promoters. Inducible promoters are also considered part of the present disclosure. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. In some embodiments, the promoter is an EF-1a promoter.

[0063] In some embodiments, 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 many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0064] Furthermore, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors include an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selection markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0065] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of a subject in vivo or ex vivo. In some embodiments, retroviral vectors are used. In some embodiments, lentiviral vectors are used. In some embodiments, adeno-associated virus (AAV) vectors can also be used.

[0066] Vectors derived from retroviruses, such as lentiviruses, are suitable tools to achieve long-term gene transfer, as they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentivirus vectors have the added advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, of being able to transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of being less immunogenic. As used herein, "lentivirus" refers to a genus in the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they are one of the most efficient methods of gene delivery vectors, as they can deliver significant amounts of genetic information into the DNA of host cells. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

[0067] Any method known in the art for delivering nucleic acids or proteins to cells can be used, such as transfection, transformation, transduction, or electroporation. As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0068] [Cells containing CD70 CAR and CD33 TEAM] In some aspects, this application discloses a cell comprising a CD70 CAR and a CD33 TEAM as described herein. In some embodiments, the cell comprises a CD70 CAR-CD33 TEAM construct as described herein. In some embodiments, the cell is an immune cell. In some embodiments, 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, 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, or a combination thereof, or any precursor, derivative, or progenitor thereof. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a human T cell.

[0069] Immune cells (e.g., T cells) can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune cells (e.g., T cells) can also be produced from induced pluripotent stem cells or hematopoietic stem or progenitor cells. In some embodiments, any number of immune cell lines available in the art can be used, including, but not limited to, Hep-2, Jurkat, and Raji cell lines. In some embodiments, immune cells (e.g., T cells) are isolated using Ficoll ELISA. TMCells can be obtained from a blood unit drawn from a subject using any number of techniques known to those skilled in the art, such as separation. In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically includes lymphocytes, including T cells, monocytes, granulocytes, B cells, NK cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution may be calcium-free, magnesium-free, or may not contain many, if not all, divalent cations. Also, surprisingly, an initial activation step in the absence of calcium leads to increased activation. As one of skill in the art can readily appreciate, the washing step can be accomplished by methods known to those of skill in the art, such as using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) following the manufacturer's instructions. After washing, the cells can be centrifuged using, for example, Ca 2+ Free, Mg 2+ The cells can be resuspended in a variety of biocompatible buffers, such as free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, the undesirable components of the apheresis sample can be removed and the cells can be resuspended directly in culture medium.

[0070] In some embodiments, the immune cells (e.g., T cells) are derived from, for example, PERCOLL TM CD3 is isolated from peripheral blood lymphocytes by lysis of red blood cells and depletion of monocytes by gradient centrifugation or counterflow centrifugal elutriation. + , CD28 + , CD4+, CD8 + , CD45RA + , and CD45RO +Specific T cell subpopulations, such as T cells, can be further isolated by positive or negative selection techniques.

[0071] Enrichment of a T cell population by negative selection can be achieved by a combination of antibodies against surface markers unique to the negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, negative selection can enrich for CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, typically CD4 + , CD25 + , CD62L hi , G.I.T.R. + , and FoxP3 + It may be desirable to enrich for or positively select for regulatory T cells expressing T. Alternatively, in some embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar selection methods.

[0072] The modified immune cells (e.g., T cells) can be autologous. "Autologous" means that the immune cells are obtained from a subject, modified to express a CAR described herein, and administered to the same subject. Administration of autologous cells to a subject may reduce immune cell rejection compared to administration of non-autologous cells. Alternatively, the modified immune cells (e.g., T cells) can be allogeneic. "Allogeneic" cells are obtained from a first subject, modified to express a CAR described herein, and administered to a second subject of the same species but different from the first subject. For example, allogeneic immune cells can be derived from a human donor and administered to a human recipient different from the donor.

[0073] In some embodiments, the application discloses a CAR-T cell comprising a CD70 CAR (e.g., a CD27 extracellular binding domain) described herein and a CD33 TEAM (e.g., comprising an anti-CD33 antibody and an anti-CD3 antibody). In some embodiments, 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.

[0074] [Treatment method] In some embodiments, the application discloses a method of treating cancer (e.g., a cancer characterized by cells expressing CD70 and CD33) comprising administering to a subject an effective amount of cells (e.g., CAR-T cells comprising a CD70 CAR and a CD33 TEAM). In some embodiments, the method is for treating a cancer characterized by cells expressing CD70, the method comprising administering to a subject in need thereof an effective amount of the modified immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) or a composition comprising the modified immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) described herein. In some embodiments, the method is for treating a cancer characterized by cells expressing CD70 and cells expressing low levels of CD70 (i.e., lower than the levels of the aforementioned cancer cells expressing CD70) or cells that do not express CD70, the method comprises administering to a subject in need thereof an effective amount of an engineered immune cell (e.g., CD70 CAR CD33 TEAM CAR-T cell) or a composition comprising an engineered immune cell (e.g., CD70 CAR CD33 TEAM CAR-T cell) described herein. In some embodiments, the method comprises administering CD70 CAR CD33 TEAM CAR-T cell to a subject having or diagnosed with a cancer that expresses CD70. In some embodiments, the method comprises administering CD70 CAR CD33 TEAM CAR-T cell to a subject having cancer cells that express CD70. In some embodiments, the method includes administering CD70 CAR CD33 TEAM CAR-T cells to a subject having cancer cells that express CD70 and CD33. In some embodiments, the method includes administering CD70 CAR CD33 TEAM CAR-T cells to a subject having cancer cells that express CD33. In some embodiments, the method includes administering CD70 CAR CD33 TEAM CAR-T cells to a subject having cancer cells that are resistant to CD70 CAR-based therapy (e.g., having reduced CD70 expression or reduced binding of the CD70 CAR to CD70 on the cancer cells due to a mutation).In some embodiments, the methods include administering CD70 CAR CD33 TEAM CAR-T cells to a subject having cancer cells with reduced CD70 expression, where reduced CD70 expression, as used herein, is reduced compared to cancer cells that express a sufficient amount of CD70 to direct the CD70 CAR T cells to kill the cancer cells (e.g., CD70-expressing cancer cells that have not yet been treated with an anti-CD70 therapeutic agent). In some embodiments, the decreased CD70 expression is decreased by 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%) compared to CD70-expressing cancer cells that have not yet been treated with an anti-CD70 therapeutic agent (e.g., a CD70 CAR) and / or CD70-expressing cancer cells that are expected to be killed by anti-CD70 CAR T cells. In some embodiments, the decreased CD70 expression is Molm13. WT is reduced by 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%) compared to CD70 expression on the cell. In some embodiments, reduced CD70 expression means no detectable CD70 expression (e.g., no detectable CD70 cell surface expression).

