Bicistronic chimeric antigen receptors and uses thereof

Abstract

One embodiment of the present invention provides a bicistronic chimeric antigen receptor (CAR) amino acid construct. Nucleic acids, recombinant expression vectors, host cells, cell populations, and pharmaceutical compositions related to the CAR construct are disclosed. Methods for detecting the presence of cancer in a mammal and methods for treating or preventing cancer in a mammal are also disclosed. Methods for making the CAR construct are disclosed. [Selection diagram] None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 506,268, filed May 15, 2017, which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Federal support by the National Institutes of Health, National Cancer Institute under Project No. Z01 BC011565. The Federal Government has certain rights in this invention.

[0003] Incorporation by reference of electronically submitted property The computer readable nucleotide / amino acid sequence listing, which was submitted contemporaneously herewith and is identified as follows, is hereby incorporated by reference in its entirety: One 180,939 byte ASCII (text) file entitled "739267_ST25.TXT", dated May 15, 2018. [Background technology]

[0004] 2. Background of the Invention Cancer is a public health concern. Despite advances in treatments such as chemotherapy, the prognosis for many cancers, including hematological malignancies, can be poor. Thus, there is an unmet need for additional treatments for cancer, particularly hematological malignancies. Summary of the Invention

[0005] Summary of the Invention One embodiment of the present invention provides a chimeric antigen receptor (CAR) amino acid construct comprising: (a) a cleavable domain; (b) a first CAR comprising a first antigen binding domain, a first transmembrane domain, and a first intracellular T cell signaling domain; and (c) a second CAR comprising a second antigen binding domain, a second transmembrane domain, and a second intracellular T cell signaling domain; wherein the first and second CARs are linked via the cleavable domain, and the first antigen binding domain comprises the antigen binding domain of the m971 antibody, and wherein when the first CAR is cleaved from the construct, the first antigen binding domain has antigen specificity for CD22.

[0006] Another embodiment of the invention provides a chimeric antigen receptor (CAR) amino acid construct comprising: (a) a cleavable domain; (b) a first CAR comprising a first antigen binding domain, a first transmembrane domain, and a first intracellular T cell signaling domain; and (c) a second CAR comprising a second antigen binding domain, a second transmembrane domain, and a second intracellular T cell signaling domain; wherein the first and second CARs are linked via the cleavable domain, and the first antigen binding domain comprises the antigen binding domain of the FMC63 antibody, and wherein when the first CAR is cleaved from the construct, the first antigen binding domain has antigen specificity for CD19.

[0007] Another embodiment of the invention provides a chimeric antigen receptor (CAR) amino acid construct comprising: (a) two or more cleavable domains; (b) a first CAR comprising a first antigen binding domain, a first transmembrane domain, and a first intracellular T cell signaling domain; and (c) a second CAR comprising a second antigen binding domain, a second transmembrane domain, and a second intracellular T cell signaling domain; wherein the first and second CARs are linked via the two or more cleavable domains.

[0008] Another embodiment of the present invention provides a method for making a chimeric antigen receptor (CAR) amino acid construct, the method comprising: (a) designing two or more cleavable domains between a first CAR comprising a first antigen binding domain, a first transmembrane domain, and a first intracellular T cell signaling domain; and (b) a second CAR comprising a second antigen binding domain, a second transmembrane domain, and a second intracellular T cell signaling domain, wherein the first and second CAR are linked via the two or more cleavable domains; and cloning a sequence comprising the first CAR, the two or more cleavable domains, and the second CAR from N-terminus to C-terminus into a plasmid.

[0009] Another embodiment of the present invention provides a CAR amino acid construct comprising an amino acid sequence described herein.

[0010] Further embodiments of the present invention provide related nucleic acids, recombinant expression vectors, host cells, cell populations, as well as pharmaceutical compositions related to the CAR amino acid constructs of the present invention.

