Car-t cells binding CD70, comprising t cell-engaging antibody molecules binding

Abstract

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

Description

[0001] Related Applications

[0002] This application claims protection under 35 U.S.C. §119(e) to the benefit of U.S. Provisional Application No. 63 / 331,758, filed on April 15, 2022, and U.S. Provisional Application No. 63 / 341,995, filed on May 13, 2022, the entire contents of each of the said U.S. Provisional Applications are incorporated herein by reference.

[0003] Government funding

[0004] 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 Art

[0005] Chimeric antigen receptor T cells (CAR-T) are highly effective against certain hematological malignancies. CD70, a member of the TNF-α family, has emerged as a promising surface target antigen in AML. However, antigen escape may occur after antigen-specific cancer therapy, leading to relapse of antigen-negative cancers. Summary of the invention

[0006] The present disclosure relates to methods and compositions for treating cancer characterized by cells expressing CD70 and optionally cells expressing lower levels of CD70 or not expressing CD70 (e.g., after anti-CD70 treatment, such that the cancer is no longer characterized by cells expressing CD70, (e.g., CD70 antigen escape)). The present disclosure relates in part to compositions and methods for overcoming such antigen escape. The present disclosure provides a dual targeting strategy in which CD70 CAR T cells are engineered to express and secrete T cell engaging antibody molecules (TEAMs). In some embodiments, the TEAM comprises a cancer binding portion (e.g., an anti-CD33 antibody) and an immune cell binding portion (e.g., an anti-CD3 antibody). Without wishing to be bound by theory, the TEAM can direct direct binding of CAR-T cells or bystander cells to target cancer cells (e.g., cancer cells expressing CD33) or myeloid-derived suppressor cells (MDSCs) (e.g., MDSCs expressing CD33) without relying on the expression of CD70 CAR.

[0007] Therefore, in one aspect, the present disclosure relates to a cell comprising a chimeric antigen receptor (CAR) and a T cell engaging antibody molecule (TEAM), wherein the CAR binds to CD70 and the TEAM binds to 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 thereof. In some embodiments, the immune cell is a T cell. In some embodiments, the cell is taken from a subject, optionally from a human subject.

[0008] 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 the CD70 binding domain of CD27. In some embodiments, the extracellular target binding domain comprises the 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 NO: 1, 8 or 9. In some embodiments, the extracellular target binding domain comprises an amino acid sequence of any one of SEQ ID NO: 1, 8 or 9. In some embodiments, the extracellular target binding domain comprises an anti-CD70 antibody, optionally scFv. In some embodiments, the transmembrane domain is the transmembrane domain of CD27. In some embodiments, the intracellular signaling domain comprises (i) a signaling domain containing an ITAM 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 domain, a CD3ε domain, a CD3δ domain, or a CD3ζ domain. In some embodiments, the intracellular signaling domain comprises a CD3ζ domain. In some embodiments, the costimulatory domain comprises a CD28 domain, a 4-1BB domain, a 2B4 domain, a KIR domain, an OX40 domain, an ICOS domain, a MYD88 domain, an IL2 receptor domain, or a 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 NO: 2-7. In some embodiments, the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 2-7.

[0009] In some embodiments, the TEAM comprises an anti-CD33 antibody or a functional fragment thereof (e.g., a VH domain and / or a VL domain 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 bifunctional antibody, a nanobody, or a monoclonal antibody. In some embodiments, the anti-CD33 antibody is a scFv. In some embodiments, the anti-CD33 antibody comprises a VH domain and / or a VL domain, the VH domain having an amino acid sequence of SEQ ID NO: 20, and the VL domain having an amino acid sequence of SEQ ID NO: 19. In some embodiments, the VH domain is located at the N-terminus of the VL domain. In some embodiments, the VL domain is located at the N-terminus of the VH domain. In some embodiments, the TEAM comprises an immune cell binding portion. In some embodiments, the immune cell binding portion binds to CD3, CD8, CD4, CXCR3, CCR4, GARP, LAP, CD25, CTLA-4 or CD16. In some embodiments, the immune cell binding portion is selected from the group consisting of: fragment antigen binding region (Fab region), single chain variable fragment (scFv), bifunctional antibody, nano antibody or monoclonal antibody. In some embodiments, the immune cell binding portion is anti-CD3 scFv. In some embodiments, the TEAM comprises a linker, which is located between the anti-CD33 antibody and the immune cell binding portion. 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 comprising an IgK secretion tag. In some embodiments, the TEAM comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 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 to 18. In some embodiments, the nucleic acid sequence encoding the TEAM is codon optimized.

[0010] In some embodiments, the cell comprises a first polynucleotide molecule and a second polynucleotide molecule, the first polynucleotide molecule comprising a nucleic acid sequence encoding the CAR, and the second polynucleotide molecule comprising a nucleic acid sequence encoding the TEAM. In some embodiments, the cell comprises a polynucleotide molecule, the polynucleotide molecule comprising a nucleic acid sequence encoding the CAR and a nucleic acid sequence encoding the TEAM. In some embodiments, the polynucleotide molecule further comprises a nucleic acid sequence encoding a linker, the nucleic acid sequence encoding the linker is located between the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the TEAM, optionally wherein the linker is a cleavable linker. In some embodiments, the cleavable linker is self-cleavable, optionally a P2A self-cleavable linker, an E2A self-cleavable linker, an F2A self-cleavable linker or a T2A self-cleavable 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, and the promoter is operably connected 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 the EF1α promoter.

[0011] In some embodiments, the sense strand of the polynucleotide molecule includes a nucleic acid sequence encoding the CAR, the linker, and a nucleic acid sequence encoding the TEAM from 5' to 3'. In some embodiments, the sense strand of the polynucleotide molecule includes a nucleic acid sequence encoding the TEAM, the linker, and a nucleic acid sequence encoding the CAR from 5' to 3'. In some embodiments, the polynucleotide molecule includes a nucleic acid sequence encoding an amino acid sequence, and the amino acid sequence is at least 85% identical to any one of SEQ ID NO: 22-25.

[0012] In some aspects, the present disclosure relates to a polynucleotide comprising a nucleic acid sequence encoding CAR and TEAM as described herein. In some aspects, the present disclosure relates to a cell comprising CAR and TEAM as described herein.

[0013] In some aspects, the disclosure relates to a method comprising administering to a subject a cell as described herein. In some aspects, the disclosure relates to a method of treating cancer characterized by cells expressing CD70, comprising administering to a subject in need thereof an effective amount of a cell as described herein. In some aspects, the disclosure relates to a method of treating cancer characterized by cancer cells expressing CD70 and CD33, comprising administering to a subject in need thereof an effective amount of a cell as described herein.

[0014] In some aspects, the disclosure relates to a method of treating cancer characterized by cancer cells expressing CD33, the method comprising administering to a subject in need thereof an effective amount of a cell as described herein.

[0015] In some aspects, the disclosure relates to a method of treating cancer characterized by cancer cells having reduced expression of CD70, the method comprising administering to a subject in need thereof an effective amount of a cell as described herein.

[0016] In some embodiments, the subject is a human. In some embodiments, administering comprises infusion. In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a myeloid cell carcinoma. In some embodiments, the cancer is acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The following drawings constitute part of this specification and are included to further illustrate certain aspects of the present disclosure, which aspects may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0018] Figure 1 A diagram of a trCD27-CD8H / TM CAR that secretes a CD33-CD3 bispecific T cell engager (or T cell engaging antibody molecule (TEAM)) is shown. The CD27 CAR can bind to acute myeloid leukemia (AML), and the CD33 TEAM can engage bystander T cells with AML cells.

[0019] Figure 2 Figure 2 shows a graph of flow cytometry data demonstrating CD70 low expression relapse following CD70 targeted CAR therapy in AML PDX. NSG mice were transplanted with 5x10 6 DFAM68555 AML patient-derived xenograft cells. On day 0, mice received 2x10 6Most mice modified with trCD8H / TM cleared their tumors, but some mice experienced recurrence of tumors with low CD70 expression.

[0020] Figures 3A-3C Shown is the paired expression of CD33 and CD70 as combinatorial CAR targets in AML. Figure 3A Co-expression of CD33 and CD70 in blood is shown. Figure 8B Shown is the expression of CD33 and CD70 individually and together in primary AML samples. Figure 3C Shown is the co-expression of the CD33+CD70 antigen pair in AML cells compared to normal BM HSCs and T cells.

[0021] Figure 4 Is to show CAR TEAM Schematic representation of the supposed advantage of: CAR TEAM The cells exhibit rapid renal clearance, sparing hepatocyte damage and having reduced hepatotoxicity.

[0022] Figures 5A-5E Diagrams of exemplary CARs and TEAMs are shown, as well as diagrams demonstrating that TEAMs are secreted by CAR-T cells and can bind to appropriate target antigens. Figure 5A : Structure of trCD27 CAR and / or CD33 TEAM constructs. Figure 5B : Transduction efficiency of activated T cells healthy donor T cells (from 3 different healthy donors and representative of at least 2 independent experiments). Figure 5C : Supernatants from the indicated cells (rows) were added to the indicated cell types (columns) and binding of anti-His tag (TEAM) was assessed. Figure 5D : Supernatants from Jurkat T cells transduced with the indicated constructs were incubated with plate-bound Molm13 tumor cells and anti-His tag AF647 antibody (red). Fluorescence intensity was measured on an Incucyte instrument 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 antibodies. The percentage of cells expressing CD69 over time is shown. PMA (phorbol 12-myristate 13-acetate) is a positive control.

[0023] Figure 6A schematic diagram of the effects of CAR-T cells and TEAMs is shown, as well as a chart indicating that CD33 TEAM mediates target-dependent cytotoxicity driven by UTD. Cells transduced with trCD27 CAR-CD33-TEAM constructs or trCD27-CD19-TEAM constructs or untransduced T cells (UTD) are added to the top of the transwell insert chamber. Untransduced T cells are added to the bottom of the transwell insert chamber together with Molm13WT cells or Molm13CD33 cells. Only the trCD27-CAR-CD33-TEAM construct can mediate the clearance of Molm13WT cells, which is target-dependent and abolished with the loss of CD33.

[0024] Figure 7A-7B Figures and micrographs are shown, demonstrating that simultaneous redirection of T cells by CAR and TEAM is effective against heterogeneous tumors and is superior to the use of a mixture of several CARs alone. Fig. 7A : CAR-T cells targeting CD70 or CD33 alone were compared with CD70CARCD33TEAM or CD70CARCD19TEAM against AML targets with different levels of CD70 or CD33 (left). In a real-time cytotoxicity assay performed on the incucyte instrument, CD70CARCD33TEAM cells showed stronger cytotoxicity and CAR expansion. Figure 7B Representative images at the end of the experiment are shown. Tumor cells are green. The data shown represent the results of CAR-T cells prepared from T cells of 3 healthy donors, each experiment was repeated twice, and the entire experiment was repeated at least twice (n=2 times).