[0075] In some embodiments, the method comprises administering the CD70 CAR CD33 TEAM CAR-T cells described herein to a subject having a cancer that expresses CD70. One of skill in the art will appreciate that after one or more administrations, some cancer cells may have reduced CD70 expression and may not bind to the CD70 CAR CD33 TEAM CAR-T cells. Thus, in some embodiments, the method of administering the CD70 CAR CD33 TEAM CAR-T cells described herein to a subject having a cancer that expresses CD70 comprises administering one or more doses of the CD70 CAR CD33 TEAM CAR-T cells to the cancer cells that have reduced CD70 expression.

[0076] In some embodiments, the methods include administering CD70 CAR CAR-T cells to a subject having a cancer that expresses CD70, and if the cancer reduces CD70 expression or is otherwise resistant to CD70 CAR-T cells, the methods further include administering CD70 CAR CD33 TEAM CAR-T cells.

[0077] The term "treating" as used herein refers to the application or administration of a composition containing one or more active agents to a subject having a target disease, a symptom of a target disease, or a predisposition to a target disease, for the purpose of curing, curing, alleviating, mitigating, altering, treating, ameliorating, improving, or affecting the disease, the symptom of the disease, or the predisposition to the disease.

[0078] In some embodiments, treating cancer in a subject with CD70 CAR CD33 TEAM CAR-T cells does not result in systemic toxicity. In some embodiments, treating cancer in a subject with CD70 CAR CD33 TEAM CAR-T cells does not result in CD33 TEAM systemic toxicity. In some embodiments, treating cancer in a subject with CD70 CAR CD33 TEAM CAR-T cells does not result in liver toxicity. In some embodiments, treating cancer in a subject with CD70 CAR CD33 TEAM CAR-T cells does not result in CD33 TEAM liver toxicity.

[0079] In some embodiments, the method is for treating a cancer characterized by cells expressing CD70, the method comprising administering to a subject in need thereof an effective amount of an engineered immune cell (e.g., CD70 CAR CD33 TEAM CAR-T cell) or a composition comprising an engineered immune cell (e.g., CD70 CAR CD33 TEAM CAR-T cell) described herein and an effective amount of an agent that enhances expression of CD70 in the cancer (e.g., azacitidine or decitabine).

[0080] Examples of cancers characterized by cells expressing CD70 include, but are not limited to, bladder cancer, invasive breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, diffuse large B-cell lymphoma (DLBC), esophageal cancer, glioblastoma (GBM), head and neck cancer, low-grade glioma (LGG), liver cancer, lung adenocarcinoma, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, gastric cancer, testicular germ cell carcinoma, 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). In some embodiments, the cancer is a lymphoma. In some embodiments, the lymphoma is B-cell non-Hodgkin's 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. In some embodiments, the cancer is leukemia. In some embodiments, the leukemia is acute myeloid leukemia (AML), small lymphocytic lymphoma (SLL), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), B-cell lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), or T-cell leukemia. In some embodiments, the cancer is bone marrow cancer. In some embodiments, the cancer is acute myeloid leukemia.

[0081] Other aspects of the disclosure provide compositions comprising any one of the immune cells described herein (e.g., CD70 CAR CD33 TEAM CAR-T cells). In some embodiments, the composition comprising the modified immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) further comprises an agent that enhances CD70 expression in cancer cells. In some embodiments, the agent results in hypomethylation of the CD-70-encoding gene in the cancer. In some embodiments, the agent is azacitidine or decitabine. In some embodiments, the composition comprises the modified immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) and azacitidine. In some embodiments, the composition comprises engineered immune cells (e.g., CD70-targeted CAR-T cells) and azacitidine, wherein the azacitidine has a concentration in the composition of 100 μM or less (e.g., 100 μM or less, 90 μM or less, 80 μM or less, 70 μM or less, 60 μM or less, 50 μM or less, 40 μM or less, 30 μM or less, 20 μM or less, 10 μM or less, 5 μM or less, 1 μM or less). In some embodiments, the composition comprises engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) and azacitidine, wherein the azacitidine has a concentration in the composition of 100 μM, 90 μM, 80 μM, 70 μM, 60 μM, 50 μM, 40 μM, 30 μM, 20 μM, 10 μM, 5 μM, or 1 μM.