[0011] Further embodiments of the present invention provide methods for detecting the presence of cancer in a mammal, and methods for treating or preventing cancer in a mammal. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 presents a schematic of an exemplary CAR construct according to an embodiment of the invention. [Figure 2-1] 2A-2C present fluorescence-activated cell sorting dot plots comparing surface expression of anti-CD19 CAR and CD3 on human T cells when T cells are transduced with a vector encoding a single anti-CD19 CAR (anti-CD19 CAR denoted as "CD19 CAR") or a V1 CAR construct (also denoted as bicistronic-V1 or bicis-V1), according to an embodiment of the invention. [Figure 2-2]2A-2C present fluorescence-activated cell sorting dot plots comparing surface expression of anti-CD22 CAR and CD3 on human T cells when T cells are transduced with a vector encoding an anti-CD22 CAR alone (anti-CD22 CAR denoted as "CD22 CAR") or a V1 CAR construct, according to an embodiment of the invention. [Figure 2-3] 2A-2C present fluorescence-activated cell sorting dot plots comparing surface expression of anti-CD19 CAR and anti-CD22 CAR on human T cells when the cells are transduced with a vector encoding the V1 CAR construct, according to an embodiment of the invention. (2C) [Diagram 3] FIG. 3 presents fluorescence-activated cell sorting dot plots comparing the surface expression of anti-CD19 CAR and anti-CD22 CAR on human T cells when transduced with vectors encoding a single anti-CD19 CAR, a single anti-CD22 CAR, LoopCAR6, or co-transduced with separate vectors encoding a single anti-CD19 CAR and a single anti-CD22 CAR. [Figure 4] FIG. 4 presents fluorescence-activated cell sorting dot plots comparing the surface expression of anti-CD19 CAR and anti-CD22 CAR on human T cells when transduced with vectors encoding the V1 CAR construct, the V5 CAR construct (also denoted as bicistronic-V5 or bicis-V5), or LoopCAR6, according to an embodiment of the invention. [Figure 5-1] Figure 5A is a bar graph showing in vitro activity based on cytokine production according to an embodiment of the invention, where Figure 5A shows IL2 levels and Figure 5B shows IFNγ levels (measured when K562 cells expressing CD19, CD22, both, or neither are contacted with T cells transduced with vectors encoding the V1 CAR construct, anti-CD19 CAR alone (CAR19), or anti-CD22 CAR alone (CAR22), according to an embodiment of the invention). [Figure 5-2]Figure 5B is a bar graph showing in vitro activity based on cytokine production according to an embodiment of the invention. Figure 5A shows IL2 levels and Figure 5B shows IFNγ levels (measured when K562 cells expressing CD19, CD22, both, or neither are contacted with T cells transduced with vectors encoding the V1 CAR construct, anti-CD19 CAR alone (CAR19), or anti-CD22 CAR alone (CAR22), according to an embodiment of the invention). [Figure 6-1] Figure 6A is a bar graph showing in vitro activity based on cytokine production according to an embodiment of the invention, where Figure 6A shows IL2 levels and Figure 6B shows IFNγ levels (measured when K562 cells expressing CD19, CD22, both, or neither are contacted with T cells transduced with vectors encoding V1 CAR constructs, V5 CAR constructs, LoopCAR6, anti-CD19 CAR alone, or anti-CD22 CAR alone according to an embodiment of the invention). [Figure 6-2] Figure 6B is a bar graph showing in vitro activity based on cytokine production according to an embodiment of the invention. Figure 6A shows IL2 levels and Figure 6B shows IFNγ levels (measured when K562 cells expressing CD19, CD22, both, or neither are contacted with T cells transduced with vectors encoding V1 CAR constructs, V5 CAR constructs, LoopCAR6, anti-CD19 CAR alone, or anti-CD22 CAR alone according to an embodiment of the invention). [Figure 7-1] Figure 7A is a bar graph showing in vitro activity based on cytokine production according to an embodiment of the invention, where Figure 7A shows IL2 levels and Figure 7B shows IFNγ levels (measured when CD19 KO and / or CD22 KO NALM6 cells are contacted with T cells transduced with vectors encoding V1 CAR constructs, V5 CAR constructs, LoopCAR6, anti-CD19 CAR alone, or anti-CD22 CAR alone according to an embodiment of the invention). [Figure 7-2]Figure 7B is a bar graph showing in vitro activity based on cytokine production according to an embodiment of the invention. Figure 7A shows IL2 levels and Figure 7B shows IFNγ levels (measured when CD19 KO and / or CD22 KO NALM6 cells are contacted with T cells transduced with vectors encoding V1 CAR constructs, V5 CAR constructs, LoopCAR6, anti-CD19 CAR alone, or anti-CD22 CAR alone according to an embodiment of the invention). [Figure 8] Figure 8 is a bar graph showing in vitro activity based on cytokine production according to an embodiment of the invention. Figure 8 shows that CAR T cells were co-incubated with NALM6 tumor cells for 18 hours and IL2 production levels were measured by ELISA in the culture supernatants ("-N": NALM6; "-N-19": NALM6-CD19neg; "-N-19-22": NALM6-CD19neg-CD22neg). [Figure 9] Figures 9-14 present bioluminescence imaging of leukemia progression in vivo following treatment with T cells transduced with vectors encoding anti-CD19 CAR alone, anti-CD22 CAR alone, LoopCAR6, V1 CAR construct, or V5 CAR construct according to embodiments of the invention compared to mock T cells (non-transduced T cells). Bioluminescence intensity indicated by increasing levels of shading represents tumor burden. "Lenti" indicates that the CAR is designed and engineered in a lentiviral backbone. [Figure 10] Figures 9-14 present bioluminescence imaging of leukemia progression in vivo following treatment with T cells transduced with vectors encoding anti-CD19 CAR alone, anti-CD22 CAR alone, LoopCAR6, V1 CAR construct, or V5 CAR construct according to embodiments of the invention compared to mock T cells (non-transduced T cells). Bioluminescence intensity indicated by increasing levels of shading represents tumor burden. "Lenti" indicates that the CAR is designed and engineered in a lentiviral backbone. [Figure 11]Figures 9-14 present bioluminescence imaging of leukemia progression in vivo following treatment with T cells transduced with vectors encoding anti-CD19 CAR alone, anti-CD22 CAR alone, LoopCAR6, V1 CAR construct, or V5 CAR construct according to embodiments of the invention compared to mock T cells (non-transduced T cells). Bioluminescence intensity indicated by increasing levels of shading represents tumor burden. "Lenti" indicates that the CAR is designed and engineered in a lentiviral backbone. [Figure 12] Figures 9-14 present bioluminescence imaging of leukemia progression in vivo following treatment with T cells transduced with vectors encoding anti-CD19 CAR alone, anti-CD22 CAR alone, LoopCAR6, V1 CAR construct, or V5 CAR construct according to embodiments of the invention compared to mock T cells (non-transduced T cells). Bioluminescence intensity indicated by increasing levels of shading represents tumor burden. "Lenti" indicates that the CAR is designed and engineered in a lentiviral backbone. [Figure 13] Figures 9-14 present bioluminescence imaging of leukemia progression in vivo following treatment with T cells transduced with vectors encoding anti-CD19 CAR alone, anti-CD22 CAR alone, LoopCAR6, V1 CAR construct, or V5 CAR construct according to embodiments of the invention compared to mock T cells (non-transduced T cells). Bioluminescence intensity indicated by increasing levels of shading represents tumor burden. "Lenti" indicates that the CAR is designed and engineered in a lentiviral backbone. [Figure 14]Figures 9-14 present bioluminescence imaging of leukemia progression in vivo following treatment with T cells transduced with vectors encoding anti-CD19 CAR alone, anti-CD22 CAR alone, LoopCAR6, V1 CAR construct, or V5 CAR construct according to embodiments of the invention compared to mock T cells (non-transduced T cells). Bioluminescence intensity indicated by increasing levels of shading represents tumor burden. "Lenti" indicates that the CAR is designed and engineered in a lentiviral backbone. [Figure 15] FIG. 15 is a line graph showing CD22 expression in patients before and after loss of CD19. [Figure 16] FIG. 16 is a dot plot showing CD19 and CD22 expression (CRISPR CD19neg and CD22neg leukemia lines compared to the parental NALM6 line). [Figure 17] Figure 17 presents images showing a comparison of in vivo progression (CRISPR CD19neg and CD22neg leukemia cells vs. parental NALM6 cells). Bioluminescence intensity as indicated by increasing shade levels represents tumor burden. [Figure 18] Figure 18 presents images showing a comparison of the therapeutic methods using CARs described herein. NSG mice were administered 2.5E5 NALM6 and a mixture of NALM6-CD19neg and NALM6-CD22neg leukemia lines on day 0. Mice in the sequential treatment group were administered 3E6 CAR+ on day 3 and 3E6 CAR+ T cells on day 9. Mice in the coinjection group were administered 3E6 anti-CD19 CAR+ and 3E6 anti-CD22 CAR+ T cells on day 3 for a total of 6E6 CAR+ T cells. Mice in the co-transduction group were administered 8E6 T cells containing 3E6 anti-CD19+ and 3E6 anti-CD22+ CAR T cells. Mice in the CD19 or CD22 groups were administered 3E6 CAR+ T cells. Bioluminescence intensity indicated by increasing shade levels represents tumor burden. It is suggested that co-injection or co-transduction of anti-CD19 and anti-CD22 CARs may reduce leukemia relapse by targeting both CD19 and CD22 simultaneously. [Figure 19] Figure 19 is a dot plot showing a comparison of single vector transduction and co-transduction with anti-CD19 and anti-CD22 CAR constructs. [Figure 20] FIG. 20 is a presentation of a graphical plot of leukemia phenotypes following CAR therapy as described in Example 7. [Figure 21] FIG. 21 diagrammatically presents the TanCAR of Example 7, according to an embodiment of the present invention. [Figure 22-1] FIG. 22A is a bar graph showing cytokine production of various CARs described herein in K562, K562-CD19, K562-CD22, and K562-CD19CD22 target cell lines, in accordance with embodiments of the invention. [Figure 22-2] FIG. 22B is a bar graph showing cytokine production of various CARs described herein in K562, K562-CD19, K562-CD22, and K562-CD19CD22 target cell lines, in accordance with embodiments of the invention. [Figure 23-1] Figure 23A presents images showing a comparison of TanCAR1 and TanCAR4 for the treatment of leukemia in vivo. NSG mice were challenged with 1E6 luciferase-expressing NALM6 leukemia on day 0. On day 3, mice were IV injected with 3E6 CAR-expressing T cells. Bioluminescence intensity, as indicated by increasing shade levels, represents tumor burden. [Figure 23-2] Figures 23B-23D present dot plots showing Incucyte killing assays with co-incubation of each CAR or mock T cell product according to an embodiment of the invention. [Figure 23-3] Figures 23B-23D present dot plots showing Incucyte killing assays with co-incubation of each CAR or mock T cell product according to an embodiment of the invention. [Figure 23-4] Figures 23B-23D present dot plots showing Incucyte killing assays with co-incubation of each CAR or mock T cell product according to an embodiment of the invention. [Figure 24]FIG. 24 presents a schematic of LoopCAR of Example 7, according to an embodiment of the present invention. [Figure 25-1] 25A-25C present bar graphs showing cytokine production of various CARs in K562, K562-CD19, K562-CD22, and K562-CD19CD22 target cell lines, according to embodiments of the invention. [Figure 25-2] 25A-25C present bar graphs showing cytokine production of various CARs in K562, K562-CD19, K562-CD22, and K562-CD19CD22 target cell lines, according to embodiments of the invention. [Figure 25-3] 25A-25C present bar graphs showing cytokine production of various CARs in K562, K562-CD19, K562-CD22, and K562-CD19CD22 target cell lines, according to embodiments of the invention. [Figure 26] FIG. 26 is a bar graph showing cytokine production of various CARs in K562, K562-CD19, K562-CD22, and K562-CD19CD22 target cell lines, in accordance with embodiments of the invention. [Figure 27] FIG. 27 is a dot plot showing an Incucyte killing assay of each CAR or mock T cell product according to an embodiment of the invention co-incubated with 10:1 NALM6:NALM6-CD19neg cells. [Figure 28] FIG. 28 is a dot plot showing an Incucyte