Chimeric Receptors with Binding Capacity for Antibodies

Abstract

Described herein are receptor configurations that allow ligation of multiple targets expressed in cancer cells via two main mechanisms: direct binding via an antibody-derived single chain variable fragment (scFv), and indirect binding through the high-affinity engagement of the Fc component of an antibody. When expressed in human T lymphocytes, these receptors (CARFcR) can trigger powerful CAR-T cell activity as well as antibody- dependent cell cytotoxicity (ADCC), either simultaneously or sequentially. CARFcR T cells are not affected by soluble antibodies, but they can be redirected and activated by antibodies bound to target cells. Therefore, such T cells can be used to redirect adoptive T cell therapy towards another tumor-associated antigen "on the fly" using clinically available antibodies. They also provide a practical way to target multiple antigens simultaneously with a single receptor, a feature that should be useful in cell therapies directed against phenotypically heterogeneous tumors.

Description

Chimeric Receptors with Binding Capacity for AntibodiesRELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 601,520, filed on November 21, 2023. The entire teachings of the above application(s) are incorporated herein by reference.INCORPORATION BY REFERENCE OF MATERIAL IN XML

[0002] This application incorporates by reference the Sequence Listing contained in the following extensible Markup Language (XML) file being submitted concurrently herewith: a) File name: 4459115700 l_Sequence_Listing.xml; created November 20, 2024, 98,243 Bytes in size.BACKGROUND

[0003] Cytotoxic lymphocytes can become powerful anti-tumor agents if they are genetically-engineered to express chimeric antigen receptors (CARs).1 2When ligated by their cognate antigen, CARs transduce signals that trigger activation of cytotoxic lymphocytes, formation of immunological synapses with cells expressing the antigen and exocytosis of lytic granules, resulting in target cell killing.1 2In T lymphocytes, CAR stimuli can also induce robust cell proliferation.3 4With high capacity for cell propagation and long lifespan, CAR-T cells can generate high effector-to-target ratios, resulting in massive antitumor responses in patients. For example, anti-CD19 CAR T cell therapy has produced dramatic and durable remissions in patients with B-cell malignancies.5'8Nevertheless, despite excellent initial responses and persistence of anti-CD19 CAR-T cells, some patients subsequently relapse with tumor lacking CD19.9 10

[0004] Another approach to cancer immunotherapy is the administration of monoclonal antibodies which can exert anti-tumor activity through a range of diverse mechanisms, including inhibition of tumor cell growth, complement fixation, macrophage phagocytosis and antibody-dependent cell cytotoxicity (ADCC).11-16The latter plays a critical role and derives from the binding of the Fc portion of the antibody to Fc receptors (FcyR), which are expressed on the surface of NK cells and other cells, such as neutrophils and macrophages; the bulk of T cells lack activating FcyR and cannot exert ADCC.17The importance of ADCCin the treatment of cancer with antibodies is underscored by the fact that allotypes of the gene encoding FcyRIIIa (FCGR3A or CD16) with higher Fc binding affinity have been associated with superior responses to antibody therapy.12 18 19SUMMARY

[0005] We sought to develop new receptors that would allow to redirect CAR-T cells after CAR-T cell infusion. Described herein are receptor configurations that allow ligation of multiple targets expressed in cancer cells via two main mechanisms: direct binding via an antibody-derived single chain variable fragment (scFv), and indirect binding through the high-affinity engagement of the Fc component of an antibody. When expressed in human T lymphocytes, these receptors (CARFcR) can trigger powerful CAR-T cell activity as well as ADCC, either simultaneously or sequentially. CARFcRT cells are not affected by soluble antibodies, but they can be activated by antibodies bound to target cells. Therefore, such T cells can be used to redirect adoptive T cell therapy towards another tumor-associated antigen “on the fly” using clinically available antibodies. They also provide a practical way to target multiple antigens simultaneously with a single receptor, a feature that should be useful in cell therapies directed against phenotypically heterogeneous tumors.

[0006] Described herein is a chimeric receptor. The chimeric receptor includes an extracellular domain, a hinge and transmembrane domain, and an intracellular domain. The extracellular domain includes an antigen binding domain; and an antibody fragment crystallizable (Fc) binding domain. The intracellular domain includes a co-stimulatory domain; and a signaling domain.

[0007] The target binding domain can adjoin the hinge and transmembrane domain. Alternatively, the antibody Fc binding domain can adjoin the hinge and transmembrane domain.

[0008] The chimeric receptor can include a linker domain joining the antigen binding domain and the antibody Fc binding domain. The linker domain can be (G4S)X, wherein x is an integer from 1 to 100; or wherein x is an integer from 1 to 10; or wherein x is an integer from 2 to 5; or wherein x is 1; or wherein x is 3. The linker domain can be (EAAAK)Z, wherein z is an integer from 1 to 100.

[0009] The Fc binding domain can be an extracellular domain of CD 16, which can have a V158F polymorphism. The extracellular domain of CD16 can include SEQ ID NO: 37.

[0010] The antigen-binding domain can be a single chain variable fragment (scFv), which can include a variable heavy (VH) domain; a variable light (VL) domain; and a linker domain joining the variable heavy domain and the variable light domain.

[0011] The antigen-binding domain can bind to CD19, CD33, CD123, or B-cell maturation antigen (BCMA).

[0012] The co-stimulatory domain can be a 4- IBB domain.

[0013] The signaling domain can be a CD3(^ domain.

[0014] Described herein is a mammalian T cell expressing the chimeric receptors described herein.

[0015] Described herein is a method of treating cancer. The method can include administering a mammalian T cell expressing a chimeric receptor described herein; and administering an anti-CD20 antibody, an anti-CD22 antibody, an anti-GD2 antibody, an anti- HER2 antibody, an anti-EGFR antibody, an anti-CD38 antibody, or a bispecific antibody binding a tumor antigen and CD 16. In some instances, the method can include withdrawing administration of the anti-CD20 antibody, the anti-CD22 antibody, the anti-GD2 antibody, the anti-HER2 antibody, the anti-EGFR antibody, the anti-CD38 antibody, or the bispecific antibody binding a tumor antigen and CD 16.

[0016] The cancer can be acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), B-cell non-Hodgkin lymphoma (NHL), B-cell chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma, breast cancer, neuroblastoma, lung cancer, or head and neck cancer.

[0017] Described herein is a vector that includes a nucleic acid. The nucleic acid encodes the following domains operably connected to each other: a chimeric receptor domain; and an expression domain. The chimeric receptor domain includes: an extracellular domain; a hinge and transmembrane domain; and an intracellular domain. The extracellular domain includes an antigen binding domain; and an antibody fragment crystallizable (Fc) binding domain. The intracellular domain includes a co-stimulatory domain; and a signaling domain. The expression domain can be an internal ribosome entry site or a self-cleaving peptide.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like referencecharacters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0019] FIG. 1 is a schematic representation of anti-CD19 CAR and anti-CD19 CARFcR. CARFcRwere made by joining the extracellular portion of CD 16 VI 58F to an anti-CD19- 41BB-CD3(^ CAR using either a flexible ((G4S)i (SEQ ID NO: 29) or (G4S)4(SEQ ID NO: 31) or a rigid (EAAAK (SEQ ID NO: 33)) linker (see SEQ ID NOs: 1-12).

[0020] FIG. 2A-C show expression of anti-CD19 CARFcRin human T cells. (FIG. 2A) Flow cytometry dot plots illustrate cell surface expression of anti-CD19 scFv (upper row) and of CD 16 (lower row) on the Y axes and of green fluorescence protein (GFP; a marker of retroviral transduction efficiency) on the X axes. The percentage of cells in each quadrant is shown. Surface anti-CD19 scFv expression was detected with a biotin-conjugated goat antimouse F(ab')2(GAM) antibody and streptavidin conjugated to allophycocyanin (APC; Jackson ImmunoResearch Laboratories); CD 16 expression was detected with APC- conjugated CD 16 antibody (BD Biosciences). (FIG. 2B) Relation between mean fluorescence intensity (MFI) of GAM and MFI of GFP of T cells shown in A. (FIG. 2C) Relation between MFI of CD 16 and MFI of GFP of T cells shown in A.

[0021] FIGs. 3 A-C show T cells expressing CARFcRacquire the capacity to bind antibody. (FIG. 3 A) Flow cytometry dot plots illustrate binding of the anti-CD20 antibody rituximab to T cells expressing CARFcR. The bound antibody was detected with goat antihuman IgG (GAH IgG) antibody conjugated to phycoerythrin (PE; Southern Biotech) and is shown on the Y axes; green fluorescence protein (GFP) is on the X axes. The percentage of cells in each quadrant is shown. (FIG. 3B) Relation between mean fluorescence intensity (MFI) of GAH IgG conjugated to PE and MFI of GFP of T cells shown in A. (FIG. 3C) Percentage of cell aggregates formed by T cells and K562 cells transduced with CD20 (K562-CD20) in the absence or presence of 1 pg / mL rituximab. Each bar represents mean (± SD) of triplicate measurements. *** P < 0.001; **** p < 0.0001.

[0022] FIGs. 4A-C show T cells expressing anti-CD19 CARFcRretain the capacity to bind CD 19. (FIG. 4A) Flow cytometry dot plots illustrate T cell binding of PE-conjugated human CD 19 protein (ACRO Biosystems) on the Y axes and GFP on the X axes. Percentage of cells in each quadrant is shown. (FIG. 4B) Relation between MFI of CD 19 protein conjugated to PE and MFI of GFP of T cells shown in A. (FIG. 4C) Percentage of cell aggregates formed by T cells and CD19+ RS4;11 cells. Each bar represents mean (± SD) of triplicate measurements. * P < 0.05; ** P < 0.01; **** P < 0.0001.