[0082] [Administration] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier, additive, or stabilizer (all of which are referred to as "additives") typically used in the art, such as a buffer, stabilizer, preservative, isotonicity agent, non-ionic surfactant, antioxidant, and / or other miscellaneous additives.

[0083] In some embodiments, any one of the engineered immune cells described herein (e.g., CD70 CAR CD33 TEAM CAR-T cells) or any one of the compositions comprising the engineered immune cells described herein are administered to a subject. Accordingly, some aspects of the present disclosure provide methods of administering any one of the engineered immune cells described herein (e.g., CD70 CAR CD33 TEAM CAR-T cells) or any one of the compositions comprising the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) to a subject.

[0084] In some embodiments, the modified immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) and the agent (e.g., azacitidine or decitabine) are administered simultaneously (e.g., the modified immune cells and the agent are formulated in a composition for administration). In some embodiments, the composition comprises modified immune cells (e.g., CD70-targeted CAR-T cells) and azacitidine, wherein the azacitidine has a concentration in the composition of 100 μM or less (e.g., 100 μM or less, 90 μM or less, 80 μM or less, 70 μM or less, 60 μM or less, 50 μM or less, 40 μM or less, 30 μM or less, 20 μM or less, 10 μM or less, 5 μM or less, 1 μM or less). In some embodiments, the composition comprises modified immune cells (e.g., CD70-targeted CAR-T cells) and azacitidine, wherein the azacitidine has a concentration in the composition of 100 μM, 90 μM, 80 μM, 70 μM, 60 μM, 50 μM, 40 μM, 30 μM, 20 μM, 10 μM, 5 μM, or 1 μM.

[0085] In some embodiments, the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) and the agent are administered sequentially. In some embodiments, the agent (e.g., azacitidine or decitabine) is administered before the engineered immune cells (e.g., CD70-targeted CAR-T cells) are administered. In some embodiments, there is a waiting period between administration of the agent (e.g., azacitidine or decitabine) and administration of the engineered immune cells. The waiting period allows the agent (e.g., azacitidine or decitabine) to enhance CD70 expression in the cancer and be cleared from the subject before the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) are administered. In some embodiments, the waiting period is 3 hours or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 12, 24 hours or more).

[0086] In some embodiments, the agent (e.g., azacitidine or decitabine) 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 that has not been exposed to the agent (e.g., azacitidine or decitabine).

[0087] In some embodiments, administration of both the engineered immune cells (e.g., CD70-targeted CAR-T cells) and the agent (e.g., azacitidine or decitabine) to a subject enhances the therapeutic effect 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 administration of either the engineered immune cells (e.g., CD70-targeted CAR-T cells) or the agent (e.g., azacitidine or decitabine) alone. The therapeutic effect may be measured by methods known in the art, such as, for example, elimination of cancer cells, increased survival of the subject, etc.

[0088] To practice the methods described herein, an effective amount of modified immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) and / or an agent that enhances CD70 expression in cancer (e.g., azacytidine or decitabine) can be administered to a subject via an appropriate route (e.g., intravenous infusion). The immune cell population may be mixed with a pharma- ceutically acceptable carrier prior to administration to form a pharmaceutical composition, which is also within the scope of the present disclosure.

[0089] The subject to be treated may be a mammal (e.g., a 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).

[0090] The term "effective amount" as used herein refers to the amount of each active ingredient required to provide a therapeutic effect to a subject, alone or in combination with one or more other active agents. As recognized by those skilled in the art, the effective amount varies depending on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex and weight, duration of treatment, route of administration, use of additives, combination with other active ingredients (if any), and similar factors within the knowledge and expertise of the medical practitioner. The amount to be administered depends on the subject being treated, including, for example, the ability of the individual's immune system to generate a cellular immune response. The exact amount of active ingredient that needs to be administered depends on the judgment of the medical practitioner. However, suitable dosage ranges can be easily determined by those skilled in the art.

[0091] The therapeutic methods described herein may be used in combination with other types of cancer treatments, such as chemotherapy, surgery, radiation therapy, gene therapy, etc. Such therapies may be administered simultaneously or sequentially (in any order) with the immunotherapy methods described herein. When co-administered with additional therapeutic agents, additive or synergistic effects may result in reduced appropriate therapeutically effective dosages for each agent.

[0092] Non-limiting examples of other anti-cancer therapeutics useful in combination with the engineered 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 inhibitor 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); VEGF antagonists (e.g., anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments); and chemotherapeutic compounds. Exemplary 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, such as vinca alkaloids; microtubule disrupting agents, such as taxanes (e.g., paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones and navelbine, and epidipodophyllotoxins; DNA damaging agents (e.g., acetylcholinesterase inhibitors ... tinomycin, amsacrine, anthracycline, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethinelamine oxaliplatin, ifosfamide, melphalan, merchlorethamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide, and etoposide.

[0093] In some embodiments, radiation, or radiation and chemotherapy, are used in combination with the cell populations comprising the modified immune cells described herein. Additional useful drugs and treatments can be found in Physician's Desk Reference, 59th Suppl., (2005), Thomson PDR, Montvale NJ; Gennaro et al., eds., Remington's The Science and Practice of Pharmacy, 20th Suppl., (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., eds., Harrison's Principles of Internal Medicine, 15th Suppl., (2001), McGraw Hill, NY; Berkow et al., eds., The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway NJ.