killing assay of each CAR or mock T cell product according to an embodiment of the invention co-incubated with 10:1 NALM6:NALM6-CD22neg cells. [Figure 29-1] Figures 29A-29F are bar graphs showing that LoopCAR6 produces various cytokines when co-incubated with target antigens. Figure 29A: Interferon gamma. [Figure 29-2] Figures 29A-29F are bar graphs showing that LoopCAR6 produces various cytokines when co-incubated with target antigens. Figure 29B: IL6. [Figure 29-3] Figures 29A-29F are bar graphs showing that LoopCAR6 produces various cytokines when co-incubated with target antigens. Figure 29C: TNF-alpha. [Figure 29-4] Figures 29A-29F are bar graphs showing that LoopCAR6 produces various cytokines when co-incubated with target antigens. Figure 29D: IL8. [Figure 29-5] Figures 29A-29F are bar graphs showing that LoopCAR6 produces various cytokines when co-incubated with target antigens. Figure 29E: IL13. [Figure 29-6] Figures 29A-29F are bar graphs showing that LoopCAR6 produces various cytokines when co-incubated with target antigens. Figure 29F: IL2. [Diagram 30] Figure 30 shows images. NSG mice were challenged with 1E6 luciferase-expressing NALM6 leukemia on day 0. On day 3, mice were IV injected with 3E6 CAR-expressing T cells. Bioluminescence intensity as indicated by increasing shade levels represents tumor burden. [Diagram 31] Figure 31 shows images. NSG mice were challenged with 1E6 luciferase-expressing NALM6 leukemia on day 0. On day 3, mice were IV injected with 9E6, 3E6 and 1E6 T cells expressing Loop F CAR (also listed herein as LoopCAR6). Bioluminescence intensity as indicated by increasing shade levels represents tumor burden. [Diagram 32]Figure 32 presents images. NSG mice were administered 1E6 NALM6 on day 0. Mice in the sequential treatment group were administered 3E6 CAR+ on day 3 and 3E6 CAR+ T cells on day 7. Mice in the coinjection group were administered 3E6 anti-CD19 CAR+ and 3E6 anti-CD22 CAR+ T cells on day 3, for a total of 6E6 CAR+ T cells. Mice in the co-transduction group were administered 10E6 T cells, containing 3E6 anti-CD19+ and 3E6 anti-CD22+ CAR T cells. Mice in the CD19 or CD22 group were administered 3E6 CAR+ T cells. Co-injection or co-transduction of anti-CD19 and anti-CD22 CARs suggests that leukemia relapse may be reduced by targeting both CD19 and CD22 simultaneously. Bioluminescence intensity, indicated by increasing shade levels, represents tumor burden. [Figure 33-1] Figure 33A shows images. NSG mice were administered a mixture of 5E5 NALM6-CD19neg and 5E5 NALM6-CD22neg luciferase-expressing leukemia on day 0. On day 3, mice were treated with 3E6 CAR-expressing T cells. Bioluminescence intensity as indicated by increasing shade levels represents tumor burden. [Figure 33-2] Figure 33B shows the images. NSG mice were administered 1E6 luciferase-expressing leukemia cells on day 0 as indicated. Leukemia in some of these groups was mixed with 1% NALM6-CD19neg or NALM6-CD22neg cells. On day 3, mice were treated with 6E6 CAR-expressing T cells. Bioluminescence intensity, as indicated by increasing shade levels, represents tumor burden. [Figure 34-1] Figure 34A shows images. NSG mice were challenged with 1E6 NALM6 leukemia on day 0. On day 7, mice were treated with 8E6 mock T cells, CD19, CD22, or Loop F CAR+ T cells. Bioluminescence intensity as indicated by increasing shade levels represents tumor burden. [Figure 34-2]Figure 34B shows images. NSG mice were challenged with 1E6 NALM6 leukemia on day 0. On day 7, mice were treated with 8E6 mock T cells, CD19, CD22, or Loop F CAR+ T cells. Bioluminescence intensity as indicated by increasing shade levels represents tumor burden. [Figure 35-1] Figure 35A is a bar graph showing IFNγ production of LoopCAR6 after co-incubation with various cell lines representative of normal tissues, according to an embodiment of the invention. [Figure 35-2] Figure 35B is a bar graph showing IFNγ production of LoopCAR6 after co-incubation with various cell lines representative of normal tissues, according to an embodiment of the invention. [Diagram 36] Figure 36 is a dot plot showing human PBMC surface expression of CD19 CAR and CD22 CAR analyzed on day 8. [Figure 37-1] Figure 37A-37G: For cytokine production, CAR T cells (1E5) were washed three times with 1XPBS and co-incubated with the same number of target cells in 200ml RPMI medium in a 96-well plate in a 37℃ incubator for 15-20 hours. For high antigen target cells, K562 expressing CD19 or CD22, or both CD19 and CD22, were used, and K562 cells served as negative control. For low target antigen lines, NALM6 and NALM6-CD19neg and NALM6-CD22neg were used, and NALM6-CD19negCD22neg was used as negative control. All tests were in triplicate. Cytokine levels of IL2 in the culture supernatant were detected by R&D ELISA kit. Figure 37A: Cytokine production of CD19 and CD22 CARs with different co-stimulation domains and different antigen density levels. [Figure 37-2]Figure 37A-37G: For cytokine production, CAR T cells (1E5) were washed three times with 1XPBS and co-incubated with the same number of target cells in 200ml RPMI medium in a 96-well plate in a 37℃ incubator for 15-20 hours. For high antigen target cells, K562 expressing CD19 or CD22, or both CD19 and CD22, were used, and K562 cells served as negative control. For low target antigen lines, NALM6 and NALM6-CD19neg and NALM6-CD22neg were used, and NALM6-CD19negCD22neg was used as negative control. All tests were in triplicate. Cytokine levels of IL2 in the culture supernatant were detected by R&D ELISA kit. Figure 37B: Cytokine production of bicistronic CARs with different co-stimulation domains and different antigen density levels. [Figure 37-3] Figure 37A-37G: For cytokine production, CAR T cells (1E5) were washed three times with 1XPBS and co-incubated with the same number of target cells in 200ml RPMI medium in a 96-well plate in a 37°C incubator for 15-20 hours. For high antigen target cells, K562 expressing CD19 or CD22, or both CD19 and CD22, were used, and K562 cells served as negative controls. For low target antigen lines, NALM6 and NALM6-CD19neg and NALM6-CD22neg were used, and NALM6-CD19negCD22neg was used as a negative control. All tests were in triplicate. Cytokine levels of IL2 in the culture supernatant were detected by R&D's ELISA kit. Figure 37C: Comparison of cytokine production of bicistronic CAR with that of bivalent CAR. [Figure 37-4]Figure 37A-37G: For cytokine production, CAR T cells (1E5) were washed three times with 1XPBS and co-incubated with the same number of target cells in 200ml RPMI medium in a 96-well plate in a 37℃ incubator for 15-20 hours. For high antigen target cells, K562 expressing CD19 or CD22, or both CD19 and CD22, were used, and K562 cells served as negative control. For low target antigen lines, NALM6 and NALM6-CD19neg and NALM6-CD22neg were used, and NALM6-CD19negCD22neg was used as negative control. All tests were in triplicate. Cytokine levels of IL2 in the culture supernatant were detected by R&D ELISA kit. Figure 37D-37F: For incucyte killing assay, 5E4 target tumor cells were co-incubated with the same amount of CAR T cells. The cells were monitored for 40 hours every 30 minutes by scanning the plates for GFP fluorescence expression. The percentage of cell death at each time point was baseline corrected. [Figure 37-5]Figure 37A-37G: For cytokine production, CAR T cells (1E5) were washed three times with 1XPBS and co-incubated with the same number of target cells in 200ml RPMI medium in a 96-well plate in a 37℃ incubator for 15-20 hours. For high antigen target cells, K562 expressing CD19 or CD22, or both CD19 and CD22, were used, and K562 cells served as negative control. For low target antigen lines, NALM6 and NALM6-CD19neg and NALM6-CD22neg were used, and NALM6-CD19negCD22neg was used as negative control. All tests were in triplicate. Cytokine levels of IL2 in the culture supernatant were detected by R&D ELISA kit. Figure 37D-37F: For incucyte killing assay, 5E4 target tumor cells were co-incubated with the same amount of CAR T cells. The cells were monitored for 40 hours every 30 minutes by scanning the plates for GFP fluorescence expression. The percentage of cell death at each time point was baseline corrected. [Figure 37-6]Figure 37A-37G: For cytokine production, CAR T cells (1E5) were washed three times with 1XPBS and co-incubated with the same number of target cells in 200ml RPMI medium in a 96-well plate in a 37℃ incubator for 15-20 hours. For high antigen target cells, K562 expressing CD19 or CD22, or both CD19 and CD22, were used, and K562 cells served as negative control. For low target antigen lines, NALM6 and NALM6-CD19neg and NALM6-CD22neg were used, and NALM6-CD19negCD22neg was used as negative control. All tests were in triplicate. Cytokine levels of IL2 in the culture supernatant were detected by R&D ELISA kit. Figure 37D-37F: For incucyte killing assay, 5E4 target tumor cells were co-incubated with the same amount of CAR T cells. The cells were monitored for 40 hours every 30 minutes by scanning the plates for GFP fluorescence expression. The percentage of cell death at each time point was baseline corrected. [Figure 37-7]Figure 37A-37G: For cytokine production, CAR T cells (1E5) were washed three times with 1XPBS and co-incubated with the same number of target cells in 200ml RPMI medium in a 96-well plate in a 37℃ incubator for 15-20 hours. For high antigen target cells, K562 expressing CD19 or CD22, or both CD19 and CD22, were used, and K562 cells served as negative control. For low target antigen lines, NALM6 and NALM6-CD19neg and NALM6-CD22neg were used, and NALM6-CD19negCD22neg was used as negative control. All tests were in triplicate. Cytokine levels of IL2 in the culture supernatant were detected by R&D ELISA kit. FIG. 37G: Incucyte killing assay with NALM6CD19negCD22neg cells. [Figure 38] Figure 38: RNA sequencing analysis demonstrates unique gene expression associated with different combinations of costimulatory domains. Bicistronic CAR T cells were co-incubated with equal numbers of NALM6 in AMV medium for 24 hours. NALM6 cells were removed with magnetic beads and tRNA was immediately extracted for RNA sequencing analysis. PCA plot shows distinct gene expression profiles associated with different combinations of costimulatory domains. [Figure 39-1] Figure 39A presents images. Bioluminescence intensity, as indicated by increasing shade levels, represents tumor burden. Figure 39A: NSG mice were administered 1E6 luciferase-expressing NALM6 leukemia on day 0. On day 3, mice were IV injected with 5E6 CAR-expressing T cells. Bioluminescence intensity represents tumor burden. [Figure 39-2] Figure 39B presents images. Bioluminescence intensity, as shown by increasing shade levels, represents tumor burden. Figure 39B: NSG mice were administered 1E5 luciferase-expressing NALM6, NALM6-CD19neg, and NALM6-CD22neg leukemia cells on day 0. On day 3, mice were IV injected with 3E6 CAR-expressing T cells. [Diagram 40] Figure 40: NSG mice were administered 2.5E5 luciferase-expressing NALM6-CD19neg and NALM6-CD22neg leukemia cells on day 0. Mice were injected IV with 3E6 CAR-expressing T cells on day 3. Bioluminescence intensity as indicated by increasing shade levels represents tumor burden. [Diagram 41] Figure 41: NSG mice were injected IV with luciferase-expressing HMB28 patient-derived ALL xenografts (CD19CD22, 1x106). On day 8, mice were injected with 3E6 CAR-expressing T cells as indicated. Bioluminescence intensity as indicated by increasing shade levels represents tumor burden. [Diagram 42] FIG. 42 is a graph showing site density as described in Example 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] One embodiment of the present invention provides a chimeric antigen receptor (CAR) amino acid construct comprising: (a) a cleavable domain; (b) a first CAR comprising a first antigen binding domain, a first transmembrane domain, and a first intracellular T cell signaling domain; and (c) a second CAR comprising a second antigen binding domain, a second transmembrane domain, and a second intracellular T cell signaling domain; wherein the first and second CARs are linked via the cleavable domain, and the first antigen binding domain comprises the antigen binding domain of the m971 antibody, and wherein when the first CAR is cleaved from the construct, the first antigen binding domain has antigen specificity for CD22.