[0025] Figures 8A-8E CD70CAR CD33TEAM Elimination of mixed tumor populations in vivo. Fig. 8A Shown is that on day -7, NSG mice were injected with 5x10 5 A mixed population of Molm13 cells (90% Molm13 CD70KO , 10% Molm13 WT , n=10 mice / group), the cells expressed the bioluminescent (BLI) reporter gene click beetle green. On day 0, mice received 2x10 6 CD70CAR CD19TEAM or CD70CAR CD33TEAM Cells (n=5 mice / single T cell donor / group). Figures 8B-8C Serial BLI monitoring revealed that CD70CARCD33TEAM The tumors of mice in the group were eliminated, but CD70CAR CD19TEAM The tumor growth of mice in the group was not controlled. P values ​​are shown as two-way ANOVA. Fig.8D Cell expansion in peripheral blood measured by flow cytometry on days 14-28 after CAR-T injection is shown. P values ​​were derived from individual Mann-Whitney U tests. Fig. 8E Phenotypic profiling of peripheral blood CAR-Ts in mouse blood by flow cytometry at day +21 after CAR-T injection compared to unstimulated and untransduced donor-matched T cells is shown. Representative examples are shown. CM (central memory), EM (effector memory), TDE (terminally differentiated effector).

[0026] Figures 9A-9C Shown in vivo with CD70CAR CD19TEAM Compared with the expression program of cells, CD70CAR CD33TEAM Cells exhibit different expression programs. Fig. 9A A volcano plot is shown, highlighting the differences in transcriptional programs between constructs. Genes with adjusted p-values ​​less than 0.05 (Benjamini-Hochberg method) are marked in red. Fig. 9B Pathway clustering analysis of all available pathways in an unbiased manner is shown (top). Heatmaps of selected pathways are shown below. Fig. 9C CD70CAR CD33TEAM (right) and CD70CAR CD19TEAM (Left) Pathway scores for TCR signaling. P values ​​were obtained by unpaired t-test.

[0027] Fig.10 Two different doses of CD70CAR administered to a patient-derived xenograft (PDX) AML mouse model are shown CD33TEAM CAR T cells. On day -4, mice were administered 5x10 6 On day 0, mice were administered 5x10 5 or 1x10 6 CD70CAR CD33TEAM CAR T cells. Untreated group and CD70CAR CD19TEAM The group was used as a control. On day 15, the concentration of tumor cells (CD33+ / CD45+) in the blood of mice was measured. The results showed that administration of 5x10 5 and 1x10 6 CD70CAR CD33TEAM CAR T cells had a similar effect on tumor cell concentrations.

[0028] Figures 11A-11E The construction of CD70-CAR-T cells secreting CD33 T cell engaging antibody molecules is shown. Fig.11A Shown, on day -4, NSG mice were transplanted with 5x10 6 DFAM68555 AML patient-derived xenograft cells. On day 0, mice received 2x10 6 Most mice treated with CAR-T cells cleared their tumors, but some mice showed signs of CD70 低 Recurrence of tumor. Fig. 11B CD70 CAR-T cells secreting CD33-CD3 T cell engaging antibody molecules are shown. Fig.11D A schematic diagram of the construct of the CAR used herein is shown. Fig.11E Transduction efficiency of primary healthy donor T cells is shown (n=3 different healthy donors and representative of at least 2 independent experiments).

[0029] Figures 12A-12D It is shown that TEAM is secreted by CAR-T cells and binds to the appropriate target antigen. Fig. 12A Supernatants from the indicated conditions (rows) were added to the indicated cell types (columns) and their binding to anti-His tag (TEAM) was assessed by flow cytometry. Fig. 12B Shown are supernatants from Jurkat T cells transduced with the indicated constructs, incubated with plate-bound K562 tumor cells expressing CD33 and anti-His tag AF647 antibody. Fluorescence intensity was measured on an Incucyte instrument and representative images are shown. Fig. 12C Shown, the supernatant from Jurkat T cells transduced with the specified constructs was co-cultured with Molm13 tumor cells, untransduced (UTD) healthy primary human T cells and anti-CD69 antibodies. The percentage of cells expressing CD69 over time is shown. PMA (phorbol 12-myristate 13-acetate) is a positive control. Fig.12D It is shown that 70 33 Construct or 70 19 Cells transduced with constructs or UTD were added to the top of the transwell insert. UTD was transduced with Molm13 WT or Molm13 CD33KO Cells were added together to the bottom of the transwell insert. Tumor proliferation was measured over time. P values ​​represent the results of two-way ANOVA between the indicated conditions. Experiments were repeated at least n=2 times.

[0030] Figures 13A-13CIt was shown that the simultaneous redirection of T cells by CAR and TEAM was effective against heterogeneous tumors and was superior to the mixed use of several CARs alone. Fig.13A It is shown that at an effector to target ratio of 2:1, the effector to target ratio is 2:1 for the engineered Molm13wt, Molm13 CD70KO 、Molm13 CD33KO AML target, combining single targeted CAR33 T cells with 70 33 or 70 19 CAR-T cells were compared. The expression of the indicated targets assessed by flow cytometry (left) and the results of real-time cytotoxicity assays ( Fig. 13B - above) and CAR-T amplification ( Fig. 13B -below). Fig. 13C Representative images at 40 hours on the incucyte instrument under the specified conditions are shown. The data presented represent the summary data of repeated experiments (repeated twice) of CAR-T cells prepared from T cells of 3 healthy donors. The experiment was repeated at least n=2 times. P values ​​represent the results of two-way ANOVA between the specified conditions.

[0031] Figures 14A-14D It was shown that in a xenograft model derived from AML patients, the effect of redirecting T cells simultaneously through CAR and TEAM was better than that of standard CAR-T cells. Fig.14A As shown, on day -4, 5x10 6 DFAM68555 AML PDX cells were injected into NSG mice (n=5 mice / group). Four days later, on day 0, 5x10 5 or 1x10 6 70 33 or 70 19 CAR-T cells. Fig. 14B It was shown that CD70 CAR-T cells secreting CD33 TEAM (70 33 ) have a higher killing efficiency on circulating CD33+ tumor cells than CD70 CAR-T cells secreting CD19 TEAM (70 19 ). Fig. 14C Shown is 70 in circulation at day +14 33 and 70 19 The number of CAR-T cells. Fig.14D : Survival rate of mice in the experiment. P value is obtained by log-rank test (Mantel Cox).

[0032] Figures 15A-15H It shows that 70 33 Elimination of mixed tumor cell populations in vivo. Fig.15A It was shown that on day -7, NSG mice were injected with 5x10 5 A mixed population of Molm13 cells (90% Molm13 CD70KO , 10% Molm13 WT , n = 10 mice per group), which expressed the bioluminescent (BLI) reporter gene click beetle green. On day 0, mice received 2x10 6 70 19 or 70CAR 33 Cells (n=5 mice / single T cell donor / group). Fig. 15B The expansion of CAR-T cells in peripheral blood measured by flow cytometry on days 14-28 after CAR-T injection is shown. Figures 15C-15D Continuous BLI monitoring of tumor growth is shown. P values ​​represent two-way ANOVA results. Fig.15E Shown is the phenotypic analysis of CAR-T cells in mouse peripheral blood by flow cytometry on day +21 after CAR-T injection and comparison with unstimulated and untransduced donor-matched T cells. Representative examples are shown. CM (central memory), EM (effector memory), TDE (terminally differentiated effector). Fig.15F Shown are CAR-T cells sorted by FACS from the spleen of mice at day +28 in a mixed tumor model, which were subjected to gene expression analysis by NanoString technology, which not only used the CAR-T feature panel, but also added a custom "drop-in" gene set. . The volcano plot highlights the differences in transcriptional programs between different constructs. Genes with adjusted p-values ​​less than 0.05 (Benjamini-Hochberg method) are marked in dark gray. Figure 15G A pathway hierarchical clustering analysis of all available pathways in an unbiased manner is shown (top), and a heat map of selected pathways is presented. Fig.15H Shows 70 33T and 70CAR 19 Pathway scores for TCR signaling. P values ​​were obtained using unpaired t-test.

[0033] Figures 16A-16G The results showed that TEAM can effectively redirect T cells from AML patients to kill tumor cells. Fig.16A Clinical characteristics of AML patients whose T cells were utilized are shown. Fig. 16B Shown is flow cytometric T cell phenotyping of AML patient T cells relative to healthy donor T cells. Fig. 16C Representative phenotypes and expression of PD-1 in T cells from AML patients are shown. Fig.16D Co-culture of TEAM producer cells with isolated AML patient's T cells and MolM13 tumor target is shown. Real-time cytotoxicity was measured on the IncuCyte instrument. Fig.16E Shown are normalized cytokine measurements taken from patients after 18 hours relative to healthy donors. Fig.16F A transwell experiment is shown with TEAM producer cells placed in the upper part and isolated AML patient T cells with tumor targets placed in the lower part. Real-time cytotoxicity (upper part) as well as activation (CD69) (lower part) were measured. Figure 16G Representative images at 40 hours of activation are shown.

[0034] Fig.17 The results showed that simultaneous redirection of T cells by CAR and TEAM is effective for heterogeneous tumors with endogenous antigen expression levels and is superior to the use of a mixture of several CARs alone. At an effector to target ratio of 2:1, single targeted CAR33 T cells were combined with 70 33 or 70 19 CAR-T cells are compared. The expression of the specified targets evaluated by flow cytometry (left) and the results of real-time cytotoxicity detection (top) and CAR-T amplification (bottom) are shown. The data shown represent the combined data obtained from repeated experiments (repeated twice) of CAR-T cells prepared from T cells of 3 healthy donors. The experiment was repeated at least n=2 times. The P value represents the two-way ANOVA results between the specified conditions.

[0035] Fig.18 A pathway hierarchical clustering analysis of all available pathways in an unbiased manner is shown (top), and a heat map of all nanostring available pathways is shown. DETAILED DESCRIPTION

[0036] Chimeric antigen receptor (CAR) binding to CD-70

[0037] In some aspects, the present disclosure provides CAR-T cells targeting CD70, a member of the tumor necrosis factor alpha family, and the use of such CAR-T cells in the treatment of hematological malignancies (e.g., acute myeloid leukemia (AML)). CD70 is continuously expressed on myeloid blasts and leukemia stem cells, but its expression is highly restricted in healthy human tissues. As previously demonstrated, CAR-T cells targeting CD70 achieve antigen-specific activation, cytokine production, and cytotoxic activity in leukemia models in vitro and in vivo, as described, for example, in PCT / US2020 / 051018, the entire contents of which are incorporated herein by reference.

[0038] 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.

[0039] "Chimeric antigen receptor (CAR)" refers to a receptor protein that can perform antigen binding and cell activation functions at the same time after engineering. In some embodiments, CAR comprises a plurality of domains with different functions and interconnected. The CAR domain includes a domain with antigen binding function, a domain with structural function, and a domain with signal transduction function. In some embodiments, CAR comprises at least an extracellular ligand domain, a transmembrane domain, and a cytoplasmic signal transduction domain (also referred to herein as an "intracellular signal transduction domain"), and the cytoplasmic signal transduction domain comprises a functional signal transduction domain derived from a stimulatory molecule as defined below. In some embodiments, CAR comprises an optional leader sequence (also referred to as a "signal peptide"), an extracellular antigen binding domain, a hinge region, a transmembrane domain, and an intracellular stimulatory domain. In some embodiments, the domains in CAR are located in the same polypeptide chain, for example, comprising a chimeric fusion protein. In some embodiments, the various domains in CAR are discontinuous with each other.

[0040] In some embodiments, the CAR described herein comprises an extracellular target binding domain, the extracellular target binding domain comprising a polypeptide that can bind to cluster of differentiation 70 (CD70). "CD70" refers to a polypeptide encoded by the human CD70 gene (NCBI gene ID: 970). As described herein, the expression of CD70 is highly restricted in normal human (non-cancer) tissues. However, CD70 is expressed in a variety of cancers, such as bladder cancer, invasive breast cancer, cervical cancer, bile duct cancer, 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 cancer, thymoma, thyroid cancer, uterine cancer, uveal melanoma, clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma, papillary renal cell carcinoma (pRCC), acute myeloid leukemia and adenoid cystic carcinoma (ACC) (Pan-Cancer Atlas 2018 edition). 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.