[0094] All patents and other publications, including literature references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by reason of prior art or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0095] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, those skilled in the relevant art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, in alternative embodiments, the functions may be performed in a different order, or may be performed substantially simultaneously. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, as appropriate, to provide still further embodiments of the present disclosure using the compositions, functions, and concepts of the above references and applications. Furthermore, considerations of biological function equivalence allow for some modifications to protein structures without affecting biological or chemical action in type or amount. These and other modifications can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0096] Specific elements of any of the foregoing embodiments can be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages in order to fall within the scope of the present disclosure.

[0097] The technology described herein is further illustrated by the following examples, which should not be construed as further limiting in any way. TIFF2025515995000002.tif248170TIFF2025515995000003.tif252170TIFF2025515995000004.tif250170TIFF2025515995000005.tif221170 EXAMPLES

[0098] Acute myeloid leukemia (AML) is the most common acute leukemia in adults and was uniformly fatal half a century ago but is now curable with intensive chemotherapy in 40% of adult cases. 1 Although this represents a significant improvement, there remains significant unmet clinical need for elderly and relapsed / refractory patients, whose cure rates rapidly fall below 10%. 1 Treatment of AML has remained largely unchanged for over 50 years since the introduction of intensive "induction" cytotoxic chemotherapy, but since 2017, eight agents have been FDA approved, including inhibitors of hedgehog, BCL-2, FLT3, and IDH1 / 2, CD33 antibody-drug conjugates, and more potent liposomal formulations of induction chemotherapy. 2 Although these interventions represent major advances, the majority of patients with AML still do not respond or relapse and die from their disease.

[0099] The immunotherapy revolution has shown dramatic responses in many malignancies 3 However, this therapy has had limited success in AML, possibly due to the role of myeloid-derived suppressor cells (MDSCs), T regulatory cells (T reg ), and effector (T eff ) This is due to a lack of neoantigens due to the low number of genetic mutations in tumors, coupled with a cell-rich immunosuppressive microenvironment. 4-8 A recent study of T cell subsets and immune checkpoint expression in newly diagnosed and relapsed AML patients showed that AML patients had significantly higher T cell counts than healthy controls. reg and exhausted T eff Cells were identified as being abundant 9 .

[0100] One way to circumvent T cell priming and the limited natural TCR repertoire is to redirect T cells using chimeric antigen receptors (CARs) and inject CAR-T cells as adoptive immunotherapy. CAR-T cell therapy was shown to be effective in treating two high-grade B cell malignancies in 2017. 10,11, now with a total of four FDA approvals, have rapidly revolutionized the treatment of lymphoid malignancies. Although these therapies result in the destruction of the malignant clone, they also produce off-target tumor effects, eliminating normal B cells and causing hypogammaglobulinemia, which fortunately can be managed by the administration of intravenous immunoglobulin.

[0101] The use of adoptive cell therapy to treat AML has been more challenging. Most of the available surface antigens present on AML blasts are also present in many bone marrow and stem cell populations, and their long-term ablation is incompatible with survival. CARs targeting many different antigens in AML have recently been described (CD123 12,13 , CD33 14,15 , FLT3 16 ), several of which are currently in phase I clinical trials, but none are as ideal as CD19 for lymphoid malignancies. 17 At least one of these caused severe side effects, including the death of the first patient to receive the treatment, which may have been due to side effects resulting from the CAR targeting CD123 on normal vasculature. 18 .

[0102] CD70 is a tumor necrosis alpha family member that functions as a ligand for CD27, which is involved in T cell signaling. CD70 has very limited expression in normal tissues, but is significantly overexpressed in many cancer types, including AML. 19,20 The first AML drug targeting CD70, an antibody-drug conjugate (ADC), ARGX-110, showed impressive response rates in a Phase I study in newly diagnosed AML patients ineligible for traditional therapies. 21 Limited experience to date with blinatumomab, a bispecific T-cell engager targeting CD19, suggests that while CAR-T cells are effective, they may also have greater clinical efficacy than traditional antibody-based therapies. 22 .

[0103] With improvements in the clinical management of CAR-T toxicity, a significant issue currently facing CAR-T cell therapy for lymphoid malignancies is relapse (Figure 2). Particularly in B-cell acute lymphoblastic leukemia (ALL), loss of target antigens can occur in up to 94% of relapsed patients. 23 The harsh reality is that even a small decrease in expression of a well-chosen target antigen may be enough to evade CAR-induced killing. 24 To address the challenge of antigen escape, several strategies have been employed: bispecific CARs that target two antigens containing linked single-chain variable fragments (tandem CARs), or two completely separate CARs expressed on the same T cell (bicistronic CARs), are currently undergoing early-phase clinical trials with varying success. 25-29 We recently developed another novel dual-targeting strategy utilizing bispecific T cell-engaging antibody molecule (TEAM)-secreting CAR-T cells (CAR-TEAM), which simultaneously targets multiple antigens and minimizes target toxicity via localized TEAM secretion restricted to the tumor site. 30 This technology has the added advantage of being able to leverage non-transduced (but infused) T cells, which typically comprise >70% of the total infused cells in clinical-grade CAR-T cell products (due to the need for low MOI to prevent multiple viral integration events) via TEAM secreted from the CAR. The lack of uniform expression of a single unique antigen, particularly in AML, suggests that strategies to enhance expression of target antigens or multi-targeting approaches may be prudent. 19 One particularly well-characterized AML antigen is CD33, which is expressed on up to 90% of leukemic blasts (although it is also expressed on normal bone marrow cells and some progenitor cells, not CD34). + Not expressed in stem cells 31 ) 32 Comprehensive AML Surfacesome analysis of AML cell lines and primary patient samples predicted that co-targeting of CD33 and CD70 in AML was feasible in >97% of patients with non-overlapping bystander tissue toxicity. 19 (Figures 3A-3C). At the same time, we addressed antigen heterogeneity andconventional , T reg , and exhausted T eff To promote local antitumor activity through recruitment of CD3 / CD33 TEAM-secreting CD70-targeted CARs and eliminate immunosuppressive MDSCs, we generated a CD3 / CD33 TEAM-secreting CD70-targeted CAR (CAR-TEAM) (Figures 1 and 5A).