[0014] CARs are artificially constructed hybrid proteins or polypeptides that contain one or more antibody antigen-binding domains (e.g., single-chain variable fragments (scFv)) linked to a T cell signaling domain. CARs are characterized by their ability to utilize the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells to selected targets in an MHC-unrestricted manner. MHC-unrestricted antigen recognition endows T cells expressing CARs with the ability to recognize antigens independent of antigen processing, thus bypassing a major tumor escape mechanism. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the alpha and beta chains of endogenous T cell receptors (TCRs). As used herein, the phrases "antigen-specific" and "elicit an antigen-specific response" mean that the CAR can specifically bind to an antigen and immunologically recognize the antigen, such that binding of the CAR to the antigen elicits an immune response.

[0015] CD22 is a lineage-restricted B cell antigen belonging to the immunoglobulin (Ig) superfamily. It is expressed on 60-70% of B cell lymphomas and leukemias (e.g., chronic B lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma) and is not present on the cell surface at early stages of B cell development or on stem cells (Vaickus et al., Crit. Rev. Oncol. / Hematol., 11:267-297 (1991); Bang et al., Clin. Cancer Res., 11:1545-50 (2005)). CD19 (also known as B lymphocyte antigen CD19, B4, and CVID3) is a cell surface molecule expressed exclusively on B lymphocytes and follicular dendritic cells of the hematopoietic lineage. It is the earliest B lineage-restricted expressed antigen and is present on most childhood B cells as well as most non-T cell acute lymphocytic leukemia cells and B cell chronic lymphocytic leukemia cells (Tedder and Isaacs, J. Immun., 143:712-717 (1989)).

[0016] In an embodiment of the invention, the invention provides multiple CARs (e.g., 2, 3, 4, 5 or more), each binding to a single antigen, each CAR being separated by a cleavable domain. In one embodiment of the invention, by cleaving the cleavable domain, each CAR, e.g., the first and second CAR, is cleaved from the CAR construct, and each cleaved CAR is present separately on the T cell surface, each with antigen specificity for its respective target, and each capable of eliciting an antigen-specific response. In one embodiment, such a CAR construct is a bicistronic CAR, e.g., capable of cleaving / cleaving two CARs. Without wishing to be bound by theory or mechanism, the cleavable domains of these CARs can be cleaved after complete translation of the complete sequence, or after translation of each CAR and the cleavable domain, such that the CAR is cleaved / cleaved before translation of the next CAR in the sequence. Examples of such CARs herein include V1, V5, V6, V7, and V8.

[0017] In an embodiment of the invention, the invention provides CARs, each of which can simultaneously bind two antigens (e.g., CD19 and CD22). These CARs have dual specificity for CD22 and CD19. As used herein with respect to CARs, the terms "bispecific," "dual specific," "bispecific," and "bivalent" mean that the same CAR can specifically bind and immunologically recognize two different antigens. Binding of the CAR to at least one of the two antigens elicits an immune response. Examples of such CARs herein include TanCAR2-4 and LoopCAR1-5. In another embodiment, the bispecific CARs can be linked by a cleavable domain.

[0018] An embodiment of the invention provides a CAR comprising an anti-CD22 antigen binding domain of the m971 antibody ("m971"). The antigen binding domain of m971 specifically binds to CD22. In this regard, a preferred embodiment of the invention provides a CAR comprising an anti-CD22 antigen binding domain comprising, consisting of, or consisting essentially of a single chain variable fragment (scFv) of the antigen binding domain of m971. The HA22 immunotoxin and the m971 antibody bind to different CD22 epitopes.

[0019] The anti-CD22 antigen-binding domain may comprise a light chain variable region and / or a heavy chain variable region. In one embodiment of the invention, the heavy chain variable region comprises a CDR1 region, a CDR2 region, and a CDR3 region. In this regard, the anti-CD22 antigen-binding domain may comprise one or more of the following: a heavy chain CDR1 region comprising the amino acid sequence of SEQ ID NO: 4; a heavy chain CDR2 region comprising the amino acid sequence of SEQ ID NO: 6; a heavy chain CDR3 region comprising the amino acid sequence of SEQ ID NO: 8. Preferably, the heavy chain of the anti-CD22 antigen-binding domain comprises all of the amino acid sequences of SEQ ID NOs: 4, 6, and 8.

[0020] In one embodiment of the invention, the light chain variable region of the anti-CD22 antigen binding domain may comprise a light chain CDR1 region, a light chain CDR2 region, and a light chain CDR3 region. In this regard, the anti-CD22 antigen binding domain may comprise one or more of the following: a light chain CDR1 region comprising the amino acid sequence of SEQ ID NO: 12; a light chain CDR2 region comprising the amino acid sequence of SEQ ID NO: 14; and a light chain CDR3 region comprising the amino acid sequence of SEQ ID NO: 16. Preferably, the light chain of the anti-CD22 antigen binding domain comprises all of the amino acid sequences of SEQ ID NOs: 12, 14, and 16. In a particularly preferred embodiment, the anti-CD22 antigen binding domain comprises all of the amino acid sequences of SEQ ID NOs: 4, 6, 8, 12, 14, and 16.

[0021] The heavy chain variable region of the anti-CD22 antigen-binding domain may comprise, consist of, or consist essentially of the amino acid sequences of SEQ ID NOs: 3 to 9. The light chain variable region of the anti-CD22 antigen-binding domain may comprise, consist of, or consist essentially of the amino acid sequences of SEQ ID NOs: 11 to 17. Thus, in one embodiment of the present invention, the anti-CD22 antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 3 to 9 and / or a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 11 to 17. Preferably, the anti-CD22 antigen-binding domain comprises the amino acid sequences of SEQ ID NOs: 3 to 9 and 11 to 17.

[0022] In an embodiment of the invention, when the second CAR is cleaved from the construct, the second antigen binding domain has antigen specificity for CD19.

[0023] In an embodiment of the invention, the second antigen-binding domain comprises an antigen-binding domain of the FMC63 antibody. In an embodiment of the invention, the second antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of FMC63 set forth below, and a light chain variable region comprising the amino acid sequence of FMC63 set forth below. In an embodiment of the invention, the second antigen-binding domain comprises the amino acid sequence of FMC63 set forth below.

[0024] Another embodiment of the present invention provides a chimeric antigen receptor (CAR) amino acid construct comprising: (a) a cleavable domain; (b) a first CAR comprising a first antigen binding domain, a first transmembrane domain, and a first intracellular T cell signaling domain; and (c) a second CAR comprising a second antigen binding domain, a second transmembrane domain, and a second intracellular T cell signaling domain; wherein the first and second CARs are linked via the cleavable domain, the first antigen binding domain comprises the antigen binding domain of the FMC63 antibody, and when the first CAR is cleaved from the construct, the first antigen binding domain has antigen specificity for CD19. In an embodiment, when the second CAR is cleaved from the construct, the second antigen binding domain has antigen specificity for CD22.

[0025] One embodiment of the present invention provides a CAR comprising an anti-CD19 antigen binding domain of the FMC63 antibody ("FMC63"). The antigen binding domain of FMC63 specifically binds to CD19. In this regard, a preferred embodiment of the present invention provides a CAR comprising an anti-CD19 antigen binding domain that comprises, consists of, or consists essentially of a single chain variable fragment (scFv) of the antigen binding domain of FMC63.