[0023] FIG. 5 shows CAR-mediated cytotoxicity of anti-CD19 CARFcR-T cells. Graphs show results of 4-hour cytotoxicity assays with T cells transduced with a MSCV retroviral vector containing GFP only (control), anti-CD19 CAR, or one of the 6 anti-CD19 CARFcR. T cells were co-cultured with CD19+ ALL cell lines Nalm6, OP-1, RS4;11, and REH, and lymphoma cell lines Ramos and Daudi at 1 : 1 effector-to-target (E:T) ratio. Each bar indicates mean (± SD) of triplicate measurements with T cells from 3 donors for control, anti-CD19 CAR, and anti-CD19 CARFcR-l, -2, -3, and -5, and 2 donors for anti-CD19 CARFcR-4 and 6.

[0024] FIGs. 6A-B show CAR-mediated long-term cytotoxicity of anti-CD19 CARFcR-T cells. (FIG. 6A) Long-term cytotoxicity of T cells transduced with a MSCV retroviral vector containing GFP only (control), anti-CD19 CAR, or one of the 6 anti-CD19 CARFcR. T cells were co-cultured with mCherry+ Nalm6 cells at 1 :4 E:T ratio. The number of mCherry+ viable Nalm6 cells was measured every 4 hours with the IncuCyte Zoom System (Essen Bioscience) and expressed as red calibrated units (RCU) x pm2 / well. Each symbol represents the mean (± SD) of triplicate measurements at the indicated time points with T cells from one donor. Data were normalized to the first time point. (FIG. 6B) Viable mCherry+ Nalm6 cells from A measured at 100 hours of co-culture. Each bar indicates the mean (± SD) of triplicate measurements. ** P < 0.01; *** P < 0.001.

[0025] FIG. 7 shows antibody-mediated cytotoxicity (ADCC) of anti-CD19 CARFcR-T cells. ADCC of T cells transduced with a MSCV retroviral vector containing GFP only (control), anti-CD19 CAR, or one of the 6 anti-CD19 CARFcRagainst SKBR3 cells with or without anti-HER2 antibody trastuzumab (10 pg / mL) after co-culture for 4 hours at 4: 1 E:T ratio. Each bar indicates the mean (± SD) of triplicate measurements with T cells from 3 donors for control, anti-CD19 CAR, anti-CD19 CARFcR-l, -2, and -5, and 2 donors for anti- CD19 CARFcR-3, -4, and 6.

[0026] FIGs. 8A-B show antibody-mediated long-term cytotoxicity of anti-CD19 CARFcR-T cells. (FIG. 8A) Long-term ADCC of T cells transduced with a MSCV retroviral vector containing GFP only (control), anti-CD19 CAR, or one of the 6 anti-CD19 CARFcRagainst HER2+ SKBR3 cells expressing mCherry, with or without the anti-HER2 antibody trastuzumab (10 pg / mL) at 1 :4 E:T ratio. The number of mCherry+ viable target cells was measured with the IncuCyte Zoom System. Each symbol represents the mean (± SD) of triplicate measurements at the indicated time points. Data were normalized to the first time point after the addition of T cells. (FIG. 8B) Number of viable mCherry+ SKBR3 cells fromA measured at 100 hours of co-culture. Each bar indicates the mean (± SD) of triplicate measurements from 1 donor. * P < 0.05; *** P < 0.001.

[0027] FIGs. 9A-D show anti-CD19 CARFcR-T cells can kill cells expressing different targets simultaneously and sequentially. (FIG. 9A) Cytotoxicity of anti-CD19 CARFcR-l T cells against CD19+ CD20+ cells Daudi and Ramos at 1 : 1 E:T ratio with or without 1 pg / mL anti-CD20 antibody rituximab. Each symbol indicates triplicate results obtained with T cells from 2 donors . *** P < 0.001; **** p < 0.0001. (FIG. 9B) Cytotoxicity of anti-CD19 CARFcR-l T cells against CD19+ CD38+ cells OP-1, REH, RS4;11, and Ramos at 1 :4 E:T ratio with or without 1 pg / mL anti-CD38 antibody daratumumab. Each symbol indicates triplicate results with T cells from 2 donors. * P < 0.05; ** P < 0.01; *** P < 0.001. (FIG. 9C) Bar graph shows percentage of ADCC against SKBR3 in the presence of 10 pg / mL anti- HER2 antibody trastuzumab after exposure to RS4;11 (scFv target). ADCC against SKBR3 is expressed as antibody-specific cytotoxicity, after subtracting background killing in the absence of trastuzumab. Mean (± SD) of triplicate measurements with T cells from 3 donors are shown. (FIG. 9D) Bar graph shows cytotoxicity of T cells against RS4;11 after exposure to K562-CD20 with 1 pg / mL anti-CD20 antibody rituximab (Ab + target). Each bar indicates mean (± SD) of triplicate measurements with T cells from 3 donors.

[0028] FIG. 10 shows that anti-CD19 CARFcR-T cells can be redirected against CD33 using a bi-specific antibody composed by an anti-CD33 scFv linked to an anti-CD16 scFv. Cytotoxicity of anti-CD19 CARFcR-l T cells against CD33+ KG-1 and Kasumi-3 cells with or without the bi-specific antibody after co-culture for 4 hours at the indicated E:T ratios. Mean (± SD) of triplicate measurements with T cells from 1 donor are shown.

[0029] FIGs. 11 A-C show that anti-CD19 CARFcR-T cells can kill ALL cells lacking CD19. (FIG. 11 A) ADCC of anti-CD19 CARFcR-l T cells against Nalm6 cells rendered CD 19-negative by CRISPR-Cas9 gene editing, with or without 1 pg / mL anti-CD38 antibody daratumumab, after co-culture for 4 hours at the indicated E:T ratios. Mean (± SD) of triplicate measurements with T cells from 1 donor is shown. (FIG. 1 IB) Long-term ADCC of anti-CD19 CARFcR-l T cells against CD19-negative Nalm6 expressing mCherry, with or without daratumumab (1 pg / mL), at 2:1 E:T ratio. The number of mCherry+ viable target cells was measured with the IncuCyte Zoom System. Data were normalized to the first time point. Each symbol represents the mean (± SD) of triplicate measurements with T cells from 1 donor at the indicated time points. (FIG. 11C) ADCC of anti-CD19 CARFcR-l T cells against CD 19-negative Nalm6 expressing mCherry after co-culture with wild type Nalm6 at2: 1 E:T ratio for 2 days. The time of addition of CD19-negative Nalm6 to the culture is indicated by the arrow. The number of viable mCherry+ target cells was measured as in FIG.1 IB. Mean (± SD) of triplicate measurements from 1 donor at the indicated time points is shown.

[0030] FIGs. 12A-B show anti-CD19 CARFcR-T cells can be activated via binding of the scFv to CD 19 and via antibody engagement. (FIG. 12A) Expression of activation markers CD25 and CD69 in unstimulated T cells. Each symbol indicates triplicate results with T cells obtained from 3 donors for CD25, and from 2 donors for CD69. ** P < 0.01. (FIG. 12B) T cells were stimulated with RS4;11 and / or 1 pg / mL immobilized rituximab. Expression of activation markers CD25 and CD69 was measured 24 hours later. Mean (± SD) of triplicate measurements with T cells from 4 donors are shown for cells stimulated with either RS4;11 or antibody; mean (± SD) of triplicate measurements with T cells from 3 donors are shown for cells stimulated with both RS4;11 and antibody. ** P < 0.01; **** P < 0.0001.

[0031] FIGs. 13A-C show exocytosis of cytotoxic granules and cytokine secretion in antiCD 19 CARFcR-T cells can be triggered by binding of the scFv to CD 19 and by antibody engagement. (FIG. 13 A) Exocytosis of cytotoxic granules was assessed by measuring levels of CD 107a after stimulating T cells for 4 hours with RS4; 11 and / or 1 pg / mL of immobilized rituximab. Mean (± SD) of triplicate measurements with T cells from 4 donors are shown for unstimulated cells and cells stimulated with either RS4;11 or antibody; data from 3 donors are shown for cells stimulated with both RS4;11 and antibody. *** P < 0.001. (FIG. 13B) TNFa secretion was measured after stimulating T cells for 6 hours as in A. Each bar represents mean (± SD) triplicate measurements with cells from 2 donors. ** P < 0.01; **** P < 0.0001. (FIG. 13C) IFNy secretion was measured after stimulating T cells for 6 hours as in A. Each bar represents mean (± SD) triplicate measurements with cells from 2 donors. * P < 0.05; **** P < 0.0001.

[0032] FIG. 14 shows proliferation of anti-CD19 CARFcR-T cells stimulated by scFv binding to CD 19 and antibody engagement. T cells transduced with a MSCV retroviral vector containing GFP only (control), anti-CD19 CAR, or anti-CD19 CARFcR-l were cultured alone, or with irradiated RS4; 11 or with 1 pg / mL rituximab in the absence or presence of irradiated K562-CD20 cells for 2 weeks. Each symbol indicates the percentage of cell recovery compared with the number of input cells. Mean (± SD) of triplicate measurements with T cells from 1 donor are shown. P-value between anti-CD19 CAR- and CARFcR-l -T cells on day 14 is shown. * P < 0.0216; ** P < 0.01.

[0033] FIGs. 15A-C are schematic representation of CAR and CARFcRtargeting CD33 (FIG. 15A), CD123 (FIG 15B), and BCMA (FIG 15C). The sequences for these CARFcRare SEQ ID NOs: 13-18.

[0034] FIGs. 16A-B show expression of anti-CD33 CARFcRin T cells. (FIG. 16A) Flow cytometry dot plots illustrate surface expression of anti-CD33 scFv (upper row) and CD 16 expression (lower row) on the Y axes and GFP on the X axes. Percentage of cells in each quadrant is shown. Anti-CD33 scFv was detected with biotin-conjugated goat anti-mouse F(ab')2(GAM) antibody and streptavidin-APC; CD 16 expression was detected with CD 16- APC. (FIG. 16B) Flow cytometry dot plots illustrate binding of anti-CD33FcR-T cells to rituximab. The bound antibody was detected with goat anti-human IgG (GAH IgG) antibody conjugated to PE. Percentage of cells in each quadrant is shown.