[0041] In some embodiments, the polypeptide that binds to CD70 comprises the CD70 binding domain of cluster of differentiation 27 (CD27) (also referred to as CD27 antigen). "CD27" refers to a polypeptide encoded by the human CD27 gene (NCBI gene ID: 939, Uniprot ID: P26842). Examples of CD27 amino acid sequences are provided below.

[0042] MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARS SQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP(SEQ ID NO:8)

[0043] 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 contains multiple cysteine-rich domains (CRDs): CDR1, CDR2, and CDR3. In some embodiments, the CD70 binding domain is located within the CRD2 domain.

[0044] In some embodiments, the CD70 binding domain in CD27 comprises a peptide having an amino acid sequence of TRPHCESCRHCN (SEQ ID NO: 9), which is located in the extracellular domain of CD27. In some embodiments, the extracellular targeting binding domain of the CAR described herein comprises a polypeptide comprising an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical) to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the extracellular targeting binding domain of the CAR described herein comprises an amino acid sequence of SEQ ID NO: 9.

[0045] In some embodiments, the extracellular targeting binding domain of the CAR described herein comprises a polypeptide comprising the extracellular domain of CD27 or a functional fragment thereof (e.g., a fragment capable of binding to CD70). In some embodiments, the functional fragment comprises SEQ ID NO: 9 or a variant thereof. In some embodiments, the extracellular targeting binding domain of the CAR described herein comprises a polypeptide comprising an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical) to the amino acid sequence of SEQ ID NO: 1, 8 or 9. In some embodiments, the extracellular targeting binding domain of the CAR described herein comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 1.

[0046] In some embodiments, the polypeptide bound to CD70 in the extracellular targeting binding domain of the CAR described herein comprises an anti-CD70 antibody. The term "antibody" used herein covers different forms 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 heavy chain variable domains (VH), light chain variable domains (VL) and variable domains (VHH) of camel-derived nanobodies) and alternative scaffolds known in the art that can be used as antigen binding domains (such as recombinant fibronectin domains), etc. In some embodiments, it is beneficial for the antigen binding domain to be derived from the same species in which the CAR will eventually be used. For example, for use in humans, it may be beneficial for the antigen binding domain of the CAR to include antigen binding domain residues of human or humanized antibodies or antibody fragments. In some embodiments, the polypeptide bound to CD70 in the extracellular targeting binding domain of the CAR described herein includes scFv bound to CD70.

[0047] In some embodiments, the CD70 antibody comprises an antibody or antigen binding domain (e.g., CDR or VH and VL) as described in US11434298, US7491390, US8124738, US11377500, or US9701752, the entire contents of each of which are incorporated herein by reference.

[0048] 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 prepared using various techniques known in the art, including but not limited to CDR transplantation (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patents No. 5,225,539, No. 5,530,101 and No. 5,585,089, the entire contents of each of which are incorporated herein by reference), veneering or surface remodeling (see, e.g., European Patent No. EP 592,106 and No. EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5): 489-498; Studnicka et al., 1994, Protein Engineering. Engineering, 7(6):805-814; and Roguska et al., 1994, Proceedings of the National Academy of Sciences (PNAS), 91:969-973, the entire contents of each of which are incorporated herein by reference), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, the entire contents of which are incorporated herein by reference), and techniques disclosed in, e.g., U.S. Patent Application Publication No. US2005 / 0042664; U.S. Patent Application Publication No. US2005 / 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 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. Res., 55(23 Suppl):5973s-5977s (1995); Couto et al., Cancer Research, 55(8):1717-22 (1995); Sandhu JS, Gene, 150(2):409-10 (1994); and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), the entire contents of each of which are incorporated herein by reference.Typically, framework residues in the framework region will be replaced by corresponding residues from the CDR donor antibody to change (e.g., improve) antigen binding ability. These framework substitutions can be identified by methods well known in the art, such as by modeling the interaction between CDR and framework residues to identify framework residues that play an important role in antigen binding, and by sequence alignment to identify abnormal framework residues in specific positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, the entire contents of which are incorporated herein by reference).

[0049] One or more amino acid residues of non-human origin are retained in a humanized antibody or antibody fragment. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from the "import" variable domain. As described herein, a humanized antibody or antibody fragment comprises one or more CDRs and a framework region from a non-human immunoglobulin molecule, wherein the amino acid residues constituting the framework are completely or mostly derived from the human germline. Various techniques for humanization of antibodies or antibody fragments are well known in the art and can be implemented essentially 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)), i.e., by replacing rodent CDRs or CDR sequences with the corresponding sequences of human antibodies, i.e., CDR transplantation (EP 239,400; 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 entire contents of which are incorporated herein by reference). In such humanized antibodies and antibody fragments, a portion of a human variable region domain is substituted with the corresponding sequence from a non-human species, wherein this portion is much less than the entire human variable region domain. Humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted with residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Mol. Immunol., 28(4 / 5):489-498; Studnicka et al., Protein Eng., 7(6):805-814 (1994); and Roguska et al., Proc. Natl. Acad. Sci. USA, 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference.

[0050] In some embodiments, the antibodies are derived from a display library. A display library is a collection of entities; each entity comprises a recoverable component that can obtain a polypeptide component and encodes or identifies the polypeptide component. The polypeptide component is varied so as to present different amino acid sequences. The polypeptide component can have any length, for example, ranging from three amino acids to more than 300 amino acids. The display library entity can contain more than one polypeptide component, such as two polypeptide chains of Fab. In an exemplary embodiment, the display library can be used to identify antigen binding domains. During the screening process, the polypeptide component of each member in 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 usually identified by being retained on a carrier.

[0051] The retained display library members can be recovered from the carrier and analyzed. The analysis can include amplification and subsequent rescreening under similar or dissimilar conditions. For example, positive screening and negative screening can be performed alternately. The analysis can also include determining the amino acid sequence of the polypeptide component and purifying the polypeptide component for detailed characterization.

[0052] The display library can be in a variety of forms. For example, phage display. In phage display, the protein component is usually covalently linked to the phage coat protein. The connection is caused by the translation of the nucleic acid encoding the protein component fused to the coat protein. The connection may include a flexible peptide linker, a protease site, or an amino acid incorporated due to suppression of a stop codon. Phage display technology is described in, for example, U.S. Patent No. 5,223,409; No. WO 92 / 18619; No. WO 91 / 17271; No. WO 92 / 20791; No. WO 92 / 15679; No. WO 93 / 01288; No. WO 92 / 01047; No. WO92 / 09690; No. WO 90 / 02809 and other documents. Phages displaying protein components can be cultured and collected using standard phage preparation methods, for example, PEG precipitation from a growth medium. After screening out a single display phage, the nucleic acid encoding the selected protein component can be isolated from cells infected with the selected phage or from the phage itself after amplification. Individual colonies or plaques can be picked, the nucleic acid isolated and sequenced. Other display formats include cell-based display (see, e.g., WO 03 / 029456), protein-nucleic acid fusion (see, e.g., U.S. Patent No. 6,207,446), ribosome display and E. coli periplasmic display.

[0053] The transmembrane domain of CAR described herein can be derived from a natural source or a recombinant source. In the case where the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. In one aspect, when CAR is bound to a target, the transmembrane domain can conduct a signal to the intracellular domain. The transmembrane domain used in particular in the present invention can include at least, for example, α, β or ζ chains of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8 (e.g., CD8α, CD8β), CD9, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, etc. The transmembrane region. In some embodiments, the transmembrane domain may include at least 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, IL2Rβ, IL2Rγ, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, rfGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(tactile), CEACAM1, CRT In some embodiments, the transmembrane domain is a transmembrane domain of CD27. In some embodiments, the transmembrane domain of CD27 comprises the amino acid sequence of ILVIFSGMFLVFTLAGALFL (SEQ ID NO: 10).

[0054] In some embodiments, the transmembrane domain can be attached to the extracellular region of the CAR (e.g., the ligand domain of the CAR) by a hinge (e.g., a hinge from a human protein). For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge, such as an IgG4 hinge or a CD8a hinge.

[0055] In some embodiments, the cytoplasmic domain or region of CAR described herein includes one or more intracellular signaling domains. The intracellular signaling domain can activate at least one normal effector function of the immune cell into which the CAR is introduced. Examples of intracellular signaling domains for CAR described herein include cytoplasmic sequences of T cell receptors (TCRs) and synergistic cytoplasmic sequences of co-receptors that initiate signal transduction after antigen receptor engagement, and also include any derivatives or variants of these sequences and any recombinant sequences with the same functional capabilities.

[0056] T cell activation can be said to be mediated by two different classes of cytoplasmic signaling sequences: one class is sequences that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains); the other class is sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic domains, such as co-stimulatory domains).

[0057] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain can generate signals to promote the immune effector functions of cells containing CAR (e.g., CAR T cells or NK cells expressing CAR). Examples of immune effector functions in CAR T cells or NK cells expressing CAR include cytolytic activity and auxiliary activity, including the secretion of cytokines. In an embodiment, the intracellular signaling domain transduces effector function signals and guides cells to perform specific functions. Although the entire intracellular signaling domain can be used, it is not necessary to use the entire chain in many cases. In terms of using a truncated portion of an intracellular signaling domain, as long as the truncated portion can conduct an effector function signal, it can be used to replace a complete chain. Therefore, the term "intracellular signaling domain" is intended to include any truncated portion of an intracellular signaling domain sufficient to conduct an effector function signal.

[0058] In some embodiments, one or more intracellular signaling domains include a primary intracellular signaling domain. An exemplary primary intracellular signaling domain includes a domain derived from a molecule responsible for primary stimulation or antigen-dependent stimulation. In some embodiments, the primary intracellular signaling domain includes a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences comprising ITAM include, but are not limited to, signaling sequences derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3θ, CD3η, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FceRI, CD66d, DAP10, and DAP12. In some embodiments, the intracellular signaling domain of CAR includes a CD3-ζ (CD3ζ) signaling domain. In some embodiments, the CD3-zeta (CD3ζ) signaling domain comprises the following amino acid sequence: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 11). In some embodiments, the CD3-zeta (CD3ζ) signaling domain of the CAR described herein comprises an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical) to the amino acid sequence of SEQ ID NO: 11.

[0059] In some embodiments, one or more intracellular signaling domains include a co-stimulatory intracellular domain. A co-stimulatory intracellular signaling domain refers to the intracellular portion of a co-stimulatory molecule. An intracellular signaling domain may include the entire intracellular portion of its source molecule, or the entire native intracellular signaling domain, or a functional fragment thereof. Exemplary co-stimulatory intracellular signaling domains include domains derived from molecules responsible for co-stimulatory signals (e.g., antigen-independent stimulation) and domains derived from cytokine receptors. In some embodiments, one or more intracellular signaling domains include a primary intracellular signaling domain and a co-stimulatory intracellular signaling domain derived from one or more co-stimulatory proteins or cytokine receptors.