[0026] The anti-CD19 antigen binding domain may comprise a light chain variable region and / or a heavy chain variable region.

[0027] In one embodiment of the invention, the light chain variable region of the anti-CD19 antigen binding domain may comprise a light chain CDR1 region, a light chain CDR2 region, and a light chain CDR3 region. In this regard, the anti-CD19 antigen binding domain may comprise one or more of a light chain CDR1 region comprising the amino acid sequence of SEQ ID NO: 24; a light chain CDR2 region comprising the amino acid sequence of SEQ ID NO: 26; and a light chain CDR3 region comprising the amino acid sequence of SEQ ID NO: 28. Preferably, the light chain of the anti-CD19 antigen binding domain comprises all of the amino acid sequences of SEQ ID NOs: 24, 26, and 28.

[0028] In one embodiment of the invention, the heavy chain variable region of the anti-CD19 antigen binding domain comprises a CDR1 region, a CDR2 region, and a CDR3 region. In this regard, the anti-CD19 antigen binding domain may comprise one or more of the following: a heavy chain CDR1 region comprising the amino acid sequence of SEQ ID NO: 32; a heavy chain CDR2 region comprising the amino acid sequence of SEQ ID NO: 34; and a heavy chain CDR3 region comprising the amino acid sequence of SEQ ID NO: 36. Preferably, the heavy chain of the anti-CD19 antigen binding domain comprises all of the amino acid sequences of SEQ ID NOs: 32, 34, and 36. In a particularly preferred embodiment, the anti-CD19 antigen binding domain comprises all of the amino acid sequences of SEQ ID NOs: 24, 26, 28, 32, 34, and 36.

[0029] The heavy chain variable region of the anti-CD19 antigen-binding domain may comprise, consist of, or consist essentially of the amino acid sequences of SEQ ID NOs: 31 to 37. The light chain variable region of the anti-CD19 antigen-binding domain may comprise, consist of, or consist essentially of the amino acid sequences of SEQ ID NOs: 23 to 29. Thus, in one embodiment of the present invention, the anti-CD19 antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 31 to 37 and / or a light chain variable region comprising the amino acid sequences of SEQ ID NOs: 23 to 29. Preferably, the anti-CD19 antigen-binding domain comprises the amino acid sequences of both SEQ ID NOs: 23 to 29 and 31 to 37.

[0030] The anti-CD22 antigen-binding domain and the anti-CD19 antigen-binding domain may comprise any antigen-binding portion of an anti-CD22 or anti-CD19 antibody, respectively. The antigen-binding portion may be any portion having at least one antigen-binding site, such as Fab, F(ab')2, dsFv, scFv, diabody, and triabody. Preferably, the antigen-binding portion is a single chain variable fragment (scFv) antibody fragment. An scFv is a truncated Fab fragment that contains the variable (V) domain of an antibody heavy chain linked to the V domain of a light antibody chain via a synthetic peptide linker, and may be generated using conventional recombinant DNA technology techniques. Similarly, disulfide-stabilized variable fragments (dsFv) may be prepared by recombinant DNA technology.

[0031] In one embodiment of the present invention, the light chain variable region and the heavy chain variable region of the anti-CD22 antigen-binding domain may be linked to each other by a linker. The linker may comprise any suitable amino acid sequence. In one embodiment of the present invention, the linker is a Gly / Ser linker having a length of about 1 to about 100, about 3 to about 20, about 5 to about 30, about 5 to about 18, or about 3 to about 8 amino acids, and is composed of consecutive glycine and / or serine residues. Thus, the Gly / Ser linker may comprise glycine and / or serine residues. Preferably, the Gly / Ser linker comprises the amino acid sequence of GGGGS (SEQ ID NO: 10), and multiple instances of SEQ ID NO: 10 may be present in the linker. In another embodiment of the present invention, the linker comprises the amino acid sequence of SEQ ID NO: 30. Any linker sequence may be used as a spacer between the antigen-binding domain and the transmembrane domain.

[0032] In an embodiment of the invention, the light chain variable region and the heavy chain variable region of the anti-CD19 antigen binding domain may be linked to each other by a linker. The linker may be any of the linkers described herein for other aspects of the invention. In one embodiment of the invention, the light chain variable region and the heavy chain variable region of the anti-CD19 antigen binding domain are linked to each other by a linker comprising the amino acid sequence of SEQ ID NO: 10 or 30.

[0033] In one embodiment, the anti-CD22 antigen binding domain comprises a light chain variable region, a heavy chain variable region, and a linker. In this regard, an embodiment of an anti-CD22 antigen binding domain comprising a light chain variable region, a heavy chain variable region, and a linker comprises, consists of, or consists essentially of all of SEQ ID NOs: 3-17.

[0034] In one embodiment, the anti-CD19 antigen binding domain comprises a light chain variable region, a heavy chain variable region, and a linker. In this regard, an embodiment of the anti-CD19 antigen binding domain comprising a light chain variable region, a heavy chain variable region, and a linker comprises, consists of, or consists essentially of all of SEQ ID NOs: 23-37.

[0035] The first CAR and the second CAR of the CAR construct of the present invention are linked to each other via one, two, three, four or more cleavable domains. The cleavable domain(s) may include one or more of any suitable cleavable domains, including domains recognized by cleavage enzymes or domains that self-cleave. Suitable domains include, for example, 2A domains, such as T2A and / or P2A, and furin cleavage sequences. Table 1 provides exemplary suitable cleavable domains.

[0036] [Table 1]

[0037] In one embodiment of the invention, the CAR construct contains more than one cleavable domain, and all of the cleavable domains are the same. In one embodiment of the invention, the CAR construct contains more than one cleavable domain that is adjacent within the CAR construct, and at least one cleavable domain is different.

[0038] In one embodiment, the antigen binding domain comprises a leader sequence. In one embodiment of the invention, the leader sequence may be located at the amino terminus of an anti-CD19 CAR in a CAR construct. In another embodiment of the invention, the leader sequence may be located at the amino terminus of an anti-CD22 CAR in a CAR construct. The leader sequence may comprise any suitable leader sequence. In one embodiment, the leader sequence comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 62. In one embodiment of the invention, the leader sequence may facilitate expression of the cleaved / truncated protein on the surface of a cell, but the presence of the leader sequence in the expressed CAR is not required for the CAR to function. In one embodiment of the invention, the leader sequence may be cleaved off from the cleaved CAR upon expression of the CAR on the cell surface. Thus, in one embodiment of the invention, the cleaved CAR lacks a leader sequence. In one embodiment of the invention, the CAR in a CAR construct lacks a leader sequence.

[0039] In one embodiment of the invention, the CAR construct comprises a hinge domain. In one embodiment of the invention, the hinge domain is a CD8 hinge domain. In a preferred embodiment, the CD8 hinge domain is human. Preferably, the CD8 hinge domain comprises, consists of, or consists essentially of SEQ ID NO: 18. In one embodiment of the invention, the hinge domain is a CD28 hinge domain. In a preferred embodiment, the CD28 hinge domain is human. Preferably, the CD28 hinge domain comprises, consists of, or consists essentially of SEQ ID NO: 40.

[0040] In one embodiment of the invention, the CAR construct comprises a transmembrane (TM) domain. In one embodiment of the invention, the TM domain is a CD8 TM domain. In a preferred embodiment, the CD8 TM domain is human. Preferably, the CD8 TM domain comprises, consists of, or consists essentially of SEQ ID NO: 19. In one embodiment of the invention, the TM domain is a CD28 TM domain. In a preferred embodiment, the CD28 TM domain is human. Preferably, the CD28 TM domain comprises, consists of, or consists essentially of SEQ ID NO: 41.

[0041] In one embodiment of the invention, the CAR construct comprises an intracellular T cell signaling domain. In one embodiment of the invention, the intracellular T cell signaling domain comprises a 4-1BB intracellular T cell signaling sequence. 4-1BB, also known as CD137, delivers a potent co-stimulatory signal to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes. Preferably, the 4-1BB intracellular T cell signaling sequence is human. In a preferred embodiment, the 4-1BB intracellular T cell signaling sequence comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:20.

[0042] In one embodiment of the invention, the intracellular T cell signaling domain comprises a CD3 zeta (ζ) intracellular T cell signaling sequence. CD3ζ binds to the TCR to generate a signal and contains an immunoreceptor tyrosine-based activation motif (ITAM). Preferably, the CD3ζ intracellular T cell signaling sequence is human. In a preferred embodiment, the CD3ζ intracellular T cell signaling sequence comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:21.

[0043] In one embodiment of the invention, the intracellular T cell signaling domain comprises a CD28 intracellular T cell signaling sequence. Preferably, the CD28 intracellular T cell signaling sequence is human. In a preferred embodiment, the CD28 intracellular T cell signaling sequence comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:42.