[0035] FIGs. 17A-B show expression of anti-CD123 CARFcRin T cells. (FIG. 17A) Flow cytometry dot plots illustrate surface expression of anti-CD123 scFv (upper row) and CD 16 expression (lower row) on the Y axes and GFP on the X axes. Percentage of cells in each quadrant is shown. Anti-CD123 scFv was detected with biotin-conjugated goat anti-mouse F(ab')2(GAM) antibody and streptavidin-APC; CD 16 expression was detected with CD 16- APC. (FIG. 17B) Flow cytometry dot plots illustrate binding of anti-CD123FcR-T cells to rituximab. The bound antibody was detected with goat anti-human IgG (GAH IgG) antibody conjugated to PE. Percentage of cells in each quadrant is shown.

[0036] FIGs. 18A-B show expression of anti-BCMA CARFcRin T cells. (FIG. 18 A) Flow cytometry dot plots illustrate surface expression of anti-BCMA scFv (upper row) and CD 16 expression (lower row) on the Y axes and GFP on the X axes. Percentage of cells in each quadrant is shown. Anti-BCMA scFv was detected with biotin-conjugated goat anti-mouse F(ab')2(GAM) antibody and streptavidin-APC; CD 16 expression was detected with CD 16- APC. (FIG. 18B) Flow cytometry dot plots illustrate binding of anti-BCMAFcR-T cells to rituximab. The bound antibody was detected with goat anti-human IgG (GAH IgG) antibody conjugated to PE. Percentage of cells in each quadrant is shown.

[0037] FIGs. 19A-B show CAR-mediated cytotoxicity of anti-CD33 CARFcR-T cells.(FIG. 19A) T cells transduced with a MSCV retroviral vector containing GFP only (control), anti-CD33 CAR, or anti-CD33 CARFcRwere co-cultured with CD33+ cells KG-1, Kasumi-3, and U937 for 4 hours at the indicated E:T ratio. Mean (± SD) of triplicate measurements with T cells from 2 donors are shown for KG-1, and mean (± SD) of triplicate measurements with T cells from 1 donor are shown for Kasumi-3 and U937. (FIG. 19B) T cells transduced as inA were co-cultured with mCherry-expressing KG-1 at 1 :2 E:T ratio for 5 days. The number of mCherry+ viable target cells was measured with the IncuCyte Zoom System. Data were normalized to the first time point. Each symbol represents the mean (± SD) of triplicate measurements from 1 donor at the indicated time points.

[0038] FIGs. 20A-B. CAR-mediated cytotoxicity of anti-CD123 CARFcR-T cells. (FIG. 20A) T cells transduced with a MSCV retroviral vector containing GFP only (control), anti- CD123 CAR, or anti-CD123 CARFcRwere co-cultured with CD123+ cells KG-1, Kasumi-3, and THP-1 for 4 hours at the indicated E:T ratios. Mean (± SD) of triplicate measurements from 2 donors are shown for KG-1 and Kasumi-3, and mean (± SD) of triplicate measurements with T cells from 1 donor are shown for THP-1. (FIG. 20B) T cells transduced as in A were co-cultured with mCherry-expressing KG-1 at 1 :2 E:T ratio for 5 days. The number of mCherry+ viable target cells was measured with the IncuCyte Zoom System. Data were normalized to the first time point. Each symbol represents the mean (± SD) of triplicate measurements with T cells from 1 donor at the indicated time points.

[0039] FIGs. 21A-C show CAR-mediated cytotoxicity of anti-BCMA CARFcR-T cells. T cells transduced with a MSCV retroviral vector containing GFP only (control), anti-BCMA CAR, or anti-BCMA CARFcRwere co-cultured with (FIG. 21 A) lymphoma cell lines Daudi and Ramos or (FIG. 2 IB) multiple myeloma cell lines RPMI8226, MMl.s, and U266-B1 for 4 hours at the indicated E:T ratios. Mean (± SD) of triplicate measurements with T cells from 2 donors are shown for Daudi, and mean (± SD) of triplicate measurements with T cells from 1 donor are shown for Ramos, RPMI8226, MMl.s, and U266-B1. (FIG. 21C) T cells transduced as in A and B were co-cultured with mCherry-expressing Daudi cells at 1 :2 E:T ratio for 5 days. The number of mCherry+ viable target cells was measured with the IncuCyte Zoom System. Data were normalized to the first time point. Each symbol represents the mean (± SD) of triplicate measurements with T cells from 1 donor at the indicated time points.

[0040] FIGs. 22A-C show antibody-mediated cytotoxicity of anti-CD33, anti-CD123 and anti-BCMA CARFcR-T cells. Graphs show data of 4-hour ADCC exerted by (FIG. 22A) anti- CD33 CAR- or CARFcR-T cells, (FIG. 22B) anti-CD123 CAR- or CARFcR-T cells, and (FIG. 22C) anti-BCMA CAR- or CARFcR-T cells against SKBR3 in the absence or presence of the anti-HER2 antibody trastuzumab (10 pg / mL) at the indicated E:T ratios. Mean (± SD) of triplicate measurements with T cells from 1 donor are shown.

[0041] FIGs. 23A-B show simultaneous CAR-mediated and antibody-mediated cytotoxicity of anti-CD123 and anti-BCMA CARFcR-T cells. (FIG. 23A) Cytotoxicity of anti-CD123 CARFcR-T cells against CD123+ CD38+ cells KG-1 and Kasumi-3 with or without 1 gg / mL anti-CD38 antibody daratumumab. Each symbol indicates data with T cells from 2 donors. ** P < 0.01; *** P < 0.001. (FIG. 23B) Cytotoxicity of anti-BCMA CARFcR-T cells against BCMA+ CD20+ cells Daudi and Ramos with or without 1 gg / mL anti-CD20 antibody rituximab (left), or BCMA+ CD38+ cells Ramos and MMl.s with or without 1 gg / mL anti-CD38 antibody daratumumab (right). Each symbol indicates data with T cells from 1 donor. * P = 0.0185; ** P < 0.01; *** P < 0.001.

[0042] FIGs. 24A-C show anti-CD33, anti-CD123 and anti-BCMA CARFcR-T cells can be stimulated by antibody engagement. Expression of activation markers CD25 and CD69 in (A) anti-CD33 CAR- or CARFcR-T cells, (B) anti-CD123 CAR- or CARFcR-T cells, and (C) anti-BCMA CAR- or CARFcR-T cells after 24-hour stimulation with 1 gg / mL of immobilized rituximab. Each bar indicates mean (± SD) of triplicate measurements with T cells from 1 donor. ** P < 0.01; *** P < 0.001; **** p < 0.0001.

[0043] FIGs. 25A-C show activation levels and proliferation rates triggered by different anti-CD19 CARFcRconfigurations. (A) Expression of activation markers CD25 and CD69 in anti-CD19 CAR- or CARFcR-T cells after 24-hour stimulation with RS4;11. Each bar indicates mean (± SD) of triplicate measurements with T cells from 3 donors. *** P < 0.001; **** p < o.OOOL (B) Expression of activation markers CD25 and CD69 in anti-CD19 CAR- or CARFcR-T cells after 24-hour stimulation with 1 gg / mL of immobilized rituximab. Each bar indicates mean (± SD) of triplicate measurements with T cells from 3 donors. **** p < 0.0001. (C) T cells expressing GFP only (control), anti-CD19 CAR, or anti-CD19 CARFcRwere cultured alone, or with irradiated RS4;11, or with 1 gg / mL rituximab in the absence or presence of irradiated K562-CD20 cells for 2 weeks. Each symbol indicates the percentage of cell recovery compared with the number of input cells. Mean (± SD) of triplicate measurements with T cells from 3 donors are shown.

[0044] FIGs. 26A-D show activity of anti-CD19 CARFcR-T cells in a xenograft model of ALL. (A) NOD-scid-IL2RGnull mice were injected IV with 0.5* 106Nalm6 cells expressing luciferase, followed by 20* 106T cells (GFP only, anti-CD19 CAR, or anti-CD19 CARFcR-5) four days later. Ventral and dorsal images show Nalm6 cell engraftment as measured by luminescence after IP injection of aqueous d-luciferin potassium salt (150 gg / g body weight). Mice images on day 4 were taken with enhanced sensitivity to detect tumor engraftment. (B) Luminescence measurements in the groups of mice shown in panel A. (C) Leukemic-freesurvival of mice for shown in A and B. * P < 0.1; ** P < 0.01; (D) Blood profile of mice taken 14 days after T-cell infusion.DETAILED DESCRIPTION

[0045] A description of example embodiments follows.Nucleic Acids

[0046] As used herein, the term “nucleic acid” refers to a polymer comprising multiple nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). “Nucleic acid” includes, for example, DNA (e.g., genomic DNA and cDNA), RNA, and DNA-RNA hybrid molecules. Nucleic acid molecules can be naturally occurring, recombinant, or synthetic. In addition, nucleic acid molecules can be single-stranded, double-stranded or triple-stranded. In certain embodiments, nucleic acid molecules can be modified. In the case of a double-stranded polymer, “nucleic acid” can refer to either or both strands of the molecule.

[0047] The terms “nucleotide” and “nucleotide monomer” refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as non-naturally occurring derivatives and analogs thereof. Accordingly, nucleotides can include, for example, nucleotides comprising naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxy cytidine) and nucleotides comprising modified bases known in the art.

[0048] As used herein, the term “sequence identity,” refers to the extent to which two nucleotide sequences, or two amino acid sequences, have the same residues at the same positions when the sequences are aligned to achieve a maximal level of identity, expressed as a percentage. For sequence alignment and comparison, typically one sequence is designated as a reference sequence, to which a test sequences are compared. The sequence identity between reference and test sequences is expressed as the percentage of positions across the entire length of the reference sequence where the reference and test sequences share the same nucleotide or amino acid upon alignment of the reference and test sequences to achieve a maximal level of identity. As an example, two sequences are considered to have 70% sequence identity when, upon alignment to achieve a maximal level of identity, the test sequence has the same nucleotide or amino acid residue at 70% of the same positions over the entire length of the reference sequence.