[0104] CD70-targeted CAR-T cells traffic to the bone marrow where they lyse AML blasts while simultaneously delivering tiny amounts of CD33 T cells locally to inhibit T conventional Engages cells and further enhances the effects of CD70-targeted CAR on AML blasts and MDSCs, but does not cause systemic toxicity due to extremely rapid clearance of TEAM in humans 22 (Figure 4).

[0105] Despite the large size of the bicistronic vector, trCD27-CAR-CD33-TEAM showed comparable transduction efficiency to single-target constructs, exceeding 60% in T cells from three healthy donors (Figure 5B). The results show that TEAM is secreted by CAR-T cells (Figure 5C). The results also show that TEAM from the supernatant of trCD27-CAR-CD33-TEAM specifically binds to target-expressing cells (Figure 5D). The results also showed that CD33TEAM reduced the percentage of cells expressing CD69 (Figure 5E).

[0106] In additional experiments, we compared the trCD27-CD33-TEAM and trCD27-CD19-TEAM constructs in treating Molm13 cells that (1) express CD33, (2) do not express CD33, and (3) do not express CD70. CD33TEAM Or CD70CAR CD19TEAM Cells transduced with the construct or non-transduced T cells (UTD) were added to the top of the transwell insert. Non-transduced T cells were transduced with Molm13 WT Or Molm13 CD33KO CD70CAR was added to the bottom of the transwell insert along with the cells. CD33TEAMOnly the construct Molm13 WT The UTD-mediated T cell clearance was able to be mediated in a target-dependent manner and was abolished by the loss of CD33. Wells without UTD at the bottom also failed to kill tumors, demonstrating that the killing was mediated by UTD+TEAM and not TEAM alone (Figure 6). These results indicate that TEAM mediates UTD-driven target-dependent cytotoxicity. The experiment also showed that simultaneous T cell redirection via CAR and TEAM was effective against heterogeneous tumors and was superior to a mixture of individual CARs. Individually targeted CD70 or CD33 CAR-T cells were used against AML targets containing different levels of CD70 or CD33, demonstrating that the CD70CAR CD33TEAM or CD70CAR CD19TEAM (Figure 7A, left). CD33TEAM The cells demonstrated excellent cytotoxicity and CAR proliferation in this real-time cytotoxicity assay in incubates at an effector-to-target ratio of 2:1 (Figures 7A-7B). CAR-T cells were generated in duplicate from T cells of three healthy donors.

[0107] Next, CD70CAR CD33TEAM were tested for efficacy in a xenograft model of AML in vivo. NSG mice were injected with 5 × 10 5 Mixed population of Molm13 cells (90% Molm13 CD70KO , 10% Molm13 WT , n=10 mice / group) on day -7 (Figure 8A). On day 0, mice were injected with 2 x 10 6 CD70CAR CD19TEAM or CD70CAR CD33TEAM Each mouse was administered either CD70CAR T cells or CD80 T cells (n=5 mice per individual T cell donor per group). Continuous BLI monitoring confirmed that CD70CAR T cells were expressed in the 100% IL-17 donor mice. CD33TEAM Tumors were eradicated in the mice, but CD70CAR CD19TEAMTumor growth became uncontrollable after CAR-T injection (Figure 8B-8C). Peripheral blood proliferation was quantified by flow cytometry on days 14-28 after CAR-T injection (Figure 8D). Peripheral blood CAR-T phenotypic profiling was also performed by flow cytometry from mouse blood on day 21 after CAR-T injection and compared to unstimulated, untransduced, donor-matched T cells (Figure 8E).

[0108] CD70CAR CD33TEAM Cells also express CD70CAR in vivo. CD19TEAM We found that CAR-T cells exhibited distinct expression programs compared to CAR-T cells. CAR-T cells were isolated by fluorescence-activated cell sorting (FACS) from the spleens of mice in the mixed tumor model. CAR-T cells were then lysed and subjected to gene expression analysis via Nanostring using a custom "drop-in" gene set plus a CAR-T characterization panel (Figure 9A-9C).

[0109] We next determined whether reducing the dose of CAR-T cells administered to patient-derived xenografted mouse models of AML altered therapeutic efficacy. 5 pcs and 1×10 6 A dose of CD70 CAR CD33 TEAM CAR-T cells was administered (Figure 10). The results showed a similar reduction in the number of CD33+ / CD45+ cells (tumor) in this mouse at both doses.

[0110] Overall, these findings demonstrate the efficacy of CD70-targeted CARs secreting CD33-targeting T cell-engaging antibody molecules against tumor targets with variable CD33 and CD70 antigen expression.