[0044] The first and second CARs can be arranged in any suitable orientation in the CAR construct. In one embodiment of the invention, the CAR construct comprises, from N-terminus to C-terminus: an anti-CD19 CAR, one or more cleavable domains, followed by an anti-CD22 CAR. In another embodiment of the invention, the CAR construct comprises, from N-terminus to C-terminus: an anti-CD22 CAR, one or more cleavable domains, followed by an anti-CD19 CAR.

[0045] FIG. 1 presents a schematic of an exemplary CAR construct according to an embodiment of the invention.

[0046] A further embodiment of the invention provides a full-length CAR construct comprising, consisting of, or consisting essentially of any one of the amino acid sequences set out in Tables 2-6.

[0047] [Table 2-1]

[0048] [Table 2-2]

[0049] [Table 3-1]

[0050] [Table 3-2]

[0051]

Table 4-1

[0052]

Table 4-2

[0053]

Table 4-3

[0054]

Table 5-1

[0055]

Table 5-2

[0056]

Table 5-3

[0057]

Table 6-1

[0058]

Table 6-2

[0059]

Table 6-3

[0060]

Table 7-1

[0061]

Table 7-2

[0062]

Table 8-1

[0063]

Table 8-2

[0064]

Table 9-1

[0065]

Table 9-2

[0066]

Table 10-1

[0067]

Table 10-2

[0068]

Table 10-3

[0069]

Table 11-1

[0070]

Table 11-2

[0071]

Table 12-1

[0072]

Table 12-2

[0073]

Table 12-3

[0074]

Table 13-1

[0075]

Table 13-2

[0076]

Table 14-1

[0077]

Table 14-2

[0078]

Table 14-3

[0079]

Table 15-1

[0080]

Table 15-2

[0081]

Table 16-1

[0082] [Table 16-2]

[0083] [Table 16-3]

[0084] In one embodiment, the CAR construct (denoted herein as V1) has the sequence:

[0085] [ka]

[0086] has.

[0087] In one embodiment, the CAR construct (denoted herein as V5) has the sequence:

[0088] [ka]

[0089] has.

[0090] In one embodiment, the CAR construct (denoted herein as V6) has the sequence:

[0091] [ka]

[0092] has.

[0093] In one embodiment, the CAR construct (denoted herein as V7) has the sequence:

[0094] [ka]

[0095] has.

[0096] In one embodiment, the CAR construct (denoted herein as V8) has the sequence:

[0097] [ka]

[0098] has.

[0099] In one embodiment, the CAR construct (denoted herein as TanCAR2) has the sequence:

[0100] [ka]

[0101] has.

[0102] In one embodiment, the CAR construct (denoted herein as TanCAR3) has the sequence:

[0103] [ka]

[0104] has.

[0105] In one embodiment, the CAR construct (denoted herein as TanCAR4) has the sequence:

[0106] [ka]

[0107] has.

[0108] In one embodiment, the CAR construct (denoted herein as LoopCAR1) has the sequence:

[0109] [ka]

[0110] has.

[0111] In one embodiment, the CAR construct (denoted herein as LoopCAR2) has the sequence:

[0112] [ka]

[0113] has.

[0114] In one embodiment, the CAR construct (denoted herein as LoopCAR3) has the sequence:

[0115] [ka]

[0116] has.

[0117] In one embodiment, the CAR construct (denoted herein as LoopCAR4) has the sequence:

[0118] [ka]

[0119] has.

[0120] In one embodiment, the CAR construct (denoted herein as LoopCAR5) has the sequence:

[0121] [ka]

[0122] has.

[0123] The CAR constructs of the present invention may provide many advantages. In one embodiment of the present invention, for example, the CAR constructs of the present invention may advantageously reduce or prevent cancer cell escape due to loss of expression of one antigen, e.g., CD19 or CD22, by the cancer cells. For example, it is believed that the CAR constructs of the present invention may reduce or prevent recurrence observed in cancer patients whose cancer has lost expression of that antigen after treatment with a CAR with antigen specificity for CD19 or CD22 only. The CAR constructs of the present invention may also be advantageous for treating patients with heterogeneous expression levels of CD19 or CD22. The CAR constructs of the present invention may also increase the patient population that can be successfully treated. For example, patients who may not respond to CAR therapy that targets only CD19 may respond to CAR therapy that targets CD22, and patients who may not respond to CAR therapy that targets only CD22 may respond to CAR therapy that targets CD19. Furthermore, with respect to the cleavable CARs of the present invention, co-transduction of T cells with two vectors, each carrying a single CAR, provides a significant number of T cells expressing only one or the other CAR, with only a small percentage of cells expressing both CARs; an advantage of using the cleavable CAR constructs of the present invention is that the expression of each CAR in each T cell that successfully incorporates the construct can be equal or substantially equal. Furthermore, by targeting both CD19 and CD22, the cleavable and non-cleavable CAR constructs of the present invention can advantageously provide a synergistic response compared to treatments that target only a single antigen, and can also provide a more broadly aggressive treatment for patients with heterogeneous expression of either or both CD19 and CD22 on cancer cells.

[0124] Thus, without being bound by a particular theory or mechanism, it is believed that by eliciting an antigen-specific response against two antigens, e.g., CD22 and CD19, the CAR constructs of the present invention provide any one or more of the following: targeting and destruction of CD22-expressing cancer cells, targeting and destruction of CD19-expressing cancer cells, reduction or elimination of cancer cells, promotion of infiltration of immune cells to the tumor site(s), and enhancement / prolongation of anti-cancer responses.

[0125] The scope of the present invention includes functional portions of the CAR constructs of the present invention described herein. When used in reference to a CAR, the term "functional portion" refers to any portion or fragment of a CAR construct of the present invention, where the portion or fragment retains the biological activity of the CAR construct of which it is a part (the parent CAR construct). A functional portion encompasses, for example, a portion of a CAR construct that retains the ability to recognize a target cell or detect, treat, or prevent a disease to a similar, equal, or greater extent than the parent CAR construct. With respect to the parent CAR construct, a functional portion may, for example, comprise about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0126] A functional portion can include additional amino acids at the amino or carboxy terminus of the portion, or at both termini, that are not found in the amino acid sequence of the parent CAR construct. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, e.g., recognizing a target cell, detecting cancer, treating or preventing cancer, etc. More desirably, the additional amino acids enhance the biological activity of the functional portion compared to the biological activity of the parent CAR construct.

[0127] The scope of the present invention includes functional variants of the CAR constructs of the present invention described herein. As used herein, the term "functional variant" refers to a CAR construct, polypeptide, or protein that has substantial or significant sequence identity or similarity with a parent CAR construct, where the functional variant retains the biological activity of the CAR of which it is a variant. Functional variants include, for example, variants of the CAR constructs described herein (parent CAR constructs) that retain the ability to recognize target cells to a similar, equal, or higher degree than the parent CAR construct. With respect to the parent CAR construct, the functional variant may, for example, be at least about 30%, about 50%, about 75%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more in amino acid sequence relative to the parent CAR construct.

[0128] A functional variant may, for example, comprise the amino acid sequence of a parent CAR with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant may comprise the amino acid sequence of a parent CAR construct with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the parent CAR construct.

[0129] The amino acid substitutions in the CAR constructs of the invention are preferably conservative amino acid substitutions, which are known in the art and include amino acid substitutions in which one amino acid having particular physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be the substitution of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid having a nonpolar side chain for another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid having a polar side chain for another uncharged amino acid having a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid having a beta-branched side chain for another beta-branched side chain (e.g., Ile, Thr, and Val), an amino acid having an aromatic side chain for another aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0130] A CAR construct can consist essentially of the specific amino acid sequence or sequences described herein, such that other components, e.g., other amino acids, do not substantially alter the biological activity of the functional variant.

[0131] The CAR constructs (including functional portions and functional variants) of the present embodiments can be of any length, i.e., can contain any number of amino acids, provided that the CAR constructs (or functional portions or functional variants thereof) retain their biological activity, e.g., the ability to specifically bind to an antigen, the ability to detect diseased cells in a mammal, or the ability to treat or prevent disease in a mammal. For example, a CAR can be about 50 to about 5000 amino acids in length, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

[0132] The CAR constructs of the present embodiments (including functional portions and functional variants of the present invention) may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine. Examples of such amino acids include β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0133] The CAR constructs (including functional portions and functional variants) of the present embodiments may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via disulfide bridges), or converted into acid addition salts and / or optionally dimerized or multimerized, or conjugated.

[0134] The CAR constructs of the present invention (including functional portions and functional variants) can be obtained by methods known in the art. The CAR constructs can be produced by any suitable method for producing polypeptides or proteins, including de novo synthesis. The CAR constructs can also be produced recombinantly using standard recombinant methods and nucleic acids described herein. See, for example, Green et al., Molecular Cloning: A Laboratory Manual (4th ed.) Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Furthermore, some parts of the CAR constructs of the present invention (including functional portions and functional variants thereof) can be isolated and / or purified from sources such as plants, bacteria, insects, mammals, (e.g., rats, humans, etc.). Methods of isolation and purification are well known in the art. Alternatively, the CAR constructs described herein (including functional portions and variants thereof) can be commercially synthesized by companies such as Synpep (Dublin, Calif.), Peptide Technologies Corp. (Gaithersburg, Md.) and Multiple Peptide Systems (San Diego, Calif.). In this regard, the CAR constructs of the present invention can be synthetic, recombinant, isolated, and / or purified.