[0060] 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 of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, and are necessary for an effective immune response. Examples of such molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD1 la / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 ( KLRFl), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, V LA-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 AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and a ligand specifically bound to CD83. For example, it has been shown that CD27 costimulation can enhance the amplification, effector function and survival of human CART cells in vitro, and improve 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 costimulatory domain of the CAR described herein comprises one or more signaling domains from one or more costimulatory proteins or cytokine receptors, and the one or more costimulatory proteins or cytokine receptors are selected from CD28, 4-1BB, 2B4, KIR, CD27, OX40, ICOS, MYD88, IL2 receptor and SynNotch. In some embodiments, the costimulatory domain of the CAR described herein comprises a 4-1BB costimulatory signaling domain. In some embodiments, the 4-1BB costimulatory signaling domain comprises the following amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 12). In some embodiments, the 4-1BB costimulatory 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.

[0061] In some embodiments, the intracellular signaling domain of the CAR described herein comprises a primary signaling domain (e.g., a domain containing an ITAM, such as a CD3-ζ signaling domain), which may be present alone or in combination with a costimulatory signaling domain (e.g., a costimulatory domain from one or more costimulatory proteins or cytokine receptors, wherein the one or more costimulatory proteins or cytokine receptors are selected from CD28, 4-1BB, 2B4, KIR, CD27, OX40, ICOS, MYD88, IL2 receptor, and SynNotch). In some embodiments, the intracellular signaling domain of the CAR described herein comprises a CD3-ζ (CD3ζ) signaling domain and a 4-1BB costimulatory signaling domain.

[0062] In some embodiments, different linker sequences can be used between different domains of CAR, such as (GGGS) n (SEQ ID NO: 35) linkers, wherein n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20). In some embodiments, the linker is (GGGS) 7 (SEQ ID NO: 36). In some embodiments, CAR comprises additional sequences from CD27, such as the handle and hinge region of CD27 located between the extracellular target binding domain and the transmembrane region. In some embodiments, the handle and hinge region of CD27 comprise the following amino acid sequence: PLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQ TLADFRQLPARTLSTHWPPQRSLCSSDFIR (SEQ ID NO: 13). In some embodiments, the CAR does not comprise additional sequences from CD27, such as the stalk and hinge regions located between the extracellular target binding domain and the transmembrane region.

[0063] In some embodiments, 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 any one of the amino acid sequences of SEQ ID NOs: 2-7. In some embodiments, the CAR described herein comprises an amino acid sequence of any one of SEQ ID NOs: 2-7.

[0064] 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, wherein the leader sequence is optionally cut from the antigen binding domain (e.g., scFv) during cell processing of the CAR and localization to the cell membrane. In some embodiments, the leader sequence is a CD27 signal peptide (e.g., a peptide having the following amino acid sequence: 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 includes the following amino acid sequence: MALPVTALLLPLALLLHAARP (SEQ ID NO: 15).

[0065] In some embodiments, CAR described herein further comprises another 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)).

[0066] T cell engaging antibody molecule (TEAM)

[0067] In some aspects, the present application discloses cells (e.g., T cells) comprising a CAR that can bind to CD70 (CD70 CAR, as described above) and a TEAM that can bind to CD33 (CD33 TEAM). As described herein, TEAM is a molecule comprising a first binding portion (e.g., an anti-CD33 antibody) and a second binding portion (e.g., an immune cell binding portion). Without wishing to be bound by theory, CD33 TEAM helps overcome cancer antigen escape (e.g., cancer cells no longer express the molecule targeted by CAR) by providing a secondary binding site (i.e., CD33) for the engagement of immune cells with cancer. In some embodiments, CD33 TEAM engages T cells with cancer. CD33 TEAM can engage both CAR-T cells and natural T cells with cancer cells. As disclosed herein, CAR-T cells expressing CD70 CAR and CD33 TEAM have improved efficacy in treating recurrent cancer cells with CD70 loss.

[0068] 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 monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies and their functional fragments (including but not limited to single-chain variable fragments (scFv)), single-domain antibodies (such as heavy chain variable domains (VH), light chain variable domains (VL) and variable domains of camel-derived nanobodies (VHH)) and alternative scaffolds known in the art that can be used as antigen-binding domains (such as recombinant fibronectin domains), etc. In some embodiments, the anti-CD33 antibody comprises a VH domain and a VL domain from the N-terminus to the C-terminus. In some embodiments, the anti-CD33 antibody comprises a VL domain and a VH domain from the N-terminus to the C-terminus. In some embodiments, the CD33 antibody is a 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.

[0069] 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 US11136390, US10556951, US10787514, US8759494, US10000566, US11174313, US10711062, US11466082, and US20210317208, the entire contents of each of which are incorporated by reference.

[0070] In some embodiments, the immune cell binding portion is a molecule that can bind to a protein expressed on the surface of 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 a molecule that binds to a protein expressed on the surface of any precursor cell, a derivative cell, or a progenitor cell of the above-mentioned cell. In some embodiments, the immune cell binding portion is a molecule (e.g., CD3) that can bind to a protein expressed on the surface of a T cell. In some embodiments, the immune cell binding portion is bound to a cell surface marker of a T cell. In some embodiments, the immune cell binding portion 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 portion is an antibody as described herein (e.g., an antibody that can bind to a protein expressed on the surface of a T cell). In some embodiments, the immune cell binding portion 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)), single domain antibodies (such as heavy chain variable domains (VH), light chain variable domains (VL) and variable domains of camel-derived nanobodies (VHH)) and alternative scaffolds known in the art that can be used as antigen binding domains (such as recombinant fibronectin domains), etc. In some embodiments, the immune cell binding portion is scFv. In some embodiments, the immune cell binding portion is an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody comprises a VH domain and a VL domain from the N-terminus to the C-terminus. In some embodiments, the anti-CD33 antibody comprises a VL domain and a VH domain from the N-terminus to the C-terminus. In some embodiments, the immune cell binding portion comprises the amino acid sequence of SEQ ID NO: 21 (anti-CD3scFv) or a variant thereof.

[0071] 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 US9657102, US20210244815, US11530275, US10759858, US20220380464, US20210253701, and US11505606.

[0072] 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 sequence (GGGS) 2 (SEQ ID NO: 37), (GGGS) 3 (SEQ ID NO: 26), (GGGS) 4 (SEQ ID NO: 38), (GGGS) 5 (SEQ ID NO: 39), (GGGS) 6 (SEQ ID NO: 40). In some embodiments, the GlySer linker comprises the amino acid sequence (GGGGS)2 (SEQ ID NO: 41), (GGGGS)3 (SEQ ID NO: 27), (GGGGS)4 (SEQ ID NO: 28), (GGGGS)5 (SEQ ID NO: 42), (GGGGS)6 (SEQ ID NO: 43). In some embodiments, the linker comprises the amino acid sequence of any one 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.

[0073] 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 an amino acid sequence of any one of SEQ ID NO: 14, 15, or 33, or comprises an amino acid sequence of any one of SEQ ID NO: 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 NO: 14, 15, or 33.

[0074] In some embodiments, the CD33 TEAM comprises CD33 scFv, a non-cleavable linker, and CD3 scFv. In some embodiments, the CD33 TEAM comprises CD33 scFv, a non-cleavable linker, CD3 scFv, a His6 tag, a T2A self-cleavable sequence, and a fluorescent protein (e.g., red fluorescent protein (mCherry).

[0075] In some embodiments, CD33 TEAM is codon optimized. Codon optimization is a process of introducing silent mutations into a nucleic acid sequence encoding a protein (e.g., a nucleic acid sequence encoding CD33 TEAM) with the purpose of increasing the expression level of the protein (e.g., CD33 TEAM). Codon optimization does not change the amino acid sequence of the protein, and methods for codon optimization are well known in the art, such as the method described by Mauro et al., BioDrugs 32.1 (2018): 69-81.

[0076] In some embodiments, the CD33 TEAM comprises an amino acid sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) identical to any one of SEQ ID NOs: 17-18. In some embodiments, the CD33 TEAM comprises an amino acid sequence of any one of SEQ ID NOs: 17-18. In some embodiments, the CD33 TEAM consists of an amino acid sequence of any one of SEQ ID NOs: 17-18.

[0077] CD70 CAR-CD33 TEAM construct

[0078] In some embodiments, the CD70 CAR CD33 TEAM construct comprises CD70 CAR as described herein and CD33 TEAM as described herein. As used herein, a construct refers to a nucleic acid sequence or amino acid sequence comprising one or more components (e.g., comprising CD70 CAR and CD33 TEAM). In some embodiments, the CD70CAR-CD33 TEAM construct comprises CD70 CAR, and the CD70 CAR comprises 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 CD70CAR and 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-cleavable 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, when the CD70 CAR CD33 TEAM construct is expressed as a fusion protein, it may be cleaved at the cleavable linker between the CD70 CAR and the CD33 TEAM. This cleavage releases the CD33 TEAM. The CD33 TEAM can be secreted by cells and can bind to cancer cells and immune cells expressing CD33 (e.g., CAR-T cells expressing CD3, untransduced T cells, endogenous T cells, and / or bystander T cells). In some embodiments, the CD33 TEAM can activate CAR-T cells expressing CD3, untransduced T cells, endogenous T cells, and / or bystander T cells.

[0079] In some embodiments, CD70 CAR and CD33 TEAM are each operably linked to a promoter, e.g., a promoter described herein. In some embodiments, CD70 CAR and CD33 TEAM are operably linked to the same promoter. In some embodiments, 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 and inducible promoter, and is described herein. In some embodiments, CD70 CAR CD33 TEAM is operably linked to the EF1α promoter.

[0080] In some embodiments, the CD70 CAR CD33 TEAM construct comprises CD70 CAR (e.g., CD27 CAR), a cleavable linker (e.g., P2A), and CD33 TEAM from N-terminus to C-terminus. In some embodiments, the CD70 CAR CD33 TEAM construct comprises CD33 TEAM, a cleavable linker (e.g., P2A), and CD70 CAR (e.g., CD27 CAR) from N-terminus to C-terminus. In some embodiments, the CD70 CAR CD33 TEAM comprises a truncated CD27 CAR and CD33 TEAM. In some embodiments, the CD70 CAR CD33 TEAM comprises a truncated CD27 (trCD27), a CD8 hinge, a CD8 transmembrane domain, a 4-1BB costimulatory signaling domain, a CD3-ζ domain, a P2A cleavable peptide, an IgK leader sequence, a CD33 scFv, and a CD3 scFv from N-terminus to C-terminus. In some embodiments, the CD70 CAR CD33 TEAM comprises, from N-terminus to C-terminus, a truncated CD27 (trCD27), a CD8 hinge, a CD8 transmembrane domain, a 4-1BB intracellular signaling domain, a CD3-ζ signaling domain, a P2A cleavable peptide, an IgK leader sequence, a CD33 scFv, a non-cleavable linker, and a CD3 scFv. In some embodiments, the CD70 CAR CD33 TEAM comprises, from N-terminus to C-terminus, a truncated CD27 (trCD27), a CD8 hinge, a CD8 transmembrane domain, a 4-1BB intracellular signaling domain, a CD3-ζ domain, a P2A cleavable peptide, an IgK leader sequence, a CD33 scFv, a non-cleavable linker, a CD3 scFv, a His6 tag, a T2A cleavable peptide, and a fluorescent protein (e.g., mCherry).

[0081] 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.

[0082] In some aspects, the present disclosure provides nucleic acid molecules (e.g., vectors) for expressing CD70 CARCD33 TEAM constructs in cells (e.g., T cells). 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, such as by screening from a cell library expressing the gene, by obtaining the gene from a vector known to contain the gene, or by isolating it directly from cells and tissues containing the gene using standard techniques. The 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., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing and Wiley-Interscience, 1987 (the entire contents of each of which are hereby incorporated by reference into this document). Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0083] In some embodiments, the desired CD70 CAR CD33 TEAM construct can be expressed in cells by means of a transposon. In some embodiments, the expression of a natural or synthetic nucleic acid CAR is typically achieved by operably linking a nucleic acid encoding a CAR to a promoter and integrating the construct into an expression vector. The vector may be suitable for replication and integration into eukaryotic organisms. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. The expression constructs disclosed herein can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Pat. Nos. 5,399,346, 5,580,859, and 5,589,466, the entire contents of which are incorporated herein by reference.