[0049] Alignment of sequences for comparison to achieve maximal levels of identity can be readily performed by a person of ordinary skill in the art using an appropriate alignment method or algorithm. In some instances, the alignment can include introduced gaps to provide for the maximal level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. AppL Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see generally Ausubel el al, Current Protocols in Molecular Biology).

[0050] When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. A commonly used tool for determining percent sequence identity is Protein Basic Local Alignment Search Tool (BLASTP) available through National Center for Biotechnology Information, National Library of Medicine, of the United States National Institutes of Health. (Altschul et al, J Mol Biol. 215(3):403-10 (1990)).

[0051] In various embodiments, two nucleotide sequences, or two amino acid sequences, can have at least, e.g., 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity. When ascertaining percent sequence identity to one or more sequences described herein, the sequences described herein are the reference sequences.Vectors

[0052] The terms “vector”, “vector construct” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein is inserted by restriction enzyme technology. A common type of vector is a “plasmid”, which generally is a self-contained molecule ofdouble-stranded DNA that can readily accept additional (foreign) DNA and which can be readily introduced into a suitable host cell. A large number of vectors, including plasmid and fungal vectors, have been described for replication and / or expression in a variety of eukaryotic and prokaryotic hosts.The terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g. the resulting protein, may also be said to be “expressed” by the cell. A polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter. Gene delivery vectors generally include a transgene (e.g., nucleic acid encoding an enzyme) operably linked to a promoter and other nucleic acid elements required for expression of the transgene in the host cells into which the vector is introduced. Suitable promoters for gene expression and delivery constructs are known in the art. Recombinant plasmids can also comprise inducible, or regulatable, promoters for expression of an enzyme in cells.

[0053] Various gene delivery vehicles are known in the art and include both viral and non-viral (e.g., naked DNA, plasmid) vectors. Viral vectors suitable for gene delivery are known to those skilled in the art. Such viral vectors include, e.g., vector derived from the herpes virus, baculovirus vector, lentiviral vector, retroviral vector, adenoviral vector, adeno- associated viral vector (AAV), and murine stem cell virus (MSCV). The viral vector can be replicating or non-replicating. Such vectors may be introduced into many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.

[0054] Non-viral vectors for gene delivery include naked DNA, plasmids, transposons, and mRNA, among others. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), pMAL plasmids (New England Biolabs, Beverly, Mass.). Such vectors may be introduced into many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.

[0055] In certain embodiments, the vector comprises an internal ribosome entry site (IRES). In some embodiments, the vector includes a selection marker, such as an ampicillinresistance gene (Amp). In some embodiments, the nucleic acid encodes a fluorescent protein, such as green fluorescent protein (GFP) or mCherry. In some embodiments, the nucleic acid is suitable for subcloning into pMSCV-IRES-GFP between EcoRI and Xhol. In some embodiments, the vector contains a multiple cloning site (MCS) for the insertion of the desired gene.

[0056] Although the genetic code is degenerate in that most amino acids are represented by multiple codons (called “synonyms” or “synonymous” codons), it is understood in the art that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. Accordingly, in some embodiments, the vector includes a nucleotide sequence that has been optimized for expression in a particular type of host cell (e.g., through codon optimization). Codon optimization refers to a process in which a polynucleotide encoding a protein of interest is modified to replace particular codons in that polynucleotide with codons that encode the same amino acid(s), but are more commonly used / recognized in the host cell in which the nucleic acid is being expressed. In some aspects, the polynucleotides described herein are codon optimized for expression in T cells.Chimeric Receptors

[0057] FIGs. 1 and 15 are schematic representations of chimeric receptors described herein. The chimeric receptors include an extracellular domain, a hinge and transmembrane domain, and an intracellular domain. The extracellular domain includes an antigen binding domain and an antibody fragment crystallizable (Fc) binding domain. The intracellular domain includes a co-stimulatory domain and a signaling domain.

[0058] The antigen binding domain binds an antigen of interest. Oftentimes, the antigen of interest is a cell-surface antigen of a tumor cell, such as CD19, CD33, CD123, or B-cell maturation antigen (BCMA). Preferably, the antigen binding domain is a single chain variable fragment (scFv) domain. Typically, an scFv domain includes an immunoglobulin variable light (VL) domain, an immunoglobulin variable heavy (VH) domain, and a linker domain joining the VLand VHdomains. The relative positions of the VLand VHdomains can be reversed.

[0059] While the embodiments described herein pertain to anti-CD19, anti-CD33, anti- CD123, and anti-BCMA constructs, a similar approach can be applied to generate constructs for other target antigens, such as CD22, mesothelin, human epidermal growth factor receptor 2 (Her2), prostate-specific membrane antigen (PSMA), disial oganglioside (GD)-2, PD-L1(CD274), CD80 or CD86. For example, based on FIGs. 1 and 15, the anti-CD19, anti-CD33, anti-CD123, and anti-BCMA VHand VLdomains can be replaced with VHand VLdomains that specifically bind to one of the other target antigens.

[0060] A variety of linker domains between VLand VHdomains are suitable. In some embodiments, the linker domain can be (G4S)X, wherein x is an integer from 1 to 100; preferably, x is an integer from 1 to 10; even more preferably, x is an integer from 2 to 5. In some embodiments, the linker domain can be (G4S)3. In other embodiments, the linker domain can be one or more glycine residues (e.g., (G)y, where y is an integer from 2 to 100. In other embodiments, the linker domain can be (EAAAK)Z, wherein z is an integer from 1 to 100. (G4S)X, (G4S)3, and (G)y are examples of flexible linkers, while (EAAAK)Zis an example of a more rigid linker.

[0061] A variety of hinge domains are suitable. In some embodiments, the hinge domain can be a IgG hinge domain. In some embodiments, the hinge can be a plurality of amino acid residues. In some embodiments, the hinge domain can be a hinge domain from IgE, IgA, IgD, or CD8a.

[0062] A variety of transmembrane domains are suitable. In some embodiments, the transmembrane domain can be a CD8a transmembrane domain. In some embodiments, the transmembrane domain can be a plurality of amino acid residues. In some embodiments, the transmembrane domain can be a transmembrane domain from CD28, CD 16, CD32, CD64, VEGFR2, FAS, FGFR2B or another transmembrane protein.

[0063] The co-stimulatory domain can be a 4-1BB / CD137 domain. In some embodiments, the co-stimulatory domain can be a stimulatory domain from CD28, OX40 / CD134, FcsRI, ICOS / CD278, ILRB / CD122, IL-2RG / CD132, and CD40.

[0064] The signaling domain can be a CD3(^ domain. In some embodiments, the signaling domain can be a signaling domain of other CD3 chains, such as CD3s, FcsRI, DAP12 or other signaling molecules containing immune-receptor tyrosine-based activation motifs (IT AMs).Methods of Making Transgenic Host Cells

[0065] Described herein are methods of making a transgenic host cell, such as transgenic natural killer (NK) cells or transgenic T cells. The transgenic host cells can be made, forexample, by introducing one or more of the vector embodiments described herein into the host cell.

[0066] In some embodiments, one or more of the nucleic acids are integrated into the genome of the host cell. In some embodiments, the nucleic acids to be integrated into a host genome can be introduced into the host cell using any of a variety of suitable methodologies known in the art, including, for example, homologous recombination, CRISPR-based systems (e g., CRISPR / Cas9; CRISPR / Cpfl) and TALEN systems.Host Cells

[0067] A variety of host cells are suitable for use in making transgenic host cells. Most commonly, the host cells are immune cells, such as natural killer (NK) cells or T lymphocyte cells.

[0068] As used herein, "natural killer cells" ("NK cells") refer to a type of cytotoxic lymphocyte of the immune system. NK cells provide rapid responses to virally infected cells and respond to transformed cells. Typically, immune cells detect peptides from pathogens presented by major histocompatibility complex (MHC) molecules on the surface of infected cells, triggering cytokine release, causing lysis or apoptosis. NK cells are unique, however, as they have the ability to recognize stressed cells regardless of whether peptides from pathogens are present on MHC molecules. They were named "natural killers" because of the initial notion that they do not require prior activation in order to kill target. NK cells are large granular lymphocytes (LGL) and are known to differentiate and mature in the bone marrow from where they then enter into the circulation. NK cell can also kill tumor cells if antigens on the surface of tumor cells are bound by antibodies; the Fc portion of the antibody bind Fc receptors (CD16) on the surface of NK cells and triggers cytotoxicity, a process known as antibody-dependent cell cytotoxicity (ADCC).