[0135] Another embodiment of the present invention provides a chimeric antigen receptor (CAR) amino acid construct comprising: (a) two or more cleavable domains; (b) a first CAR comprising a first antigen binding domain, a first transmembrane domain, and a first intracellular T cell signaling domain; and (c) a second CAR comprising a second antigen binding domain, a second transmembrane domain, and a second intracellular T cell signaling domain; wherein the first and second CARs are linked via two or more cleavable domains. In one embodiment, the two or more cleavable domains are directly adjacent or have at least one linker between at least two of the cleavable domains. In one embodiment, there are exactly two cleavable domains.

[0136] Another embodiment of the present invention provides a method for making a chimeric antigen receptor (CAR) amino acid construct, comprising: (a) designing two or more cleavable domains between a first CAR comprising a first antigen-binding domain, a first transmembrane domain, and a first intracellular T cell signaling domain; and (b) a second CAR comprising a second antigen-binding domain, a second transmembrane domain, and a second intracellular T cell signaling domain, wherein the first and second CARs are linked via the two or more cleavable domains; and cloning a sequence comprising the first CAR, the two or more cleavable domains, and the second CAR from N-terminus to C-terminus into a plasmid. In one embodiment, the two or more cleavable domains are directly adjacent or have at least one linker between at least two of the cleavable domains. In one embodiment, there are exactly two cleavable domains.

[0137] Further provided according to one embodiment of the present invention is a nucleic acid comprising a nucleotide sequence encoding any of the CAR constructs described herein (including functional portions and functional variants thereof). The nucleic acid of the invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen binding domains, transmembrane domains, linkers and / or intracellular T cell signaling domains described herein.

[0138] In one embodiment, the nucleic acid comprises a nucleotide sequence encoding any of the CAR constructs described herein. In one embodiment of the present invention, the nucleic acid may comprise, consist of, or consist essentially of the nucleotide sequence of SEQ ID NO:53 (anti-CD19 / anti-CD22 V1 CAR), SEQ ID NO:54 (anti-CD19 / anti-CD22 V5 CAR), SEQ ID NO:55 (anti-CD19 / anti-CD22 V6 CAR), SEQ ID NO:56 (anti-CD19 / anti-CD22 V7 CAR), SEQ ID NO:57 (anti-CD19 / anti-CD22 V8 CAR), SEQ ID NO:71 (TanCAR2), SEQ ID NO:72 (TanCAR3), SEQ ID NO:73 (TanCAR4), SEQ ID NO:74 (LoopCAR1), SEQ ID NO:75 (LoopCAR2), SEQ ID NO:76 (LoopCAR3), SEQ ID NO:77 (LoopCAR4), or SEQ ID NO:78 (LoopCAR5).

[0139] As used herein, "nucleic acid" includes "polynucleotides", "oligonucleotides" and "nucleic acid molecules" and generally refers to polymers of DNA or RNA, which may be single-stranded or double-stranded, may be synthetic or obtained from natural sources (e.g., isolated and / or purified), may contain natural, non-natural or modified nucleotides, and may contain modified internucleotide linkages, such as, for example, phosphoramidite or phosphorothioate linkages, instead of the natural linkages, non-natural linkages or phosphodiesters found between nucleotides of unmodified oligonucleotides. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions and / or substitutions. However, as discussed herein, in some instances, it may be preferred for the nucleic acid to contain one or more insertions, deletions, inversions and / or substitutions. In some embodiments, the nucleic acid may encode additional amino acid sequences that do not affect the function of the CAR construct and may or may not be translated upon expression of the nucleic acid by a host cell.

[0140] The nucleic acid of one embodiment of the present invention may be recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed by joining a natural or synthetic nucleic acid segment outside a living cell to a nucleic acid molecule capable of replicating in the living cell, or (ii) a molecule resulting from the replication of the one described in (i) above. For purposes of this specification, the replication may be in vitro replication or in vivo replication.

[0141] Recombinant nucleic acids may have sequences that do not occur in nature or that are created by the artificial combination of two otherwise separate segments of sequence. This artificial combination is frequently accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acid, e.g., by genetic engineering techniques such as those described in Green et al. (supra). Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, e.g., Green et al. (supra). For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides (e.g., phosphorothioate derivatives and acridine substituted nucleotides) designed to increase the biological stability of the molecule or to enhance the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N 6 -Isopentenyladenine (N 6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -Substituted adenine (N 6 -substituted adenine), 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -Isopentenyladenine (2-methylthio-N 6-isopentenyladenine, uracil-5-oxyacetic acid(v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the invention can be purchased commercially from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0142] The nucleic acid may comprise any isolated or purified nucleotide sequence encoding a CAR construct or any of its functional portions or variants. Alternatively, the nucleotide sequence may comprise a nucleotide sequence that is degenerate to any of the sequences, or a combination of degenerate sequences.

[0143] Embodiments of the present invention also provide isolated or purified nucleic acids comprising a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein, or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0144] Nucleotide sequences that hybridize under stringent conditions may hybridize under high stringency conditions. By "high stringency conditions" is meant that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of a nucleic acid described herein) in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions that distinguish polynucleotides with exactly complementary sequences or polynucleotides containing only scattered minor mismatches from random sequences that happen to have only a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity melt more easily than full-length complements of 14-17 bases or more, and are easily distinguished by high stringency hybridization. Relatively high stringency conditions include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at temperatures of about 50-70°C. Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the CAR constructs of the present invention. It is generally understood that conditions can be made more stringent by adding increasing amounts of formamide.

[0145] The present invention also provides nucleic acids comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any of the nucleic acids described herein.

[0146] In one embodiment, the nucleic acid of the invention may be incorporated into a recombinant expression vector. In this regard, one embodiment of the invention provides a recombinant expression vector comprising any of the nucleic acids of the invention. For purposes of this specification, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that allows expression of the mRNA, protein, polypeptide, or peptide by a host cell when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and the vector is contacted with the cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the cell. The vector of the invention is not naturally occurring as a whole. However, portions of the vector may be naturally occurring. The recombinant expression vector of the invention may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vector may contain naturally occurring internucleotide bonds, non-naturally occurring internucleotide bonds, or both types of bonds. Preferably, the non-naturally occurring or modified nucleotides or internucleotide bonds do not interfere with the transcription or replication of the vector. An exemplary vector backbone is the backbone of the lentivector of SEQ ID NO:58.

[0147] In one embodiment, the recombinant expression vector of the present invention may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and expansion or for expression, or both, such as plasmids and viruses. The vector may be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burine, MD), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clonetech, Palo Alto, CA). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 may also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clonetech). Examples of animal expression vectors include pEUK-C1, pMAM and pMAMneo (Clonetech). The recombinant expression vector can be a viral vector, e.g., a retroviral vector or a lentiviral vector.

[0148] In one embodiment, the recombinant expression vector of the invention can be prepared using standard recombinant DNA techniques, for example as described in Green et al. (supra). The expression vector construct, which can be circular or linear, can be prepared to contain a replication system that is functional in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, ColE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, etc.

[0149] Recombinant expression vectors can include control sequences, such as transcriptional and translational initiation and termination codons, that are specific to the type of host cell (e.g., bacterial, fungal, plant or animal) into which the vector will be introduced, as appropriate, considering whether the vector is DNA- or RNA-based. Recombinant expression vectors can also include restriction enzyme recognition sites to facilitate cloning.

[0150] The recombinant expression vector may contain one or more marker genes that allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. Suitable marker genes for the expression vector of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0151] The recombinant expression vector may comprise a native or non-native promoter operably linked to a nucleotide sequence encoding a CAR construct (including functional parts and functional variants thereof) or a nucleotide sequence that is complementary or hybridizes to the nucleotide sequence encoding the CAR construct. The selection of a promoter, e.g., strong, weak, inducible, tissue-specific, and developmental stage-specific, is within the ordinary skill of the art. Similarly, the combination of a nucleotide sequence with a promoter is within the ordinary skill of the art. The promoter may be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long-terminal repeat of the murine stem cell virus.

[0152] The recombinant expression vectors of the invention can be designed for either transient expression, stable expression, or both. The recombinant expression vectors can also be made for constitutive or inducible expression.

[0153] Additionally, the recombinant expression vector can be made to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that kills a cell that expresses the suicide gene. A suicide gene can be a gene that confers sensitivity to a reagent, e.g., a drug, on a cell that expresses the gene, causing the cell to die when the cell is contacted or exposed to the reagent. Suicide genes are known in the art and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine daminase, purine nucleoside phosphorylase, and nitroreductase.