[0084] The frequency of transcription initiation can be regulated by additional promoter elements (e.g., enhancers). Typically, promoter elements are located in the region of 30-110bp upstream of the start site, but recently many promoters have been found to also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, so when these elements are inverted or moved relative to each other, the function of the promoter can still be retained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50bp apart before the activity begins to decline. Depending on the promoter, individual elements can work collaboratively or independently to activate transcription.

[0085] An example of a suitable promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operatively 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 the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) promoter, the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus (Epstein-Barr virus) immediate early promoter, the Rous sarcoma virus (Rous sarcomavirus) promoter, and human gene promoters, such as but not limited to the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the present disclosure is not limited to the use of constitutive promoters. Inducible promoters are also included as part of the present disclosure. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked thereto when it is desired to express it, and can turn off the expression when it is not desired to express it. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. In some embodiments, the promoter is an EF-1a promoter.

[0086] In some embodiments, the nucleic acid comprising the nucleotide sequence encoding the CD70 CAR CD33 TEAM construct described herein is a vector. The nucleic acid can be cloned into a variety of 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.

[0087] Further, expression vector can be provided to cell in the form of viral vector.Viral vector technology is well known in the art, and is described in, for example, Sambrook et al. (2001, " Molecular Cloning: Laboratory Manual ", Cold Spring Harbor Laboratory in New York) and other virology and molecular biology manuals.Viruses that can be used as vectors include but are not limited to retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses.In general, suitable vectors are included in at least one organism, a replication origin, a promoter sequence, a convenient restriction endonuclease site and one or more selectable markers (for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0088] A variety of virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of a subject in vivo or in vitro. In some embodiments, a retroviral vector is used. In some embodiments, a lentiviral vector is used. In some embodiments, an adeno-associated virus (AAV) vector can also be used.

[0089] Vectors derived from retroviruses (such as lentiviruses, etc.) are suitable tools for achieving long-term gene transfer because they enable transgenes to achieve long-term, stable integration and can be propagated in daughter cells. Compared with vectors derived from oncogenic retroviruses (such as murine leukemia viruses, etc.), lentiviral vectors have additional advantages because they can transduce non-proliferating cells, such as hepatocytes, etc. The lentiviral vector also has the additional advantage of low immunogenicity. As used herein, "lentivirus" refers to a genus of the Retroviridae family. Among retroviruses, lentiviruses are unique in that they can infect non-dividing cells; they can deliver a large amount of genetic information to the DNA of host cells, so lentiviruses are one of the most effective methods in gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a way to achieve high levels of gene transfer in vivo.

[0090] Any method known in the art for delivering nucleic acids or proteins into cells, such as transfection, transformation, transduction or electroporation, can be used. As used herein, the term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acids into host cells. "Transfected" or "transformed" or "transduced" cells are cells that have been transfected, transformed or transduced with exogenous nucleic acids. The cells include primary subject cells and their progeny cells.

[0091] Cells containing CD70 CAR and CD33 TEAM

[0092] In some aspects, the present application discloses cells comprising CD70 CAR and 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. "Immune cells" can be T cells, NK cells, dendritic cells, macrophages, B cells, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells, mesenchymal stem cells, or a combination thereof, or any precursor cell, derivative, or progenitor cell thereof. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a human T cell.

[0093] Immune cells (e.g., T cells) can be obtained from a variety 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 by induced pluripotent stem cells or hematopoietic stem cells or progenitor cells. In some embodiments, any number of immune cell lines available in the art can be used, including but not limited to T cell lines, including, for example, Hep-2, Jurkat, and Raji cell lines. In some embodiments, any number of techniques known to those skilled in the art (such as Ficoll) can be used. TM Immune cells (e.g., T cells) are obtained from a unit of blood collected from a subject by separation. In some embodiments, cells in the circulating blood of an individual can be obtained by apheresis. The product of apheresis generally comprises lymphocytes (T cells), monocytes, granulocytes, B cells, NK cells, other nucleated leukocytes, erythrocytes, and platelets. In some embodiments, the cells collected by apheresis can be washed to remove the plasma fraction, and the cells are placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution does not contain calcium, may not contain magnesium, or may not contain many (if not all) divalent cations. Moreover, surprisingly, performing the initial activation step without calcium results in enhanced activation. As will be readily appreciated by one of ordinary skill in the art, the washing step can be accomplished by methods known to those of skill in the art, such as using a semi-automatic "flow-through" centrifuge (e.g., Baxter CytoMate's Cobe 2991 cell processor or Haemonetics' cell preserver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca-free2+ Mg-free 2+ PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, the apheresis sample can be depleted of unwanted components and the cells resuspended directly in culture medium.

[0094] In some embodiments, immune cells (eg, T cells) are isolated by lysing red blood cells and removing monocytes (eg, by PERCOLL TM Gradient centrifugation or by counterflow centrifugal elutriation) from peripheral blood lymphocytes. Specific T cell subsets, such as CD3 + T cells, CD28 + T cells, CD4 + T cells, CD8 + T cells, CD45RA + T cells and CD45RO + T cells, etc., can be further isolated by positive or negative selection techniques.

[0095] Enrichment of T cell populations by negative selection can be accomplished with the aid of a combination of antibodies against surface markers specific to the negatively selected cells. One approach is to perform cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against surface markers present on the negatively selected cells. For example, to enrich CD4 + Cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be necessary to enrich or positively select for cells that normally express CD4 + 、CD25 + 、CD62L hi GITR + and FoxP3 + Alternatively, in some embodiments, regulatory T cells are depleted by anti-C25 conjugated beads or other similar selection methods.

[0096] Engineered immune cells (e.g., T cells) can be autologous. "Autologous" means that immune cells are taken from a subject, engineered to express CAR described herein and applied to the same subject. Compared with the administration of non-autologous cells, the administration of autologous cells to a subject can reduce the rejection of immune cells. Alternatively, engineered immune cells (e.g., T cells) can be allogeneic cells. "Allogeneic" means that the cell is obtained from a first subject, modified to express CAR described herein, and applied to a second subject, which is different from the first subject but belongs to the same species. For example, allogeneic immune cells can be derived from human donors and applied to human recipients different from donors.

[0097] In some embodiments, the present application discloses a CAR-T cell comprising a CD70 CAR (e.g., a CD27 extracellular binding domain) and a CD33 TEAM (e.g., comprising an anti-CD33 antibody and an anti-CD3 antibody) as described herein. 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.

[0098] Treatment

[0099] In some embodiments, the present application discloses a method for treating cancer (e.g., a cancer characterized by cells expressing CD70 and CD33), the method comprising administering an effective amount of cells (e.g., CAR-T cells comprising CD70 CAR and CD33TEAM) to a subject. In some embodiments, the method is used to treat cancer characterized by cells expressing CD70, and the method comprises administering an effective amount of an engineered immune cell described herein (e.g., CD70 CAR CD33 TEAM CAR-T cell) or a composition comprising the engineered immune cell (e.g., CD70 CARCD33 TEAM CAR-T cell) to a subject in need thereof. In some embodiments, the method is used to treat cancer characterized by cells expressing CD70 and cells expressing low levels of CD70 (i.e., lower than the level of the cancer cells expressing CD70 mentioned above) or cells that do not express CD70, and the method comprises administering an effective amount of the engineered immune cells described herein (e.g., CD70 CAR CD33 TEAM CAR-T cells) or a composition comprising the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) to a subject in need thereof. In some embodiments, the method comprises administering CD70 CAR CD33 TEAM CAR-T cells to a subject suffering from or diagnosed with a cancer expressing CD70. In some embodiments, the method comprises administering CD70 CAR CD33 TEAM CAR-T cells to a subject having cancer cells expressing CD70. In some embodiments, the method comprises administering CD70 CAR CD33 TEAM CAR-T cells to a subject having cancer cells expressing CD70. In some embodiments, the method comprises administering CD70 CAR CD33 TEAM CAR-T cells to a subject having cancer cells expressing CD70 and CD33. In some embodiments, the method includes administering CD70 CAR CD33 TEAM CAR-T cells to a subject with cancer cells expressing CD33. In some embodiments, the method includes administering CD70 CAR CD33 TEAM CAR-T cells to a subject with cancer cells that are resistant to CD70CAR-based treatment (e.g., CD70 expression is reduced or mutations reduce the binding of CD70 CAR to CD70 of cancer cells). In some embodiments, the method includes administering CD70 CAR CD33 TEAM CAR-T cells to a subject with cancer cells with reduced CD70 expression. As used herein, "reduced CD70 expression" is a decrease in expression relative to cancer cells expressing CD70 that can be directed to be killed by CD70 CAR T cells (e.g., cancer cells expressing CD70 that have not been treated with anti-CD70 therapeutic agents).In some embodiments, CD70 expression 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-expressing cancer cells that have not been treated with an anti-CD70 therapeutic agent (e.g., CD70 CAR) and / or CD70-expressing cancer cells that are expected to be killed by anti-CD70 CAR T cells. In some embodiments, with Molm13. WT In some embodiments, "reduced CD70 expression" means that CD70 expression is not detected (e.g., CD70 expression on the surface of a cell is not detected).

[0100] In some embodiments, the method includes administering the CD70CAR CD33 TEAM CAR-T cells described herein to a subject having a cancer expressing CD70. It will be appreciated by those skilled in the art that after one or more administrations, some of the cancer cells may reduce the expression of CD70 to avoid being bound by the CD70 CAR CD33 TEAM CAR-T cells. Therefore, in some embodiments, the method of administering the CD70CAR CD33TEAM CAR-T cells described herein to a subject having a cancer expressing CD70 includes administering one or more rounds of CD70 CARCD33 TEAM CAR-T cells to cancer cells in which CD70 expression has been reduced.

[0101] In some embodiments, the method comprises administering CD70 CAR CAR-T cells to a subject having a cancer that expresses CD70, and where the cancer reduces CD70 expression or otherwise becomes resistant to CD70 CAR-T cells, the method further comprises administering CD70 CAR CD33 TEAM CAR-T cells.

[0102] As used herein, the term "treatment" refers to the application or administration of a composition comprising one or more active agents to a subject suffering from a target disease, experiencing symptoms of a target disease, or having a susceptibility to a target disease, with the purpose of treating, curing, alleviating, alleviating, altering, remedying, modifying, improving, or affecting the disease, symptoms of the disease, or susceptibility to the disease.

[0103] In some embodiments, cancer treatment of a subject using CD70 CAR CD33 TEAM CAR-T cells does not result in systemic toxicity. In some embodiments, cancer treatment of a subject using CD70 CAR CD33 TEAM CAR-T cells does not result in CD33 TEAM systemic toxicity. In some embodiments, cancer treatment of a subject using CD70 CAR CD33 TEAM CAR-T cells does not result in hepatotoxicity. In some embodiments, cancer treatment of a subject using CD70 CAR CD33 TEAM CAR-T cells does not result in CD33 TEAM hepatotoxicity.