[0069] As used herein, “T lymphocytes” or “T cells” refers to lymphocytes that mature in the thymus. T cells can be further characterized into subpopulations, including T helper (TH) cells, T cytotoxic (Tc) cells, and T regulatory (Treg) cells. THand Tccells can be characterized according to the presence or absence of membrane glycoproteins CD4 and CD8. Generally, THcells express CD4 on their surface, while Tccells express CD8 on their surface. T helper cells can be further characterized as TH1 cells and TH2 cells. T cells can also exert ADCC if transduced with a receptor encoding CD 16 and signaling molecules.21

[0070] In some aspects, the NK cell or the T cells are mammalian cells. Examples of "mammalian" or "mammals" include primates (e.g., human), canines, felines, rodents, porcine, ruminants, and the like. Specific examples include humans, dogs, cats, horses, cows, sheep, goats, rabbits, guinea pigs, rats and mice. In a particular aspect, the mammalian T or NK cell is a human T or NK cell.Values and Ranges

[0071] Unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in various embodiments, unless the context clearly dictates otherwise. “About” in reference to a numerical value generally refers to a range of values that fall within ±8%, in some embodiments ±6%, in some embodiments ±4%, in some embodiments ±2%, in some embodiments ±1%, in some embodiments ±0.5% of the value unless otherwise stated or otherwise evident from the context.EXEMPLIFICATIONIntroduction

[0072] Cytotoxic lymphocytes can become powerful anti-tumor agents if they are genetically-engineered to express chimeric antigen receptors (CARs).1 2When ligated by their cognate antigen, CARs transduce signals that trigger activation of cytotoxic lymphocytes, formation of immunological synapses with cells expressing the antigen and exocytosis of lytic granules, resulting in target cell killing.1 2In T lymphocytes, CAR stimuli can also induce robust cell proliferation.3 4With high capacity for cell propagation and long lifespan, CAR-T cells can generate high effector-to-target ratios, resulting in massive antitumor responses in patients. For example, anti-CD19 CAR T cell therapy has produced dramatic and durable remissions in patients with B-cell malignancies.5'8Nevertheless, despite excellent initial responses and persistence of anti-CD19 CAR-T cells, some patients subsequently relapse with tumor lacking CD19.9 10In such patients, it should be beneficial to redirect the infused T cells against other targets retained by the relapsing cells, as another infusion of CAR-T cells with a different specificity might be clinically and / or practically impossible.

[0073] Another approach to cancer immunotherapy is the administration of monoclonal antibodies which can exert anti-tumor activity through a range of diverse mechanisms, including inhibition of tumor cell growth, complement fixation, macrophage phagocytosis and antibody-dependent cell cytotoxicity (ADCC).11-16The latter plays a critical role and derives from the binding of the Fc portion of the antibody to Fc receptors (FcyR), which are expressed on the surface of NK cells and other cells, such as neutrophils and macrophages; the bulk of T cells lack activating FcyR and cannot exert ADCC.17The importance of ADCC in the treatment of cancer with antibodies is underscored by the fact that allotypes of the gene encoding FcyRIIIa (FCGR3A or CD16) with higher Fc binding affinity have been associated with superior responses to antibody therapy.12 18 19

[0074] We sought to develop new receptors that would allow to redirect CAR-T cells after CAR-T cell infusion. To this end, we reasoned that endowing CARs with the capacity to bind the Fc portion of antibodies may achieve this objective but it was unclear whether a) CAR function would be affected by modifications to the CAR sequence; b) the presence of soluble antibodies would impair it; and c) T cells bearing such receptor could exert ADCC in the presence of an antibody. To answer these questions, we tested multiple receptor configurations that might allow direct binding via an antibody-derived single chain variable fragment (scFv), and indirect binding through the high-affinity engagement of the Fc component of an antibody. When expressed in human T lymphocytes, these receptors (CARFcR) could trigger various degrees of CAR-T cell activity as well as ADCC, either simultaneously or sequentially. CARFcRT cells were not affected by soluble antibodies, but they could be activated by antibodies bound to target cells. Therefore, such T cells can be used to redirect adoptive T cell therapy towards another tumor-associated antigen “on the fly” using clinically available antibodies. They also provide a practical way to target multiple antigens simultaneously with a single receptor, a feature that should be useful in cell therapies directed against phenotypically heterogeneous tumors.Materials and MethodsCells and culture conditions

[0075] The cell lines RS4;11, REH and Nalm6 derived from acute lymphoblastic leukemia (ALL); KG-1, Kasumi-3, U937, THP-1 and K562 from acute myeloid leukemia (AML); Daudi and Ramos from B-cell non-Hodgkin lymphoma (NHL); RPMI8226, U266- Bl, and MMl.s from multiple myeloma; and SKBR3 from breast cancer were obtained fromthe American Type Culture Collection (ATCC; Rockville, MD). The B-lineage ALL cell line OP-1 was previously developed in our laboratory.20CD19-negative Nalm6 was generated by electroporating CRISPR-CD19 gRNA (Genscript, Piscataway, NJ) targeting exon 3 into wild type Nalm6 using Amaxa Cell Line Nucleofector Kit V (Lonza, Basel, Switzerland) and Nucleofector II program C-005 (Lonza); the CRISPR-CD19 gRNA-electroprated Nalm6 was subsequently sorted using MoFlo Astrios cell sorter (Beckman Coulter, Brea, CA) to obtain pure population of CD 19-negative Nalm6. The genes encoding mCherry, firefly luciferase, CD20 or an anti-CD33 and anti-CD16 bispecific antibody32were expressed using a murine stem cell virus (MSCV) retroviral vector (Vector Development and Production Shared Resource Laboratory, St. Jude Children’s Research Hospital, Memphis, TN). Cell lines were maintained in RPML1640 (HyClone, Marlborough, MA) supplemented with 10 - 20% fetal bovine serum (FBS) (HyClone) and 1% penicillin-streptomycin (Gibco, Waltham, MA).

[0076] Peripheral blood mononucleated cells were isolated by density gradient centrifugation from discarded anonymized byproducts of platelet donations from the Health Sciences Authority Blood Bank (Singapore) with the approval of the Institutional Review Board of the National University of Singapore. T cells were cultured in RPMI-1640 with 10% FBS and 1% penicillin-streptomycin, and stimulated with TransAct (Miltenyi Biotec, Bergisch Gladbach, Bonn); 120 lU / mL interleukin-2 (IL-2; Proleukin, Novartis, Basel, Switzerland) was added to the cultures every 2 - 3 days.Gene cloning and retroviral transduction

[0077] The anti-CD19-41BB-CD3^ CAR was previously made in our laboratory.3The extracellular portion of CD16 with V158F polymorphism was previously generated in our laboratory using site-directed mutagenesis by PCR from the FCRG3A (CD16) cDNA obtained from Origene (Rockville, MD).21It was joined to the single-chain variable fragment (scFv) of anti-CD19 (clone FMC63) in various configurations by using splicing by overlapping extension PCR (FIG. I).3A similar approach was used to generate receptors directed against CD33 (FIG. 15 A), CD 123 (FIG. 15B), and B-cell maturation antigen (BCMA; FIG. 15C) using scFv derived from antibodies targeting CD33 (clone hP67.6), CD123 (clone 26292), or BCMA (clone C11D5.3), respectively. All constructs were subcloned into an MSCV-IRES-GFP vector between EcoRI and Xhol restriction sites.

[0078] Retroviral transduction was performed as previously described.21Briefly, MSCV retroviral vector-conditioned medium was added to RetroNectin (Takara, Otsu, Japan)-coatedpolypropylene tubes; after centrifugation and removal of the supernatant, T cells were added to the tubes and left at 37°C for 24 hours; fresh viral supernatant was added again after 24 hours. After 48 hours, the transduced T cells were washed twice with RPMI-1640 and maintained in RPMI-1640 with 10% FBS, 1% penicillin-streptomycin, and 200 lU / mL IL-2 until the time of the experiments, 7-21 days after transduction.Detection of surface receptor expression and target binding

[0079] Surface expression of scFv was detected using a biotin-conjugated goat antimouse F(ab’)2antibody followed by streptavidin conjugated to allophycocyanin (APC; both from Jackson ImmunoResearch, West Grove, PA). Binding of CD 19 antigen to the antiCD 19 scFv was tested with human CD 19 protein conjugated to phycoerythrin (PE; ACRO Biosystems, Newark, DE).

[0080] Surface expression of CD 16 was detected using a CD16-APC antibody (clone B73.1, BD Biosciences, Franklin Lakes, NJ). Antibody-binding capacity of CD16 was assessed by exposing T cells to 1 pg / mL of anti-CD20 antibody rituximab (Selleckchem, Houston, TX) followed by goat anti-human IgG conjugated to PE (Southern Biotech, Birmingham, AL).

[0081] Cell staining was analyzed using a Fortessa flow cytometer (BD Biosciences) with BDFACS Diva software (BD Biosciences).Cell aggregation and cytotoxicity

[0082] To measure aggregation between T cells and target cells, T cells were co-cultured for 1 hour at 1 : 1 ratio with calcein red-orange AM (Thermo Fisher Scientific, Waltham, MA)-labeled RS4;11 or CD20 transduced-K562 (K562-CD20) in the presence of 1 pg / mL anti-CD20 antibody rituximab. Cell aggregates were quantified as the percentage of GFP and calcein red-orange AM double positive cells using Accuri C6 plus flow cytometer (BD Biosciences).

[0083] To assess cytotoxicity, target cells were labeled with calcein red-orange AM and co-cultured with T cells for 4 hours. In some experiments, 1 pg / mL anti-CD20 antibody rituximab or 1 pg / mL anti-CD38 antibody daratumumab (Selleckchem) or anti-CD33 and anti-CD16 bi-specific antibody were also added to the co-culture. Viable target cells were counted by Accuri C6 plus flow cytometer. To measure cytotoxicity against adherent target cells (e.g., SKBR3), we used target cells transduced with firefly luciferase. After plating thecells for 16 hours, T cells were added, with or without 10 pg / mL anti-HER2 antibody trastuzumab (MedChemExpress, Monmouth Junction, NJ). Bright-Glo luciferase reagent (Promega, Madison, WI) was added after 4 hours of co-culture. Luminescence signal was measured 5 minutes later using BioTek FLx800 plate reader (BioTek, Winooski, VT) and analyzed with BioTek Gen5 2.0 data analysis software.

[0084] To test sequential cytotoxicity, T cells were first co-cultured with target cells for CAR-mediated killing at 1 : 1 ratio for 4 hours. The co-culture was subsequently transferred to different target cells for ADCC testing and cultured for another 24 hours. In other experiments, the order of the co-culture was reversed. The number of viable target cells at the end of each incubation was measured by Accuri C6 plus flow cytometer or BioTek FLx800 plate reader. In another experiment, T cells were first co-cultured with wild-type Nalm6 for 2 days at 2: 1 E:T ratio before re-challenged with CD19-negative Nalm6 at 2: 1 : 1 T cells : wildtype Nalm6 : CD 19-negative Nalm6 ratio in the presence or absence of 1 pg / mL anti-CD38 antibody daratumumab.