[0111] One of the most well-characterized AML antigens is CD33, which is expressed on up to 90% of leukemic blasts. 31 (Expressed on normal bone marrow cells and some progenitor cells, but not on CD34+ stem cells) 32Importantly, treatment with monoclonal antibodies prior to bone marrow autotransplantation to remove CD33+ cells results in the elimination of committed myeloid progenitors while allowing delayed but normal trilinear hematopoiesis. 33 Surprisingly, CD33 was also found on hepatocytes, and treatment with the CD33 ADC gemtuzumab ozogamicin (GO) caused fatal hepatotoxicity in the form of hepatic veno-occlusive disease (VOD). 34 CD33 can also be found on immunosuppressive MDSCs in the bone marrow of AML patients. Recently, synergistic targeting of these MDSCs and AML using a CD3 / CD33 bispecific T cell engager has been successfully demonstrated in vitro. 35 Comprehensive AML Surfacesome analysis of AML cell lines and primary patient samples predicted that co-targeting of CD33 and CD70 in AML would be feasible in >97% of patients with non-overlapping bystander tissue toxicity. 19 .

[0112] At the same time, addressing antigen heterogeneity and T conventional , T reg , and the exhausted T eff Based on our previously optimized CD70-targeted CAR-T platform, we generated and tested a CD3 / CD33 TEAM-secreting CD70-targeted CAR (CAR-TEAM) to promote local antitumor activity through recruitment of CD3+ / CD33+ and eliminate immunosuppressive MDSCs. 36 .

[0113] In an AML patient-derived xenograft model, a second-generation ligand-based CD70-targeted CAR construct containing a modified hinge and transmembrane domain was previously demonstrated to be superior to the unmodified form in abrogating protease-mediated CAR decapitation (Leick et al. 2022). However, CD70 antigen expression was lost in some mice (Figure 11A). CD33 is a myeloid antigen highly expressed on the majority of AML blasts (indeed, it was expressed at high levels in the PDX model (Figure 11B)), and it has been suggested that dual targeting of CD33 and CD70 by CAR-T cells eliminates the majority of AML with off-tumor toxicity by non-overlapping bystanders (Perna et al. 2017). Therefore, an optimized CD70-targeted CAR was utilized as a platform for developing a dual-targeting strategy by modifying it to secrete a CD33-targeted T cell engaging antibody molecule (TEAM) called 70 33 (Figure 11C). In downstream tests, this construct was compared to a control type that secreted a CD33-targeted CAR (CAR33) and a CD19-targeted TEAM70 19 that is an antigen not seen in typical AML cases (Figure 11D). Despite the additional payload of the secreted TEAM, transduction efficiency was sufficient and equivalent to CAR33 across healthy donor T cells (Figure 11E).

[0114] To ensure proper target binding of the TEAM molecule to its intended antigen, supernatants were collected from Jurkat T cells transduced with each construct, incubated with target cells expressing the relevant antigen, and the presence of TEAM was assayed by flow cytometry (Figure 12A). 70 33 The supernatants bound to CD33-expressing K562 as well as untransduced T (UTD) cells, and 70 19 the supernatants were found to bind to CD19-expressing K562 and UTD. In a separate confirmation assay, 70 33 or 70 19 the supernatants were added to immobilized molm13 tumor targets, followed by addition of an anti-His-PE antibody to enable visualization of TEAM binding by microscopy (Figure 12B).

[0115] Next, to assess the ability of TEAM molecules to activate bystander T cells, we plated 70 μg of T cells into wells containing immobilized tumor targets, UTD T cells, and an antibody against the activation marker CD69. 33 , 70 19 , or UTD supernatant was added. 33 The supernatant was indistinguishable from the UTD supernatant. 19 TEAM-producing cells were found to result in significantly more UTD CD69 expression than TEAM-producing cells (p=.0001) (Figure 12C). Finally, to confirm the cytolytic potential of TEAM, a transwell assay was performed in which TEAM-producing cells were placed on the top of a transwell insert that was too small for cells to pass through but large enough for proteins to pass through, while tumor targets and UTDs were placed on the bottom (Figure 12D). 33 Tumors were eliminated only when producer cells were placed at the top of the transwell and UTD was included at the bottom (p=.018). This phenomenon was target specific and consistent with Molm13 CD33KO 70 cm on top of the transwell insert 33 Or 70 19 They grew at the same rate regardless of whether they had a tumor or not (p=.97).

[0116] After confirming the specificity and cytolytic ability of the CD33 TEAM molecule, we investigated how the whole construct functions under different levels of CD33 and CD70 antigen expression. 19 , 70 33 The killing activity of 70% of wild-type and modified molm13 cells was compared. 33 CAR-T cells are 19 (p=.064) WT molm13 versus CAR33 (p=.007) CD33KO , and 70 19 (p<.0001) CD70KO It was found that the IL-16 mediated excellent cytotoxicity against 70 33CAR-T cells also had the highest proliferation levels across each sort (Figure 13B). 33 Similar results were observed when using (Figure 17).

[0117] double target 70 33 After establishing the in vitro efficacy of the system, we returned to the original PDX model to see if this strategy was an improvement over the original optimized CD70 CAR. In the stress version of the PDX model, NSG mice were injected with 5 × 10 6 5 x 10 fresh PDX cells were injected on day 0, followed by 10 x 10 5 pcs or 1×10 6 70 pieces 33 Or 70 19 The patients were then injected with CAR-T cells (1 / 2 and 1 / 4 of the previous therapeutic dose, respectively) (Figure 14A). 33 CAR-T cells at both dose levels provided superior tumor control by day +14 (p=.0028, p=.0059, respectively; Figure 14B), numerically higher proliferation (p=.78, p=.13, respectively; Figure 14C), and extended survival (both p=.027; Figure 14D).