[0104] In some embodiments, the method is for treating a cancer characterized by cells expressing CD70, and the method comprises administering to a subject in need thereof an effective amount of an engineered immune cell described herein (e.g., CD70 CARCD33 TEAM CAR-T cell) or a composition comprising the engineered immune cell (e.g., CD70 CAR CD33 TEAM CAR-T cell) and an effective amount of an agent that enhances the expression of CD70 in cancer (e.g., azacitidine or decitabine).

[0105] Examples of cancers characterized by cells expressing CD70 include, but are not limited to, bladder cancer, invasive breast cancer, cervical cancer, bile duct cancer, colorectal cancer, diffuse large B-cell lymphoma (DLBC), esophageal, 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 cancer, thymoma, thyroid cancer, uterine cancer, uveal melanoma, clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma, papillary renal cell carcinoma (pRCC), acute myeloid leukemia, and adenoid cystic carcinoma (ACC). In some embodiments, the cancer is a lymphoma. In some embodiments, the lymphoma is B cell non-Hodgkin lymphoma (NHL), mantle cell lymphoma, Burkitt's lymphoma, B cell lymphoblastic lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma or T cell lymphoma. In some embodiments, the cancer is leukemia. In some embodiments, the leukemia is acute myeloid leukemia (AML), small lymphocytic lymphoma (SLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), B cell lymphoblastic leukemia, chronic lymphocytic leukemia (CLL) or T cell leukemia. In some embodiments, the cancer is myeloid cell carcinoma. In some embodiments, the cancer is acute myeloid leukemia.

[0106] Other aspects of the present disclosure provide compositions comprising any of the immune cells described herein (e.g., CD70CAR CD33 TEAM CAR-T cells). In some embodiments, the composition comprising engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) further comprises an agent that can enhance CD70 expression in cancer cells. In some embodiments, the agent causes low methylation of the coding CD-70 in cancer. In some embodiments, the agent is azacitidine or decitabine. In some embodiments, the composition comprises engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) and azacitidine. In some embodiments, the composition comprises engineered immune cells (e.g., CAR-T cells targeting CD70) and azacitidine, wherein the concentration of azacitidine in the composition is 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 CD33TEAM CAR-T cells) and azacitidine, wherein the concentration of azacitidine in the composition is 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.

[0107] Application

[0108] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient or stabilizer (all of which are referred to as "excipients") commonly used in the art, such as a buffer, stabilizer, preservative, isotonic agent, nonionic detergent, antioxidant and / or other various additives.

[0109] In some embodiments, any of the engineered immune cells described herein (e.g., CD70 CARCD33 TEAM CAR-T cells) or any of the compositions comprising the engineered immune cells described herein are administered to a subject. Therefore, some aspects of the present disclosure provide methods of administering to a subject any of the engineered immune cells described herein (e.g., CD70 CAR CD33 TEAM CAR-T cells) or any of the compositions comprising the engineered immune cells (e.g., CD70 CARCD33 TEAM CAR-T cells).

[0110] In some embodiments, the engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) and the agent (e.g., azacitidine or decitabine) (e.g., the engineered immune cells and the agent are formulated in the form of a composition for administration) are administered simultaneously. In some embodiments, the composition comprises engineered immune cells (e.g., CAR-T cells targeting CD70) and azacitidine, wherein the concentration of azacitidine in the composition is 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., CAR-T cells targeting CD70) and azacitidine, wherein the concentration of azacitidine in the composition is 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.

[0111] In some embodiments, engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) and agents are administered sequentially. In some embodiments, agents (e.g., azacitidine or decitabine) are administered before the engineered immune cells (e.g., CAR-T cells targeting CD70). In some embodiments, there is a waiting period between the administration of agents (e.g., azacitidine or decitabine) and the administration of engineered immune cells. The waiting period is to allow agents (e.g., azacitidine or decitabine) to enhance CD70 expression in cancer and to 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 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 24 hours or more).

[0112] 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 not exposed to the agent (e.g., azacitidine or decitabine).

[0113] In some embodiments, the simultaneous administration of engineered immune cells (e.g., CAR-T cells targeting CD70) and agents (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 times, at least 5 times, at least 10 times or more) compared to when the engineered immune cells (e.g., CAR-T cells targeting CD70) or agents (e.g., azacitidine or decitabine) are administered alone. The therapeutic effect can be measured by methods known in the art, such as clearance of cancer cells, prolonged survival of the subject.

[0114] To practice the methods described herein, an effective amount of engineered immune cells (e.g., CD70 CAR CD33 TEAM CAR-T cells) and or agents that enhance CD70 expression in cancer (e.g., azacitidine or decitabine) can be administered to a subject by a suitable route (e.g., intravenous infusion). Prior to administration, the immune cell population can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.

[0115] The subject to be treated can be a mammal (e.g., a human, mouse, pig, cow, rat, dog, guinea pig, rabbit, hamster, cat, goat, sheep, or monkey). The subject can suffer from cancer or an immune disease (e.g., an autoimmune disease).

[0116] As used herein, the term "effective amount" refers to the amount of each active agent required to impart a therapeutic effect to a subject, either alone or in combination with one or more active agents. As recognized by those skilled in the art, the effective amount varies according to the specific condition being treated, the severity of the condition, the various parameters of the individual patient (including age, physical condition, size, sex and weight), the duration of treatment, the route of administration, the use of excipients, the combined use with other active agents (if any), and similar factors within the knowledge and expertise of the health practitioner. The amount to be administered depends on the subject to be treated, for example, including the ability of the individual's immune system to produce a cell-mediated immune response. The precise amount of active ingredient to be administered depends on the judgment of the practitioner. However, a suitable dosage range can be easily determined by those skilled in the art.

[0117] The treatment methods described herein can be used in combination with other types of therapies for cancer (such as chemotherapy, surgery, radiation, gene therapy, etc.). Such therapies can be administered simultaneously or sequentially (in any order) with the immunotherapy described herein. When co-administered with additional therapeutic agents, the appropriate therapeutically effective dose of each agent may be reduced due to additive or synergistic effects.

[0118] Non-limiting examples of other anti-cancer therapeutics that can be used in combination with the modified immune cells described herein include, but are not limited to, immune checkpoint inhibitors (e.g., PDL1, PD1 and CTLA4 inhibitors), anti-angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteinases, prolactin, angiostatin, endostatin, bFGF soluble receptor, transforming growth factor β, interferon α, soluble KDR and FLT-1 receptors and placenta proliferator-activated protein kinase), VEGF antagonists (e.g., anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments), 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. alkaloids; microtubule disrupting agents such as taxanes (e.g., paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones and vinorelbine, and epidipodophyllotoxin; DNA damaging agents (e.g., actinomycin, amsacrine, anthracycline, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, carbosporin, chloramphenicol ... (including but not limited to oxaliplatin, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamine, oxaliplatin, ifosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide, and etoposide).

[0119] In some embodiments, radiation or radiation and chemotherapy are used in combination with a population of cells comprising the modified immune cells described herein. Additional useful agents and therapies may be found in Physician's Desk Reference, 59th Supplement, (2005), Thomson PDR, Montvale NJ; Gennaro et al., Remington's The Science and Practice of Pharmacy, 20th Supplement, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Harrison's Principles of Internal Medicine, 15th Supplement, (2001), McGraw Hill, NY; Berkow et al., The Merck Manual of Diagnosis and Therapy, 1996. Therapy, (1992), Merck Research Laboratories, Rahway NJ.

[0120] All patents and other publications cited throughout this application, including references, issued patents, published patent applications, and co-pending patent applications, are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, methods described in such publications that may be used in connection with the technology described herein. These publications are provided solely because they were published prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors do not have the right to antedate such disclosures by virtue of prior art or for any other reason. All statements regarding the dates of these documents or descriptions of their contents are based on information available to the applicant and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0121] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the exact form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications can be made within the scope of the present disclosure as will be appreciated by those skilled in the relevant art. For example, although the method steps or functions are presented in a given order, alternative embodiments can perform functions in different orders or can perform functions substantially in parallel. The teachings of the present disclosure provided herein can be appropriately applied to other programs or methods. The various embodiments described herein can be combined to provide additional embodiments. If necessary, various aspects of the present disclosure can be modified to provide more embodiments of the present disclosure using the compositions, functions and concepts in the above-mentioned references and applications. In addition, for the consideration of biological functional equivalence, some changes can be made in the protein structure without affecting the nature or amount of biological or chemical effects. According to the detailed description, these and other changes can be made to the present disclosure. All such modifications are intended to be included within the scope of the appended claims.

[0122] The specific elements in any of the foregoing embodiments may be combined with or replaced with elements in other embodiments. In addition, although the advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages to fall within the scope of the present disclosure.

[0123] The techniques described herein are further illustrated by the following examples, which should in no way be construed as further limiting.

[0124] Table 1: Sequences

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131] Example

[0132] Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Although it was fatal half a century ago, 40% of adult patients can now be cured through intensive chemotherapy. 1While this represents substantial progress, significant unmet clinical needs remain for elderly patients and relapsed / refractory patients, in whom cure rates rapidly decline to less than 10 percent. 1 In the more than 50 years since the advent of intensive “induction” cytotoxic chemotherapy, the treatment of AML has changed little, however, eight FDA drugs have been approved since 2017, including hedgehog inhibitors, BCL-2 inhibitors, FLT3 inhibitors, IDH1 / 2 inhibitors, CD33 antibody-drug conjugates, and liposomal formulations of more potent induction chemotherapy. 2 Although these interventions represent substantial progress, the majority of AML patients remain unresponsive to treatment or relapse and die from their disease.

[0133] The immunotherapy revolution has seen significant efficacy in many malignancies 3 However, this treatment approach has had limited success in AML, likely due to the low tumor mutational burden that results in a lack of neoantigens, coupled with the presence of a large number of myeloid-derived suppressor cells (MDSCs), regulatory T cells (T 调节 ) and exhausted effector T cells (T 效应 ) immunosuppressive microenvironment 4-8 Recent studies on T cell subsets and immune checkpoint expression in newly diagnosed and relapsed AML patients have found that T 调节 and depleted T 效应 Cells are enriched in AML patients 9 .

[0134] One way to circumvent T cell priming and the limited natural TCR repertoire is to redirect T cells using chimeric antigen receptors (CARs) and infuse CAR-T cells as adoptive immunotherapy. CAR-T cell therapy has rapidly revolutionized the treatment of lymphoid malignancies, with two FDA approvals for aggressive B-cell malignancies in 2017. 10,11 , and now there are four. These therapies destroy the malignant clone, however, there are also off-target (on target, off tumor) effects that lead to the elimination of normal B cells, thus causing hypogammaglobulinemia, which fortunately can be controlled by administering intravenous immunoglobulin.

[0135] It has become more difficult to treat AML using adoptive cell therapy. Most of the available surface antigens present on AML blasts are also present in many myeloid and stem cell populations, and their prolonged elimination is incompatible with patient survival. Recently, CARs targeting many different antigens in AML have emerged (CD123 12,13、CD33 14,15 、FLT3 16 ), some of which are currently in phase I clinical trials, but none is as ideal as CD19 for lymphoid malignancies. 17 At least one of these CARs has caused serious side effects, including the death of the first patient treated, which may be caused by side effects of the CAR targeting CD123 on normal blood vessels. 18 .

[0136] CD70 is a member of the tumor necrosis alpha family that acts as a ligand for CD27 involved in T cell signaling. CD70 expression is extremely restricted on normal tissues but is significantly overexpressed in many cancer types, including AML. 19,20 The first AML drug to target CD70, ARGX-110, an antibody-drug conjugate (ADC), showed impressive response rates in a Phase I trial in newly diagnosed AML patients who were ineligible for traditional therapies. 21 Although experience is limited, previous experience with the CD19-targeted bispecific T-cell engager blinatumomab suggests that while effective, CAR-T cells may have a stronger clinical efficacy than traditional antibody-based therapies. 22 .