[0085] For long-term cytotoxicity, target cell lines expressing mCherry were co-cultured with T cells and monitored with the IncuCyte Zoom System (Essen BioScience, Ann Arbor, MI) set to collect whole-well imaging data every 4 hours.Cell activation, proliferation, degranulation, and cytokine production

[0086] To measure T cell activation, T cells were cultured alone or with target cells for 24 hours. Expression levels of activation markers were measured using antibodies against CD25-PE (clone 2A3, BD Biosciences) and CD69-APC (clone L78, BD Biosciences). In some experiments, activation marker expression was measured after culturing T cells in rituximab-coated plates for 24 hours. To prepare the plate, 100 pL of 1 pg / mL rituximab was added to 96-well flat bottom plates (Costar, Coming, NY) and incubated at 4°C overnight; the plate was washed once with lx DPBS (Gibco) before use.

[0087] To measure T cell proliferation, T cells were cultured alone or in the presence of irradiated (100 Gy) target cells at 1 : 1 ratio; 100 lU / mL IL-2 was added every 2 -3 days, irradiated target cells were added at the beginning of the culture and every 7 days thereafter. In parallel experiments, 1 pg / mL rituximab was added to the culture once a week. The number of viable GFP-positive T cells was counted using Accuri C6 plus flow cytometer.

[0088] Exocytosis of lytic granules was assessed by measuring levels of CD 107a after culturing T cells with target cells at 1 : 1 ratio. In some experiments, T cells were cultured inrituximab-coated plates prepared as mentioned above. Anti -human CD107a-PE (H4A3; BD Biosciences) was added at the beginning of the cultures. After 1 hour, monensin (BD GolgiStop) was added, and the cultures were continued for another 3 hours before acquisition by flow cytometer.

[0089] To measure TNFa and IFNy secretion, T cells were cultured with target cells at 1 : 1 ratio or in rituximab-coated plates as above. After 1 hour, brefeldin A (BD GolgiPlug) was added and the cultures were continued for another 5 hours. The cells were then stained intracellularly using anti-TNFa-APC (clone 6401.1111; BD Biosciences) or anti-IFNy-PE (clone 25723.11; BD Biosciences) and analyzed with a flow cytometer.Xenograft model

[0090] To assess anti-leukemic in vivo, 0.5* 106Nalm6 expressing luciferase were injected intravenously (IV) in NOD.Cg-PrkdcscldIL2rgtmlSzJ (NOD-scid-IL2RGnull) mice (Jackson Laboratory, Bar Harbor, ME). Four days later, mice were distributed to 4 groups with equivalent tumor load and injected IV with 20* 106T cells expressing GFP alone, CAR, or CARFcR; 1 group of mice received culture media only. All mice received 20,000 IU of IL-2 intraperitoneally (IP) every 2 to 3 days. Tumor progression was determined by measuring luminescence signal with Xenogen IVIS-200 System (PerkinElmer, Waltham, MA), after IP injection of aqueous d-luciferin potassium salt (150 pg / g body weight;PerkinElmer). Luminescence was analyzed with Living Image 3.0 software. Mice were euthanized when luminescence reached 1 x 1011photons / second, or earlier if physical signs warranted euthanasia.

[0091] Mouse blood was obtained via cheek prick, and cells were counted with Celltac a automated hematology analyzer (Nihon Kohden, Tokyo, Japan). After treatment with red blood cell lysis solution (MilliporeSigma, Burlington, MA), cells were stained with antihuman CD45-APC (2D1; BioLegend, San Diego, CA), anti-human CD3-PE / Cy7 (HI30; BD Pharmingen, San Diego, CA), and anti-mouse CD45-PE (30-F11; BD Pharmingen). These studies were approved by the Institutional Animal Care and Use Committee of the National University of Singapore.ResultsDesign and expression of CARs with antibody-binding capacity

[0092] To test whether a CAR could be endowed with the capacity to bind the Fc portion of antibodies while retaining the capacity to bind its cognate antigen, we inserted the extracellular portion of CD16 with V158F polymorphism into the anti-CD19-41BB-CD3^ (anti-CD19 CAR) sequence. We generated six different configurations of the receptor by joining CD16 and anti-CD19 scFv with either a flexible ((G4S)4(SEQ ID NOs: 28 and 29) and (G4S)4(SEQ ID NOs: 30 and 31)) or a rigid (EAAAK) (SEQ ID NOs: 32 and 33) linker (“CARFcR”). The receptors aCD19-(G4S)1-CD16vbbz (“CARFcR-l”; SEQ ID NOs: 1 and 2), aCD19-(G4S)4-CD16vbbz (“CARFcR-2”; SEQ ID NOs: 3 and 4), and aCD19-EAAAK- CD16vbbz (“CARFcR-3”; SEQ ID NOs: 5 and 6), and aCD19-CD16vbbz (“CARFcR-4”; SEQ ID NOs: 7 and 8) had CD 16 downstream of the anti-CD19 scFv, while the receptor CD16v- (G4S)!-aCD19bbz (“CARFcR-5”; SEQ ID NOs: 9 and 10) and CD16v-aCD19bbz (“CARFcR- 6”; SEQ ID NOs: 11 and 12) had it upstream (FIG. 1). Regardless of receptor design, transduction of CARFcRconstructs into human T cells resulted in high expression of both scFv and CD 16, and similar percentages of scFv-positive cells were measured in T cells transduced with the standard anti-CD19 CAR and with the CARFcR(FIG. 2 A). To account for differences in transduction efficiency, we used GFP as reference and compared expression levels of scFv. CARFcr-5 and -6 had lower scFv expression compared to the standard CAR, while the other CARFcrhad similar expression (FIG. 2B). Similar analysis was also performed for CD 16 expression. CD 16 was expressed at similar levels for all CARFcr, except for CARFcr-l which showed slightly lower CD16 expression (FIG. 2C).

[0093] To determine whether addition of CD 16 had conferred capacity to bind antibodies to the CAR, we exposed T cells expressing the receptors to the anti-CD20 antibody rituximab. As shown in FIG. 3A, binding of rituximab was detectable only in T cells transduced with CARFcR. To precisely compare the antibody-binding capacity of the different receptors, which can be affected by differences in transduction efficiency, we used levels of GFP as a reference. As shown in FIG. 3B, at any level of GFP, CARFcR-l, -5, and -6 showed higher binding to rituximab. All six CARFcRyielded to T cells a significantly higher capacity to aggregate with K562-CD20 cells when rituximab was added to the culture, whereas the antibody had no effect if T cells were transduced with either CAR or GFP alone (FIG. 3C).

[0094] We then compared the antigen binding capacity of the anti-CD19 scFv in the CARFcRusing human CD 19 protein. CD 19 binding was detected for all six CARFcR(FIG.4A). Of note, using GFP as a reference for levels of transduction, T cells expressing CARFcR- 2 and -3 had higher binding than the standard CAR at any given level of GFP, while CARFcR- 5 and -6 had lower binding (FIG. 4B). T cells expressing either CAR or CARFcRhad higher capacity to form aggregates with the CD 19+ RS4;11 cells than control T cells. Interestingly, there was a significantly greater aggregate formation with T cells expressing CARFcR-l, and - 2 as compared to those with the standard CAR, while CARFcR-3, 4, -5, and -6 had fewer aggregates (FIG. 4C). In sum, CARFcRhad antibody binding capacity while retaining the capacity to bind the scFv cognate antigen.T cells expressing CARFcRcan trigger both CAR-mediated cytotoxicity and ADCC

[0095] Although CARFcRretained the capacity to bind the cognate antigen, it was important to ensure that CAR-mediated T-cell cytotoxicity was not perturbed. To this end, we performed 4-hour cytotoxicity assays with the CD 19+ target cells, including the ALL cell lines Nalm-6, OP-1, RS;11, and REH, and the lymphoma cell lines Ramos and Daudi. As shown in FIG. 5, cytotoxicity of anti-CD19 CARFcRwas similar to that mediated by the standard anti-CD19 CAR. We noted, however, that the cytotoxicity exerted by T cells expressing CARFcR-2 was lower compared to that of cells expressing the other CARFcR. (FIG. 5). In 5-day cultures with Nalm6 at 1 :4 E:T ratio, T cells expressing CARFcR-l had more powerful anti-tumor activity that those expressing the other five CARFcR(FIGs. 6A-B), indicating that the construct with the CD 16 V158F joined downstream of the anti-CD19 scFv with a short flexible linker had the least interference with CAR function.

[0096] In parallel experiments, we tested whether the antibody -binding capacity of the CARFcRcould also mediate ADCC. To this end, we cultured the HER2+ SKBR3 breast cancer cell line with T cells expressing CARFcRor CAR in the presence of anti-HER2 antibody trastuzumab. Only T cells expressing CARFcRexerted specific cytotoxicity against SKBR3 (FIG. 7).

[0097] CARFcR-T cells also killed SKBR3 cells in long-term cultures (FIG. 8A). Like in the experiments targeting CD 19+ cells with the anti-CD19 scFv, we observed a greater tumor cell killing with T cells expressing CARFcR-l (FIG. 8B). In sum, different CARFcRconfigurations resulted in functional difference (Table 1). We selected CARFcR-l for further studies.Table 1. Ranking Scoring of CARFcRaccording to performance in different assaysCARFcRallows cytotoxicity against multiple targets

[0098] A unique property of CARFcRis the recognition of multiple targets through scFv and antibody binding. Therefore, it was important to ascertain that addition of antibody would not impair CAR function and determine whether it was possible to increase the cytotoxicity of CARFcRT cells with an antibody directed against a surface molecule expressed in target cells besides that recognized by the scFv. As shown in FIG. 9A, the addition of anti-CD20 antibody rituximab increased cytotoxicity of anti-CD19 CARFcR-l T cells against CD19+ CD20+ cell lines Daudi and Ramos. Likewise, anti-CD19 CARFcR-l Tcells exerted significantly higher cytotoxicity against CD19+ CD38+ cell lines OP-1, REH, RS4;11, and Ramos in the presence of anti-CD38 antibody daratumumab (FIG. 9B).