[0118] To more accurately model the CD70 antigen escape previously identified following CD70 CAR monotherapy, 5 Molm13 WT Cells and Molm13 CD70KO The mixture of cells was injected into NSG mice on day -7, followed by 2 × 10 6 70 pieces 19 , 70 33 , or UTD cells were injected (Figure 15A). 33 CAR-T cells were 70% effective at all time points. 19Tumors from both healthy donors showed significantly higher proliferation by 70% by day +18 compared to 10% by day +14 (day +14; p=.053, day +21; p=.13, day +28; p=.00087). 33 It was eradicated in mice, but 19 CAR-T cells failed to control tumor growth (p=0.0003, Figures 15C-15D). At day +21, both constructs adopted a predominantly central memory-like phenotype (Figure 15E).

[0119] To understand whether there were distinct transcriptional programs driving the behavior of these two CAR-T cells, we collected the spleens of mice from each group on day +28 and performed FACS sorting on CAR-T cells, followed by targeted gene expression analysis (Figure 15F). Key players in CAR-T cell proliferation signaling, such as JAK2 and MYC, are involved in the expression of CAR-T cells. 33 and negative regulators of T cell function such as CISH and PDCD1 (PD-1) were 70 19 Interestingly, the adoption of an NK cell-like phenotype was associated with CAR-T dysfunction (Good et al. 2021), so 19 70% compared to CAR-T cells 33 The gene with the highest fold change in expression was the NK cell receptor NCR1. Unbiased pathway clustering resulted in clustering of CAR constructs with significant differences in signature scores for activation, cytotoxicity, and persistence (Figure 15G, Figure 18). 33 The significantly higher pathway score of TCR signaling in CAR-T cells suggests simultaneous CAR-T activation via CAR and TCR (by engaging with TEAM) (Figure 15H).

[0120] One limitation of CAR-T cell therapy in cancer patients has been the inherently defective T cells used as manufacturing substrates, due to the immunosuppressive effects of the cancer itself or the damage of T cells by intensive chemo-immunotherapy (Das et al. 2019; Fraietta et al. 2018). To evaluate the ability of the TEAM described here to successfully harness AML patient T cells to target AML, PBMCs were collected from AML patients who underwent various intensive chemotherapy and immunotherapy including allogeneic bone marrow transplantation (Figure 16A). T cells were isolated and phenotypic analysis by flow cytometry was performed. T cells from AML patients were found to be more differentiated and exhausted phenotype compared to healthy donor T cells (Figure 16B-16C). AML patient cells showed significant activation in the presence of tumor (p=.036), but at lower levels compared to healthy donor T cells (p=.0052, Figure 16G). To evaluate the cytotoxicity of AML patient T cells acting as bystanders redirected via TEAM, their isolated T cells were co-cultured with TEAM-producing cells and AML targets. AML patients uniformly demonstrated significant cytotoxicity (p=.0002, less potent than healthy donor T cells (p=.017), Figure 16D). AML patient T cells produced proinflammatory cytokines, but interestingly in lower numbers than healthy donor T cells, with the exception of IFNg (Figure 16E).

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Claims

1. A cell containing a chimeric antigen receptor (CAR) that binds to CD70 and a T-cell engagement antibody molecule (TEAM) that binds to CD33.

2. The cell according to claim 1, which is an immune cell.

3. The cell according to claim 2, wherein the immune cell is a T cell, NK cell, dendritic cell, macrophage, B cell, neutrophil, eosinophil, basophil, mast cell, myeloid-derived suppressor cell, mesenchymal stem cell, their precursors, or a combination thereof.

4. The cell according to claim 3, wherein the immune cell is a T cell.

5. Cells according to any one of claims 1 to 4, which are optionally collected from a human subject.

6. CAR, (i) Extracellular target binding domain containing a polypeptide that binds to CD70; (ii) Transmembrane domains; and (iii) Intracellular signaling domain A cell according to any one of claims 1 to 4, comprising

7. The cell according to claim 6, wherein the extracellular target binding domain includes the CD70 binding domain of CD27.

8. The cell according to claim 7, wherein the extracellular target binding domain includes the extracellular domain of CD27.

9. The cell according to claim 8, wherein the extracellular target binding domain comprises an amino acid sequence that is at least 80% identical to any one of the amino acid sequences of SEQ ID NO: 1, 8, or 9.

10. The cell according to claim 9, wherein the extracellular target binding domain comprises any one amino acid sequence of SEQ ID NO: 1, 8, or 9.

11. The cell according to claim 6, wherein the extracellular target binding domain comprises an anti-CD70 antibody, or optionally scFv.

12. The cell according to claim 6, wherein the transmembrane domain is the transmembrane domain of CD27.

13. The cell according to claim 6, wherein the intracellular signaling domain comprises (i) an ITAM-containing signaling domain and / or (ii) one or more signaling domains derived from a costimulatory protein or cytokine receptor.

14. The cell according to claim 13, wherein the intracellular signaling domain includes a CD3γ, CD3ε, CD3δ, or CD3ζ domain.

15. The cell according to claim 14, wherein the intracellular signaling domain includes a CD3ζ domain.

16. The cell according to claim 6, wherein the co-stimulatory domain comprises CD28, 4-1BB, 2B4, KIR, OX40, ICOS, MYD88, IL2 receptor, or SynNotch domain.