[0137] With improvements in the clinical management of CAR-T toxicity, the key issue facing CAR-T cell therapy for lymphoid malignancies is relapse ( Figure 2 ). Especially in B-cell acute lymphoblastic leukemia (ALL), up to 94% of relapsed patients may have target antigen loss. 23 Alarmingly, even a modest decrease in the expression of a carefully selected target antigen may be sufficient to evade CAR killing. 24 To address the challenge of antigen escape, several strategies have been used: bispecific CARs targeting two antigens and linked with single-chain variable fragments (tandem CARs) or two completely independent CARs expressed in the same T cell (bicistronic CARs) are currently in early clinical trials with varying success rates. 25-29 Another novel dual-targeting strategy has recently been pioneered, which utilizes CAR-T cells (CAR-TEAMs) that secrete bispecific T cell-engaging antibody molecules (TEAMs) that simultaneously target multiple antigens and minimize on-target toxicity by confining TEAM secretion to tumor sites. 30This technology has the added advantage of utilizing untransduced but infused T cells, which typically comprise >70% of all infused cells in clinical-grade CAR-T cell products (due to the need for low MOI to exclude multiple viral integration events), via TEAMs secreted by the CAR. Particularly in AML, the lack of uniform expression of any single unique antigen suggests that strategies to increase target antigen expression or a multitargeted approach may be prudent. 19 One particularly well-characterized AML antigen is CD33, which is expressed on up to 90% of leukemic blasts (also expressed on normal myeloid cells and some progenitor cells, but not CD34). + Expression on stem cells 31 ) 32 Comprehensive AML surfaceomics analysis of AML cell lines and primary patient samples predicts that simultaneous targeting of CD33 and CD70 in AML will be feasible in more than 97% of patients without overlapping bystander tissue toxicity 19 ( Figures 3A-3C ). In order to simultaneously resolve antigenic heterogeneity, by recruiting T 常规 , T 调节 and depleted T 效应 To promote local antitumor activity and eliminate immunosuppressive MDSCs, we constructed a CD3 / CD33 TEAM (CAR-TEAM) that secretes a CD70-targeting CAR ( Figure 1 and Figure 5A ).

[0138] The CD70-targeted CAR-T cells are transported to the bone marrow where they lyse AML blasts and simultaneously deliver a very small amount of CD33 TEAM locally to engage the T 常规 cells, and further enhance the effect of CD70-targeted CAR on AML blasts and MDSCs, but because TEAM is cleared very quickly in the human body, it does not cause systemic toxicity 22 ( Figure 4 ).

[0139] Despite the larger size of the bicistronic vector, the transduction efficiency of trCD27-CAR-CD33-TEAM in T cells from three healthy donors was comparable to that of the single-targeting constructs, both exceeding 60% ( Figure 5B ). The results showed that TEAM is secreted by CAR-T cells ( Figure 5C The results also showed that TEAM from the supernatant of trCD27-CAR-CD33-TEAM specifically bound to cells expressing the target ( Figure 5D The results also showed that CD33 TEAM reduced the percentage of cells expressing CD69 ( Figure 5E ).

[0140] Additional experiments compared the trCD27-CD33-TEAM construct with the trCD27-CD19-TEAM construct in treating Molm13 cells that (1) express CD33, (2) do not express CD33, and (3) do not express CD70. CD33TEAM Construct or CD70CAR CD19TEAM Construct-transduced cells or untransduced T cells (UTD) were added to the top of the transwell insert. Untransduced T cells were incubated with Molm13 WT Cell or Molm13 CD33KO The cells were added together to the bottom of the transwell insert. CD70CAR only CD33TEAM The construct is able to mediate Molm13 WT Cell clearance was target-dependent and abolished with the loss of CD33. Wells lacking UTD on the bottom were also unable to kill tumors, suggesting that the killing effect was mediated by UTD+TEAM rather than TEAM alone. ( Figure 6 ). These results suggest that TEAM mediates UTD-driven target-dependent cytotoxicity. The experiments also showed that simultaneous redirection of T cells by CAR and TEAM is effective against heterogeneous tumors and is superior to the use of a mixture of several CARs alone. Targeting AML targets with different levels of CD70 or CD33 ( Fig. 7A , left) CAR-T cells targeting CD70 or CD33 alone were combined with CD70CAR CD33TEAM or CD70CAR CD19TEAM In this real-time cytotoxicity assay performed on the incucyte instrument, CD70CAR CD33TEAM The cells showed superior cytotoxicity and CAR expansion capabilities at an effector-to-target ratio of 2:1. Figure 7A-7B ). CAR-T cells were prepared in duplicate from T cells of three healthy donors.

[0141] Next, testing CD70CAR CD33TEAM In vivo efficacy in a xenograft model of AML. NSG mice were injected with 5x10 5 A mixed population of Molm13 cells (90% Molm13 CD70KO , 10% Molm13 WT , n = 10 mice per group), the cells expressed the bioluminescent (BLI) reporter gene beetle green ( Fig. 8A). On day 0, mice received 2x10 6 CD70CAR CD19TEAM or CD70CAR CD33TEAM Cells (n=5 mice per individual T cell donor in each group). Continuous BLI monitoring revealed that CD70CAR CD33TEAM Tumors were eradicated in the mouse group, but CD70CAR CD19TEAM Uncontrolled tumor growth in the Figures 8B-8C ). On days 14-28 after CAR-T injection, the proliferation of peripheral blood CAR-T cells was determined by flow cytometry ( Fig.8D ). At day +21 after CAR-T injection, peripheral blood CAR-T phenotypic profiles were analyzed by flow cytometry in mouse blood and compared with unstimulated and untransduced donor-matched T cells ( Fig. 8E ).

[0142] It was also found that in vivo, CD70CAR CD19TEAM Compared with the expression program of cells, CD70CAR CD33TEAM The cells exhibited different expression programs. CAR-T cells were isolated from the spleen of mixed tumor model mice by fluorescence activated cell sorting (FACS). Then, the CAR-T cells were lysed and gene expression analysis was performed using the CAR-T cell characterization panel by nanostring technology in addition to the custom 'insert' gene set ( Figures 9A-9C ).

[0143] Next, we determined whether reducing the dose of CAR-T cells administered to a patient-derived xenograft AML mouse model would alter the therapeutic effect. 5 and 1x10 6 CD70 CAR CD33 TEAM CAR-T cells ( Fig.10 The results showed that the number of CD33+ / CD45+ cells (tumor cells) in mice treated with both doses was reduced to a similar extent.

[0144] Overall, these findings demonstrate the efficacy of a CD70-targeted CAR that secretes a CD33-targeted T cell-engaging antibody molecule against tumor targets with variable expression of CD33 and CD70 antigens.

[0145] One well-characterized AML antigen is CD33, which is expressed on up to 90% of leukemic blasts. 31 (Also expressed on normal myeloid cells and some progenitor cells, but not on CD34+ stem cells32 Importantly, depletion of CD33+ cells by treatment of autologous bone marrow transplants with monoclonal antibodies resulted in the elimination of committed myeloid progenitors, but normal trilineage hematopoiesis occurred, albeit with a delay. 33 Unexpectedly, CD33 is also present on hepatocytes, and treatment with the CD33 ADC, gemtuzumab ozogamicin (GO), has resulted in 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 patients with AML. Recently, studies have demonstrated the success of synergistic targeting of these MDSCs and AML using CD3 / CD33 bispecific T cell engagers in vitro. 35 Comprehensive AML surfaceomics profiling of AML cell lines and newly diagnosed patient samples predicted that simultaneous targeting of CD33 and CD70 in AML would be feasible in more than 97% of patients without overlapping toxicities involving bystander tissues. 19

[0146] To simultaneously resolve antigenic heterogeneity, T 常规 , T 调节 and depleted T 效应 To promote local anti-tumor activity and eliminate immunosuppressive MDSCs, the researchers constructed and tested a CD70-targeting CAR (CAR-TEAM) that secretes CD3 / CD33 TEAM based on the previously optimized CD70-targeting CAR-T platform. 36

[0147] Second-generation ligand-based CD70-targeting CAR constructs with modified hinge and transmembrane domains have previously been shown to abrogate protease-mediated CAR cleavage in patient-derived xenograft models of AML relative to unmodified versions (Leick et al. 2022). However, in some mice, CD70 antigen expression is lost ( Fig.11A CD33 is a myeloid cell antigen that is highly expressed on most AML blasts (and, in fact, is also expressed at high levels in PDX models, Fig. 11B ), and it has been shown that the combination of CD33 and CD70 targeting by CAR-T cells can eliminate the majority of AML without non-overlapping off-target toxicity involving bystanders (Perna et al. 2017). Therefore, using the optimized CD70-targeting CAR as a platform, a dual targeting strategy was developed by modifying it to secrete a CD33-targeted T cell engaging antibody molecule (TEAM), which was named 70 33 ( Fig. 11CFor downstream testing, this construct was compared with a CD33-targeted CAR (CAR33) and a control version that secreted TEAM 70 targeting CD19. 19 , (CD19 is an antigen not found in typical AML cases) Fig.11D Despite the addition of the secreted TEAM payload, the transduction efficiency was sufficient in T cells from healthy donors and comparable to that of CAR33 ( Fig.11E ).

[0148] To ensure proper target binding of TEAM molecules to their intended antigens, supernatants of Jurkat T cells transduced with each construct were collected and incubated with target cells expressing the relevant antigen and the presence of TEAM was determined by flow cytometry ( Fig. 12A ). It was found that 70 33 The supernatant showed binding to CD33-expressing K562 and untransduced T (UTD) cells, whereas 70 19 The supernatant was combined with K562 and UTD expressing CD19. In a separate confirmatory assay, 70 33 The supernatant or 70 19 The supernatant was added to the immobilized molm13 tumor target, followed by the addition of anti-His-PE antibody, allowing visualization of TEAM binding by microscopy ( Fig. 12B ).

[0149] Next, to evaluate the ability of TEAM molecules to activate bystander T cells, 70 33 Supernatant, 70 19 Supernatants or UTD supernatants were added to wells containing immobilized tumor targets, UTD T cells, and antibodies against the activation marker CD69. 33 The supernatant resulted in a higher UTD CD69 expression than 70 19 The expression of UTD CD69 was significantly higher (p = .0001). 19 The UTD CD69 expression caused by the UTD supernatant was indistinguishable from that caused by the UTD supernatant ( Fig. 12C Finally, to determine the cytolytic capacity of TEAM, a transwell assay was performed in which TEAM-producing cells were placed in the top portion of a transwell insert (too small for cells to pass through, but large enough for protein to pass through), while the tumor target and UTD were placed in the bottom portion ( Fig.12D ). Only if 70 33Tumors were eliminated only when 70 cells were placed on the top of the transwell and the bottom contained UTD (p = .018). This phenomenon was target specific because no matter whether the top of the transwell insert contained 70 33 Still 70 19 ,Molm13 CD33KO (CD33 knockout Molm cells) all grew at the same rate (p=.97).