[0099] We also tested whether scFv-mediated killing affected the capacity of anti-CD19 CARFcR-l-T cells to subsequently exert ADCC. For this purpose, we co-cultured T cells alone or with CD 19+ RS4;11 cells for 4 hours and then transferred them to cultures containing HER2+ SKBR3 cells in the presence of trastuzumab. Only T cells expressing the anti-CD19 CARFcR-l exerted ADCC against SKBR3, and the degree of cytotoxicity was similar regardless of whether T cells had been previously exposed to RS4;11 (FIG. 9C). Conversely, prior co-culture with K562-CD20 and rituximab for 24 hours did not affect killing of CD 19+ RS4;11 cells (FIG. 9D).A bi-specific antibody directed against CD33 and CD 16 can trigger NK cell activation via ligation of CD 16 and significantly increase their cytotoxicity against target cells expressing CD33. To evaluate whether anti-CD19 CARFcR-l T cells could be activated by the bi-specific antibody, we produced it by transducing Jurkat cells with the corresponding cDNA and collecting the cells’ supernatant. This was then added to cultures of anti-CD19 CARFcR-l T cells and CD33+ KG-1 and Kasumi-3 cells. As shown in FIG. 10, the bi-specific antibody triggered cytotoxicity against CD33+ cell lines.CARFcRcan prevent reoccurrence of CD 19-negative ALL

[0100] Loss of target expression is one of the mechanisms by which tumor cells escape killing by CAR-T cells. With its ability to bind both CAR-cognate antigen and antibody, anti- CD19 CARFcRmight be able to prevent disease recurrence from CD 19-negative escape by targeting another antigen recognizable by the antibody. To test this, we first generated CD19- negative Nalm6 by CRISPR-Cas9 and evaluated its killing by anti-CD19 CARFcR-l T cells. As shown in FIG. 11 A and B, anti-CD19 CARFcR-l T cells exerted strong cytotoxicity against CD 19-negative Nalm6 in the presence of anti-CD38 antibody daratumumab, whereas no killing was observed in the absence of the antibody. We subsequently tested the cytotoxicity of anti-CD19 CARFcR-l in a CD 19-negative relapse model by first co-culturing anti-CD19 CARFcR-l T cells with wild type Nalm6 and adding CD19-negative Nalm6 after all the wild type Nalm6 had been eliminated. The growth of CD 19-negative Nalm6 was completely suppressed by anti-CD19 CARFcR-l T cells with the addition of daratumumab to the culture (FIG. 11C).Functional characteristics of CARFcR

[0101] To further examine CARFcRfunction, we measured levels of the activation markers CD25 and CD69 before and after co-culturing T cells expressing the anti-CD19 CARFcR-l with the CD 19+ cell line RS4;11. Interestingly, levels of both CD25 and CD69 were significantly lower in anti-CD19 CARFcR-l than in the standard anti-CD19 CAR-T cells in the absence of stimulation (FIG. 12A), but both markers increased to similar levels in T cells expressing either receptor after exposure to CD 19+ cells (FIG. 12B). We also assessed whether cross-linking of CARFcRwith an immobilized antibody (rituximab) could induce T- cell activation. As shown in FIG. 12B, both CD25 and CD69 expression increased in T cells expressing anti-CD19 CARFcR-l but not in anti-CD19 CAR-T cells. Likewise, stimulation via anti-CD19 CAR scFv by RS4;11 or CD 16 by immobilized rituximab resulted in significantly higher exocytosis of lytic granules and cytokine secretion (FIGs. 13A-C). Of note, culturing T cells expressing anti-CD19 CARFcR-l with RS4;11 on rituximab-coated plates did not interfere with CAR signaling (FIGs. 12A-B and 13A-C).

[0102] Proliferation of T cells expressing anti-CD19 CARFcR-l was triggered by coculture with irradiated RS4;11 and occurred at a rate similar to that of standard anti-CD19 CAR-T cells (FIG. 14). By contrast, K562-CD20 cells coated with rituximab stimulated the proliferation of anti-CD19 CARFcR-l-T cells but not that of anti-CD19 CAR-T cells, while unbound rituximab had no significant effects on cell proliferation (FIG. 14).CARFcRcan be directed against antigens beyond CD 19

[0103] It was important to determine whether the functional gains brought about by the CARFcRtechnology were confined to the anti-CD19 specificity or were more generally applicable. To test this issue, we designed CARs directed against CD33, CD123, or BCMA with a similar configuration to that of the anti-CD19-41BB-CD3^ CAR used in the previous experiments and then generated the corresponding CARFcR-l. The schematic representation of anti-CD33, anti-CD123, and anti-BCMA CARFcRis shown in FIGs. 15A-C (SEQ ID NOs: 13-18).

[0104] Transduction of these three CARFcRin T cells resulted in high expression of both scFv and CD 16 on the cell surface, whereas only scFv expression was detected in T cells transduced with corresponding CARs lacking CD 16 (FIGs. 16A-B, 17A-B, and 18A-B). Levels of scFv expression were similar in CARFcRand CAR. Expression of these CARFcRinT cells also allowed the T cells to bind to Fc portion of antibody (FIGs. 16A-B, 17A-B, and 18A-B).

[0105] The cytotoxicity of T cells expressing anti-CD33 CARFcRagainst the CD33+ cell lines KG-1, Kasumi-3, and U937 was similar to that of T cells expressing anti-CD33 CAR (FIGs. 19A-B). Likewise, anti-CD123 CARFcR- and anti-CD123 CAR-T cells had similar cytotoxicity against CD123+ KG-1, Kasumi-3 and THP-1 (FIGs. 20A-B); anti-BCMA CARFcR- and anti-BCMA CAR-T cells exerted similar cytotoxicity against BCMA+ lymphoma cell lines Daudi and Ramos and multiple myeloma cell lines RPMI8226, MMl.s, and U266-Bl (FIGs. 21A-C).

[0106] Importantly, the inclusion of CD16 into the CAR constructs did not impair the Fc binding properties of the CD16 and its subsequent function. T cells expressing anti-CD33, anti-CD123, or anti-BCMA CARFcRexerted strong ADCC against HER2+ SKBR3 cells in the presence of anti-HER2 antibody trastuzumab, while no cytotoxicity was observed with the corresponding CARs lacking CD 16 (FIGs. 22A-C). Moreover, addition of an antibody did not interfere with the cytotoxic function of CARFcRand improved target cell killing was seen when the target antigens of the scFv and of the antibody were expressed simultaneously in the same cell. T cells expressing anti-CD123 CARFcRwere co-cultured with CD123+ CD38+ KG1 and Kasumi3 cells, and anti-BCMA CARFcR-T cells with BCMA+ CD38+ Ramos, and MMl.s; in all cultures, cytotoxicity was assessed with or without the anti-CD38 antibody daratumumab. In parallel experiments, anti-BCMA CARFcRT cells were also tested in co-culture with BCMA+ CD20+ Daudi and Ramos cells with or without rituximab. In all experiments, the addition of the antibody improved killing of target cells with CARFcR(FIGs. 23A-B). As expected, stimulation of T cells expressing all three CARFcRwith immobilized antibody resulted in increased expression of activation markers CD25 and CD69 (FIGs. 24A- C).Comparison of activation levels of different anti-CD19 CARFcRconfigurations

[0107] To further assess the effect of different CARFcRconfigurations on T-cell activation, we stimulated T cells expressing the various anti-CD19 CARFcRconstructs with CD 19+ RS4;11 for 24 hours. All anti-CD19 CARFcRconstructs induced upregulation of CD25 and CD69 expression similar to that induced by anti-CD19 CAR when stimulated with target antigen (FIG. 25A). The increased in CD25 and CD69 expression varied among the different anti-CD19 CARFcRconstructs, with anti-CD19 CARFcR-5 showing the highestincrease (FIG. 25 A and Table 2) . In a parallel experiments, we stimulated anti-CD19 CARFcR-T cells with immobilized monoclonal antibody rituximab. Only T cells expressing anti-CD19 CARFcRhad increased expression of CD25 and CD69 (FIG. 25B). Similarly, the highest levels of activation markers expression were seen in anti-CD19 CARFcR-5 T cells (FIG. 25B and Table 2).

[0108] We subsequently evaluated the proliferation of T-cells expressing the various anti- CD19 CARFcRconstructs in co-cultures with irradiated target cells (FIG. 25C). Both anti- CD19 CAR- and anti-CD19 CARFcR-T cells had increased proliferation in the presence of irradiated CD 19+ RS4;11, but only anti-CD19 CARFcR-T cells had higher proliferation rate when the cells were co-cultured with irradiated K562-CD20 and antibody (FIG. 25C). The proliferation rate was different among the various anti-CD19 CARFcRconstructs (Table 2), although the difference was not statistically significant.Table 2. Scoring of CARFcRbased on activation levels in response to stimulation.*Score: 3 = 25th percentile; 2 = 50th percentile; 1 = 75th percentileAnti-leukemic activity of anti-CD19 CARFcR-T cells in a murine model of ALL

[0109] To assess the anti-leukemic activity of CARFcR-T cells in vivo, we engrafted NOD-scid-IL2RGnull mice with Nalm6 cells and treated the mice with anti-CD19 CAR- or anti-CD19 CARFcR-5-T cells four days later. Nalm6 cells rapidly expanded in untreated mice and in mice that received control T cells, whereas growth of Nalm6 cells was markedly reduced in mice injected with anti-CD19 CAR- or anti-CD19 CARFcR-5-T cells (FIGs. 26A- B). Survival of mice treated with anti-CD19 CARFcR-5-T cells was similar to that of anti- CD19 CAR-T cells (FIG. 26C). The percentage of T cells (hCD3+ hCD45+) in peripheral blood of mice that received anti-CD19 CAR- or anti-CD19 CARFcR-5-T cells was also similar, and there was no difference in the levels of platelet, red blood cell, and hemoglobin between the two groups (FIG. 26D).Discussion

[0110] This study aimed at developing a receptor that could redirect CAR-T cell specificity with an antibody, thus retaining CAR-mediated function and gaining ADCC capacity. To construct such multi-functional CAR, the extracellular portion of CD16 with the V158F polymorphism, which encodes a high-affinity Fc receptor, was inserted into the anti- CD19-41BB-CD3^ (anti-CD19 CAR) sequence in six different configurations. The resulting chimeric receptors, named CARFcR, could be highly expressed in human T cells, had scFv- mediated CD 19 binding as well as CD16-mediated antibody binding. In turn, these dual capacities triggered both CAR-mediated cytotoxicity and ADCC, although there were functional differences among CARFcRwith different configurations. The unique properties of CARFcRwere not limited to the specific antigen recognized by the anti-CD19 scFv; CARFcRwith scFvs directed against CD33, CD 123 and BCMA could be efficiently expressed in T cells and all elicited CAR-mediated cytotoxicity as well as ADCC.