17. The cell according to claim 16, wherein the co-stimulatory domain includes a 4-1BB domain.

18. The cell according to any one of claims 1 to 4, wherein the extracellular target binding domain further comprises a signal peptide, and optionally the signal peptide comprises a CD27 signal peptide.

19. A cell according to any one of claims 1 to 4, wherein CAR comprises an amino acid sequence that is at least 80% identical to any one amino acid sequence of sequence numbers 2 to 7.

20. The cell according to any one of claims 1 to 4, wherein CAR comprises one amino acid sequence of sequence numbers 2 to 7.

21. The cell according to any one of claims 1 to 4, wherein the TEAM comprises an anti-CD33 antibody or a functional fragment thereof.

22. The cell according to claim 21, wherein 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.

23. The cell according to claim 21, wherein the anti-CD33 antibody is scFv.

24. The cell according to claim 21, wherein 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.

25. The cell according to claim 24, wherein the VH domain is located at the N-terminus of the VL domain.

26. The cell according to claim 24, wherein the VL domain is located at the N-terminus of the VH domain.

27. The cell according to any one of claims 1 to 4, wherein the TEAM includes an immune cell binding portion, and the immune cell binding portion optionally binds to CD3, CD8, CD4, CXCR3, CCR4, GARP, LAP, CD25, CTLA-4, or CD16.

28. The cell according to claim 27, wherein the immune cell binding region 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.

29. The cell according to claim 27, wherein the immune cell binding site is anti-CD3 scFv.

30. The cell according to claim 27, wherein the TEAM includes a linker between the anti-CD33 antibody and the immune cell binding portion.

31. The cell according to claim 30, wherein the linker is a non-cleaving linker, optionally a (GGGGS)3 (Sequence ID: 27) linker.

32. The cell according to any one of claims 1 to 4, wherein the TEAM further comprises a secretion tag, optionally an IgK secretion tag.

33. A cell according to any one of claims 1 to 4, wherein TEAM comprises an amino acid sequence that is at least 85% identical to any one of sequence numbers 17 to 18.

34. A cell according to any one of claims 1 to 4, comprising a polynucleotide molecule containing a nucleic acid sequence encoding any one of the amino acid sequences of sequence number 17 to 18.

35. A cell according to any one of claims 1 to 4, wherein the nucleic acid sequence encoding TEAM is codon-optimized.

36. A cell according to any one of claims 1 to 4, comprising a first polynucleotide molecule containing a nucleic acid sequence encoding CAR and a second polynucleotide molecule containing a nucleic acid sequence encoding TEAM.

37. A cell according to any one of claims 1 to 4, comprising a polynucleotide molecule containing a nucleic acid sequence encoding CAR and a nucleic acid sequence encoding TEAM.

38. The cell according to claim 37, wherein the polynucleotide molecule further comprises a nucleic acid sequence encoding a linker between a nucleic acid sequence encoding CAR and a nucleic acid sequence encoding TEAM, and optionally the linker is a cleavable linker.

39. The cell according to claim 38, wherein the cleavage linker is self-cleaving and is optionally a P2A, E2A, F2A, or T2A self-cleaving linker.

40. The cell according to claim 37, wherein the cleavage linker contains a protease motif.

41. The cell according to claim 38, wherein the linker includes an intra-sequence ribosome entry site (IRES).

42. The cell according to claim 34, comprising a promoter in which a polynucleotide molecule is ligated to act on a nucleic acid sequence encoding CAR and a nucleic acid sequence encoding TEAM.

43. The cell according to claim 42, wherein the promoter is a constitutively active promoter.

44. The cell according to claim 43, wherein the promoter is the EF1 alpha promoter.

45. The cell according to claim 38, wherein the sense strand of the polynucleotide molecule comprises a nucleic acid sequence encoding CAR, a linker, and a nucleic acid sequence encoding TEAM from 5' to 3'.

46. The cell according to claim 38, wherein the sense strand of the polynucleotide molecule comprises a nucleic acid sequence encoding TEAM, a linker, and a nucleic acid sequence encoding CAR from 5' to 3'.

47. The cell according to claim 34, wherein the polynucleotide molecule comprises a nucleic acid sequence encoding an amino acid sequence that is at least 85% identical to any one of sequence numbers 22 to 25.

48. A polynucleotide comprising nucleic acid sequences encoding CAR and TEAM as described in claim 37.

49. A polypeptide comprising CAR and TEAM according to any one of claims 1 to 4.

50. A pharmaceutical product comprising the cells described in claim 1.

51. A pharmaceutical agent for the treatment of cancer characterized by cancer cells expressing CD70, comprising an effective amount of the cells described in claim 1.

52. A pharmaceutical agent for the treatment of cancer characterized by cancer cells expressing CD70 and CD33, comprising an effective amount of the cells described in claim 1.

53. A pharmaceutical agent for the treatment of cancer characterized by cancer cells expressing CD33, comprising an effective amount of the cells described in claim 1.

54. A pharmaceutical agent for the treatment of cancer characterized by cancer cells in which CD70 expression is reduced, comprising an effective amount of the cells described in claim 1.

55. A pharmaceutical product according to any one of claims 50 to 54, wherein the subject is a human.

56. A pharmaceutical product according to any one of claims 50 to 54, wherein administration includes infusion.

57. The pharmaceutical product according to any one of claims 51 to 54, wherein the cancer is a blood cancer.

58. The pharmaceutical product according to any one of claims 51 to 54, wherein the cancer is bone marrow cancer.

59. The pharmaceutical product according to any one of claims 51 to 54, wherein the cancer is acute myeloid leukemia.