[0150] After confirming the specificity and cytolytic capacity of the CD33 TEAM molecule, the performance of the whole construct at different levels of CD33 and CD70 antigen expression was studied. 19 , 70 33 The killing ability of 70 33 CAR-T cells mediate superior cytotoxicity against: molm13 WT Compared to 70 19 (p=.064), molm13 CD33KO Compared with CAR33 (p = .007) and molm13 CD70KO Compared to 70 19 (p<.0001)( Figures 13A-13C ). 70 33 CAR-T cells also had the highest levels of expansion in different combinations of each condition ( Fig. 13B ). When using 70 33 Similar results were observed when targeting different native levels of CD33 and CD70 in AML cell lines Kasumi1, Monomac1, and OCI-AML3 ( Fig.17 ).

[0151] After establishing the dual-targeting 70 33 After evaluating the in vitro efficacy of the system, we returned to the original PDX model to determine if this strategy was an improvement over the original optimized CD70 CAR. In the stressed version of the PDX model, NSG mice were injected with 5x10 6 fresh PDX cells, followed by 5x10 5 or 1x10 6 70 33 or 70 19 CAR-T cells (previous treatment dose, respectively 1 / 2 and 1 / 4)( Fig.14A ). At both dose levels, by day +14, 70 33CAR-T cells produced superior tumor control (p = .0028, p = .0059, Fig. 14B ), numerically higher amplification (p = .78, p = .13, Fig. 14C ) and prolonged survival (p = .027 for both, Fig.14D ).

[0152] To more accurately mimic the CD70 antigen escape previously identified after CD70 CAR monotherapy, 5x10 5 Molm13 WT Cells and Molm13 CD70KO The mixture of cells was injected into NSG mice, and 2x10 6 70 19 , 70 33 or UTD cells ( Fig.15A ). Compared with 70 19 , 70 33 The expansion of CAR-T cells was significantly higher at each time point (day +14, p = .053; day +21, p = 0.13; day +28, p = .00087). 33 Tumors in mice were inoculated with 70% leukemia cells from two healthy donors on day +18. 33 CAR-T cells completely eliminated, while 70 19 CAR-T cells failed to control tumor growth (p = 0.0003, Figures 15C-15D At day +21, both constructs showed mainly a central memory-like phenotype ( Fig.15E ).

[0153] To understand whether there are different transcriptional programs driving the behavior of these two CAR-T cells, spleens from mice in each group were collected on day +28 and FACS sorted for CAR-T cells, which then underwent targeted gene expression analysis ( Fig.15F ). 33 In CAR-T, the expression of key factors involved in CAR-T cell tissue signal transduction, such as JAK2 and MYC, was high, while in 70 19 In CAR-T cells, expression of negative regulators of T cell function, such as CISH and PDCD1 (PD-1), was higher. Interestingly, since the adoption of an NK cell-like phenotype has been associated with CAR-T dysfunction (Good et al. 2021), in 70 33 Compared to 70 19The gene with the highest expression fold change in CAR-T cells was the NK cell receptor NCR1. Unbiased pathway clustering analysis showed that the constructs clustered separately and had key differences in the gene set scores (signature scores) for activation, cytotoxicity, and persistence ( Figure 15G , Fig.18 ). 70 33 The score of TCR signaling pathway in CAR-T cells was significantly higher, indicating that CAR-T cells can be activated by both CAR and TCR (through engagement with TEAM) ( Fig.15H ).

[0154] One limitation of CAR-T cell therapy in cancer patients is that the T cells used to prepare the matrix themselves are inherently defective due to the immunosuppressive effects of the cancer itself or due to damage to T cells caused by intensive chemoimmunotherapy (Das et al. 2019; Fraietta et al. 2018). To evaluate the ability of the TEAM described in this article to successfully use T cells from AML patients to target AML, PBMCs were obtained from AML patients who had received multiple intensive chemotherapy and immunotherapy (including allogeneic bone marrow transplantation) Fig.16A ). T cells were isolated and phenotyped by flow cytometry. The results showed that compared with T cells from healthy donors, T cells from AML patients showed a more differentiated and exhausted phenotype ( Figures 16B-16C AML patients' cells showed significant activation in the presence of tumor (p = .036), although the level of activation was lower than that of T cells from healthy donors (p = .0052, Figure 16G To evaluate the cytotoxicity of T cells from AML patients as bystander cells redirected by TEAM, T cells isolated from these patients were co-cultured with TEAM producer cells and AML targets. AML patients uniformly showed significant cytotoxicity (p = .0002, although the potency was lower than that of healthy donor T cells, p = .017, Fig.16D ). T cells from AML patients produced inflammatory cytokines, albeit in lower amounts than those produced by T cells from healthy donors, with the exception of IFNg ( Fig.16E ).

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Claims

1. A cell comprising a chimeric antigen receptor (CAR) and a T cell engaging antibody molecule (TEAM), wherein the CAR binds to CD70 and the TEAM binds to CD33.

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

3. The cell according to claim 2, wherein 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 thereof.

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

5. The cell according to any one of claims 1 to 4, wherein the cell is taken from a subject, optionally a human subject.

6. The cell according to any one of claims 1 to 5, wherein the CAR comprises: (i) an extracellular target binding domain, the extracellular target binding domain comprising a polypeptide that binds to CD70; (ii) a transmembrane domain; and (iii) Intracellular signaling domain.

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

8. The cell of claim 7, wherein the extracellular target binding domain comprises the extracellular domain of CD27.

9. The cell of claim 8, wherein 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 NO: 1, 8 or 9.

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

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

12. The cell according to any one of claims 6 to 11, wherein the transmembrane domain is the transmembrane domain of CD27.

13. The cell of any one of claims 6 to 12, wherein the intracellular signaling domain comprises (i) an ITAM-containing signaling domain and / or (ii) one or more signaling domains from one or more co-stimulatory proteins or cytokine receptors.

14. The cell of claim 13, wherein the intracellular signaling domain comprises a CD3γ domain, a CD3ε domain, a CD3δ domain, or a CD3ζ domain.

15. The cell of claim 14, wherein the intracellular signaling domain comprises a CD3 zeta domain.

16. according to the cell described in any one of claims 6 to 15, wherein the costimulatory domain comprises CD28 domain, 4-1BB domain, 2B4 domain, KIR domain, OX40 domain, ICOS domain, MYD88 domain, IL2 receptor domain or SynNotch domain.

17. The cell of claim 16, wherein the co-stimulatory domain comprises a 4-1BB domain.

18. The cell according to any one of claims 1 to 17, wherein the extracellular target binding domain further comprises a signal peptide, optionally wherein the signal peptide comprises a CD27 signal peptide.

19. The cell of any one of claims 1 to 18, wherein 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.

20. The cell of any one of claims 1 to 19, wherein the CAR comprises the amino acid sequence of any one of SEQ ID NOs: 2-7.

21. The cell according to any one of claims 1 to 20, wherein the TEAM comprises an anti-CD33 antibody or a functional fragment thereof.

22. The cell of 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 of claim 21 or claim 22, wherein the anti-CD33 antibody is a scFv.

24. The cell according to any one of claims 21 to 23, wherein the anti-CD33 antibody comprises a VH domain and / or a VL domain, the VH domain having the amino acid sequence of SEQ ID NO: 20, and the VL domain having the amino acid sequence of SEQ ID NO:

19.

25. The cell of claim 24, wherein the VH domain is N-terminal to the VL domain.

26. The cell of claim 24, wherein the VL domain is N-terminal to the VH domain.

27. The cell of any one of claims 1 to 26, wherein the TEAM comprises an immune cell binding portion, optionally the immune cell binding portion binds to CD3, CD8, CD4, CXCR3, CCR4, GARP, LAP, CD25, CTLA-4 or CD16.

28. The cell of claim 27, wherein 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.

29. A cell according to claim 27 or claim 28, wherein the immune cell binding moiety is an anti-CD3 scFv.

30. The cell of any one of claims 27 to 29, wherein the TEAM comprises a linker located between the anti-CD33 antibody and the immune cell binding moiety.

31. The cell of claim 30, wherein the linker is a non-cleavable linker, optionally a (GGGGS)3 (SEQ ID NO: 27) linker.

32. The cell of any one of claims 1 to 31, wherein the TEAM further comprises a secretion tag, optionally comprising an IgK secretion tag.

33. The cell of any one of claims 1 to 32, wherein the TEAM comprises an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 17-18.

34. The cell of any one of claims 1 to 33, wherein the cell comprises a polynucleotide molecule comprising a nucleic acid sequence encoding the amino acid sequence of any one of SEQ ID NOs: 17-18.

35. The cell of any one of claims 1 to 34, wherein the nucleic acid sequence encoding the TEAM is codon optimized.

36. The cell according to any one of claims 1 to 35, comprising a first polynucleotide molecule and a second polynucleotide molecule, wherein the first polynucleotide molecule comprises a nucleic acid sequence encoding the CAR and the second polynucleotide molecule comprises a nucleic acid sequence encoding the TEAM.

37. The cell according to any one of claims 1 to 35, comprising a polynucleotide molecule comprising a nucleic acid sequence encoding the CAR and a nucleic acid sequence encoding the TEAM.

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

39. The cell of claim 38, wherein the cleavable linker is self-cleavable, optionally a P2A self-cleavable linker, an E2A self-cleavable linker, a F2A self-cleavable linker or a T2A self-cleavable linker.

40. The cell of claim 37, wherein the cleavable linker comprises a protease motif.

41. The cell of claim 38, wherein the linker comprises an internal ribosome entry site (IRES).

42. The cell according to any one of claims 34 to 41, wherein the polynucleotide molecule comprises a promoter, which is operably linked to the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the TEAM.

43. The cell of any one of claims 42, wherein the promoter is a constitutively active promoter.

44. The cell of any one of claims 43, wherein the promoter is the EF1α promoter.

45. The cell according to any one of claims 39 to 44, wherein the sense strand of the polynucleotide molecule comprises, from 5' to 3', the nucleic acid sequence encoding the CAR, the linker, and the nucleic acid sequence encoding the TEAM.

46. ​​The cell according to any one of claims 39 to 44, wherein the sense strand of the polynucleotide molecule comprises, from 5' to 3', the nucleic acid sequence encoding the TEAM, the linker, and the nucleic acid sequence encoding the CAR.

47. The cell of any one of claims 34 to 46, wherein the polynucleotide molecule comprises a nucleic acid sequence encoding an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 22-25.

48. A polynucleotide comprising a nucleic acid sequence encoding CAR and TEAM according to any one of claims 37 to 44.

49. A polypeptide comprising the CAR according to any one of claims 1 to 47 and a TEAM.

50. A method comprising administering to a subject a cell according to any one of claims 1 to 47.

51. A method of treating cancer characterized by cancer cells expressing CD70, the method comprising administering to a subject in need thereof an effective amount of the cells of any one of claims 1 to 47.

52. A method of treating cancer characterized by cancer cells expressing CD70 and CD33, the method comprising administering to a subject in need thereof an effective amount of a cell according to any one of claims 1 to 47.

53. A method of treating cancer characterized by cancer cells expressing CD33, the method comprising administering to a subject in need thereof an effective amount of the cells of any one of claims 1 to 47.

54. A method of treating cancer characterized by cancer cells having reduced CD70 expression, the method comprising administering to a subject in need thereof an effective amount of a cell according to any one of claims 1 to 47.

55. The method of any one of claims 50 to 54, wherein the subject is a human.

56. The method of any one of claims 50 to 55, wherein administering comprises infusion.

57. The method of any one of claims 51 to 56, wherein the cancer is a hematological cancer.

58. The method of any one of claims 51 to 57, wherein the cancer is medullary cell carcinoma.

59. The method of any one of claims 51 to 58, wherein the cancer is acute myeloid leukemia.

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