[0111] The CARs that were tested in this study were directed against known targets for CAR T-cell therapy of leukemia, lymphoma and multiple myeloma.5'8’22Four of the six currently approved CAR-T cell products are directed against CD 19 and two against BCMA; two of anti-CD19 CARs and both anti -BCMA CARs have 2ndgeneration signaling properties delivered by a 4-lBB-mediated co-stimulus. Anti-CD33 and anti-CD123 CARs have been tested clinically in AML although results have been generally disappointing;23 24lack of antigen expression in a substantial proportion of cells may have been a factor contributing to poor response and / or relapse. The CARFcRdesign developed in this study allows the receptorto have similar activity as a standard CAR and also to target other antigens beyond the CAR cognate antigen. This quantum increase in function can be triggered “on the fly”, i.e., postinfusion, and has a clear clinical application. For instance, CD19-clonal escape is a well- known cause of relapse in patients with ALL and B-NHL treated with anti-CD19 CAR T cell therapy.9 10Anti-CD19 CAR-T cells may persist but are ineffective against the CD 19- negative relapsing cells. In such cases, administration of clinically available antibodies directed against CD20, CD22, and / or CD38 antigens often expressed in B-lymphoid cancers, should reactivate the anti-tumor effect of the infused T cells without the need of a new CAR- T cell manufacturing and lymphodepleting chemotherapy. In some instances, a bi-specific antibody binding CD 16 and an antigen expressed in B-lymphoid cancers, such as CD20, CD22, and / or CD38 can be administered. CARFcR-T cells and antibodies could also be used to target multiple antigens as initial therapy, an approach that should reduce the risk of clonal escape. Among the many possible combinations, anti-CD19 CARFcRcould be used with the anti-CD20 antibody rituximab in NHL, and anti-BCMA CARFcRwith the anti-CD38 antibody daratumumab in multiple myeloma. Importantly, our study showed that CARFcRT cells can exert CAR-mediated and ADCC function simultaneously as well as sequentially.

[0112] Targeting multiple antigens simultaneously might also be achieved by infusing separate cell populations each expressing a different CAR. Besides multiplying the costs of cell manufacturing, this approach increases the risk of CAR-related toxicities by increasing the number of infused cells. Alternatively, multiple CARs could be expressed in the same cell but this approach often results in a diminished expression and / or activity of the individual CARs.25To this end, a focus of this study was to identify receptor configurations that would allow ADCC gain-of-function without affecting the expression and the function of the original CAR. In the selected configurations, CAR-mediated cytotoxicity and proliferation was often indistinguishable to that of the original CAR. Expression of CARs with dual scFvs or scFv with cytokine receptors linked to a single signaling cassette has been proposed as a means to target multiple antigens simultaneously.25 26If the function of the CAR with individual specificities is maintained, this approach has merit. We think, however, that our CAR with ADCC properties would be advantageous. First, it offers much more flexibility regarding the second antigen that can be targeted, which can be adapted to the immunophenotype of the cancer cells in each patient. Second, it allows new specificities to be added to CAR-T cells that have already been infused. Thirdly, ADCC activity can be activated and shut down on demand by initiating or withdrawing antibody administration.Importantly, autologous T cells expressing a receptor with ADCC function only was previously developed in our laboratory,21and was tested in combination with rituximab in patients with rituximab-resistant B-cell NHL.27’28Infusion of such cells led to marked clinical responses accompanied by long-term persistence of the receptor-expressing T cells, limited toxicity and no evidence of antibody-mediated autoimmunity. Others have sought to achieve similar objectives with CARs that recognize binders attached to antibodies or bispecific antibodies that recognize scFv linkers.29 30These approaches, however, requires the use of conjugated antibodies or bispecific engagers not readily available in the clinic, and whose capacity to fix complement and engage NK cells and macrophages might be inferior to that of naked antibodies or not existent at all.

[0113] CAR-T cell therapy has revolutionized the treatment of ALL and contributed major advances in the treatment of B-NHL and multiple myeloma. With technological improvements, therapeutic impact could extend to other blood cancers and solid tumors. To this end, a major obstacle to CAR-T cell success is represented by expression of individual targets on only a fraction of tumor cells, leading to inadequate tumor reduction and / or targetnegative escape.31A second limitation is that the design of CARs directed against different specificities might not results in CARs with T cell activation properties on par to those of the prototype CAR, due to protein-folding, receptor clustering and other variables difficult to predict. The technology developed in this study allows targeting multiple tumor antigens simultaneously or sequentially while maintaining the functional integrity of the original CAR. Moreover, it opens the possibility of combining the known anti-tumor mechanisms of antibodies therapy, including complement fixation and recruitment of NK and macrophage activity, to cooperate with T cell cytotoxicity. Although we tested CARFcRin T lymphocytes, it is likely that their functions could expand the therapeutic potential of other cytotoxic lymphocytes, such as NK cells and gamma delta T cells.Summary of Sequence ListingTable 2. Summary of sequence of the sequence listingREFERENCES

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[0121] 8. Turtle CJ, Hanafi LA, Berger C, et al. CD19 CAR-T cells of definedCD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest 2016;126:2123-38.

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[0146] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0147] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A chimeric receptor comprising: a) an extracellular domain comprising: i) an antigen binding domain; and ii) an antibody fragment crystallizable (Fc) binding domain; b) a hinge and transmembrane domain; and c) an intracellular domain comprising: i) a co-stimulatory domain; and ii) a signaling domain.

2. The chimeric receptor of claim 1, wherein the antigen binding domain adjoins the hinge and transmembrane domain.

3. The chimeric receptor of claim 1, wherein the antibody Fc binding domain adjoins the hinge and transmembrane domain.

4. The chimeric receptor of claim 1, further comprising a linker domain joining the antigen binding domain and the antibody Fc binding domain.

5. The chimeric receptor of claim 4, wherein the linker domain is (G4S)X, wherein x is an integer from 1 to 100; or wherein x is an integer from 1 to 10; or wherein x is an integer from 2 to 5; or wherein x is 1; or wherein x is 3.

6. The chimeric receptor of claim 4, wherein the linker domain is (EAAAK)Z, wherein z is an integer from 1 to 1007. The chimeric receptor of claim 1, wherein the Fc binding domain is an extracellular domain of CD 16.

8. The chimeric receptor of claim 7, wherein the extracellular domain of CD 16 has a V158F polymorphism.

9. The chimeric receptor of claim 8, wherein the extracellular domain of CD 16 comprises SEQ ID NO: 37.

10. The chimeric receptor of claim 1, wherein the antigen-binding domain is a single chain variable fragment (scFv).

11. The chimeric receptor of claim 10, wherein the scFv comprises: a) a variable heavy (VH) domain; b) a variable light (VL) domain; and c) a linker domain joining the variable heavy domain and the variable light domain.

12. The chimeric receptor of any one of claims 1 through 11, wherein the antigen-binding domain binds to CD19, CD33, CD123, or B-cell maturation antigen (BCMA).

13. The chimeric receptor of any one of claims 1 through 11, wherein the co-stimulatory domain is a 4-1BB domain.

14. The chimeric receptor of any one of claims 1 through 11, wherein the signaling domain is a CD3(^ domain.

15. A mammalian T cell expressing the chimeric receptor of any one of claims 1 through 11.

16. A method of treating cancer, the method comprising: a) administering a mammalian T cell expressing a chimeric receptor of any one of claims 1 through 14; and b) administering an anti-CD20 antibody, an anti-CD22 antibody, an anti-GD2 antibody, an anti-HER2 antibody, an anti-EGFR antibody, an anti-CD38 antibody, or a bispecific antibody binding a tumor antigen and CD 16.

17. The method of claim 16, further comprising: a) withdrawing administration of the anti-CD20 antibody, the anti-CD22 antibody, the anti-GD2 antibody, the anti-HER2 antibody, the anti-EGFR antibody, the anti-CD38 antibody, or the bispecific antibody binding a tumor antigen and CD 16.

18. The method of claim 16 or 17, wherein the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), B-cell non-Hodgkin lymphoma (NHL), B-cell chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), multiple myeloma, breast cancer, neuroblastoma, lung cancer, or head and neck cancer19. A vector comprising a nucleic acid, wherein the nucleic acid encodes the following domains operably connected to each other: a) a chimeric receptor domain comprising: i) an extracellular domain comprising:1) an antigen binding domain; and2) an antibody fragment crystallizable (Fc) binding domain; ii) a hinge and transmembrane domain; iii) an intracellular domain comprising:1) a co-stimulatory domain; and2) a signaling domain; b) a expression domain.

20. The vector of claim 19, wherein the expression domain is an internal ribosome entry site or a self-cleaving peptide.

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