[0154] The scope of the present invention includes conjugates, e.g., bioconjugates, comprising any of the CAR constructs of the invention (including any of their functional portions or variants), nucleic acids, recombinant expression vectors, host cells, or host cell populations. Conjugates and methods for synthesizing conjugates are generally known in the art.

[0155] An embodiment of the present invention further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any species of cell that may contain a recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, e.g., a plant, an animal, a fungus, or an alga, or a prokaryotic cell, e.g., a bacterium or a protozoan. The host cell may be a cultured cell or a primary cell, i.e., directly isolated from an organism, e.g., a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell that grows in a suspension. Suitable host cells are known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For purposes of amplifying or replicating a recombinant expression vector, the host cell may be a prokaryotic cell, e.g., a DH5α cell. For purposes of producing a recombinant CAR construct, the host cell may be a mammalian cell. The host cell may be a human cell. The host cell may be of any cell type, may be derived from any type of tissue, and may be at any stage of development, but the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell or a natural killer cell (NK cell).

[0156] For purposes herein, the T cells may be any T cell, such as a cultured T cell, e.g., primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cells may be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells may also be enriched or purified. The T cells may be human T cells. The T cells may be T cells isolated from a human. The T cells may be CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD8 + T cells can be of any type and at any developmental stage, including, but not limited to, T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, etc. T cells are CD8 +T cells or CD4 + It may be a T cell.

[0157] Also provided according to one embodiment of the present invention is a cell population comprising at least one host cell described herein. The cell population can be a heterogeneous population comprising host cells comprising any of the described recombinant expression vectors in addition to at least one other cell, e.g., a host cell not comprising any of the recombinant expression vectors (e.g., a T cell), or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the cell population can be a substantially homogenous population, where the population primarily comprises (e.g., consists essentially of) host cells comprising the recombinant expression vector. The population can also be a clonal population of cells, where all cells of the population are clones of a single host cell comprising the recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment of the present invention, the cell population is a clonal population comprising host cells comprising the recombinant expression vector described herein.

[0158] The CAR constructs (including functional portions and variants thereof), nucleic acids, recombinant expression vectors, and host cells (including populations thereof), all of which are hereinafter collectively referred to as the "anti-CAR construct material of the invention", may be isolated and / or purified. As used herein, the term "isolated" means removed from its natural environment. The terms "purified" or "isolated" do not require complete purification or isolation; rather, they are intended as relative terms. Thus, for example, a purified (or isolated) host cell preparation is one in which the host cells are more pure than the cells in their natural environment in the body. Such host cells may be produced, for example, by standard purification techniques. In some embodiments, a preparation of host cells is purified such that the host cells represent at least about 50%, e.g., at least about 70%, of the total cell content of the preparation. For example, the purity may be at least about 50%, may be greater than about 60%, about 70%, or about 80%, or may be about 100%.

[0159] The CAR construct material of the present invention can be formulated into a composition, such as a pharmaceutical composition. In this regard, one embodiment of the present invention provides a pharmaceutical composition comprising any of the CAR construct materials of the present invention described herein and a pharma- ceutically acceptable carrier. A pharmaceutical composition of the present invention containing any of the CAR construct materials of the present invention can contain more than one CAR construct material of the present invention, e.g., a CAR construct and a nucleic acid, or two or more different CAR constructs. Alternatively, the pharmaceutical composition may comprise the CAR construct material of the invention in combination with other pharmacologic active agents or drugs, such as, for example, chemotherapeutic agents such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In a preferred embodiment, the pharmaceutical composition comprises the host cell of the invention or a population thereof.

[0160] For pharmaceutical compositions, the pharma- ceutically acceptable carrier may be any of those conventionally used, limited only by chemical and physical considerations, such as solubility and lack of reactivity with the active agent(s), and the route of administration. Pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients and diluents, are well known to those skilled in the art and are readily available to the public. It is preferred that the pharma-ceutically acceptable carrier has no adverse side effects or toxicity under the conditions of use.

[0161] The choice of carrier is determined in part by the specific CAR construct material of the present invention and by the specific method used to administer the CAR construct material of the present invention.Therefore, there are various suitable formulations of the pharmaceutical composition of the present invention.Methods for preparing administrable (e.g., parenterally administrable) compositions are known or obvious to those skilled in the art and are described in detail in, for example, Remington: The Science and Practice of Pharmacy, Pharmaceutical Press; 22nd Edition (2012).

[0162] The CAR construct material of the present invention may be administered in any suitable manner. Preferably, the CAR construct material of the present invention is administered by injection (e.g., subcutaneously, intravenously, intratumorally, intraarterially, intramuscularly, intradermally, intraperitoneally, or intrathecally). Preferably, the CAR construct material of the present invention is administered intravenously. Suitable pharmacologic acceptable carriers for the CAR construct material of the present invention for injection may include any isotonic carrier, such as, for example, normal saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or lactated Ringer's solution. In one embodiment, the pharmacologic acceptable carrier is supplemented with human serum albumin.

[0163] An "effective amount" or "therapeutically effective amount" refers to a dose sufficient to prevent or treat cancer in an individual. The amount that is effective for therapeutic or prophylactic use will depend, for example, on the stage and severity of the disease or condition being treated, the age, weight, and general health of the patient, and the judgment of the prescribing physician. The size of the dose will also be determined by the selected activity, the method of administration, the timing and frequency of administration, the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular activity, and the desired physiological effect. It will be understood by those skilled in the art that various diseases or disorders may require extended treatment, possibly involving multiple administrations, with the CAR construct material of the present invention, at each or various rounds of administration. By way of example, and not intended to limit the invention, when the CAR construct material of the present invention is a host cell, an exemplary dose of host cells is at least 1 million cells (1x10 6 cells / dose).

[0164] For purposes of the present invention, the amount or dose of the CAR construct material of the present invention administered should be sufficient to produce a therapeutic or prophylactic response in a subject or animal over a reasonable time frame. For example, the dose of the CAR construct material of the present invention should be sufficient to bind to an antigen or detect, treat or prevent cancer for a period of about 2 hours or more, e.g., about 12 to about 24 hours or more, from the time of administration. In certain embodiments, the period may be longer. The dose is determined by the efficacy of the particular CAR construct material of the present invention and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.

[0165] For purposes of the present invention, an assay that involves, for example, comparing the extent to which target cells are lysed and / or IFN-γ is secreted by T cells expressing an uncoupled CAR of a CAR construct of the present invention upon administration of a particular dose of such T cells between a set of mammals each receiving different doses of T cells can be used to determine a starting dose to administer to a mammal. The extent to which target cells are lysed and / or IFN-γ is secreted upon administration of a particular dose can be assayed by methods known in the art.

[0166] When the CAR construct material of the present invention is administered together with one or more additional therapeutic agents, the one or more additional therapeutic agents may be co-administered to a mammal. "Co-administration" refers to administering the one or more additional therapeutic agents and the CAR construct material of the present invention close enough in time that the CAR construct material of the present invention can enhance the effect of the one or more additional therapeutic agents, or vice versa. In this regard, the CAR construct material of the present invention may be administered first, and the one or more additional therapeutic agents may be administered second, or vice versa. Alternatively, the CAR construct material of the present invention and the one or more additional therapeutic agents may be co-administered. An exemplary therapeutic agent that may be co-administered with the CAR construct material is IL-2. It is believed that IL-2 enhances the therapeutic effect of the CAR construct material of the present invention.

[0167] It is contemplated that the CAR construct material of the present invention may be used in a method of treating or preventing disease in a mammal. Without being bound to a particular theory or mechanism, the CAR construct of the present invention has biological activity (e.g., the ability to detach / cleave a CAR that recognizes an antigen (e.g., one or both of CD19 and CD22)) such that when expressed by a cell, the detached CAR can mediate an immune response against a cell expressing the antigen, e.g., one or both of CD19 and CD22. In this regard, one embodiment of the present invention provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal any of the CAR constructs, nucleic acids, recombinant expression vectors, host cells, cell populations, and / or pharmaceutical compositions of the present invention in an amount effective to treat or prevent cancer in the mammal.

[0168] One embodiment of the invention further comprises lymphodepleting the mammal prior to administering the CAR construct material of the invention. Examples of lymphodepletion include, but are not limited to, non-myelosuppressive lymphodepleting chemotherapy, myelosuppressive lymphodepleting chemotherapy, total body irradiation, and the like.

[0169] For the purposes of the methods of the present invention, a host cell or cell population is administered, which may be allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0170] The mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, rodent mammals such as mice and hamsters, and lagomorph mammals such as rabbits. The mammal may be from the order Carnivora, including Felines (cats) and Canidae (dogs). The mammal may be from the order Bovidae, including Bovidae (cows) and Suidae (pigs), or from the order Equidae, including Equidae (horses). The mammal may be from the order Primates, Ceboids, or Simoides (monkeys), or from the order Anthropoids (humans and apes). Preferably, the mammal is a human.

[0171] In relation to the therapeutic method of the present invention, the cancer may be any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal or anorectum, eye cancer, cancer of the intrahepatic bile duct, joint cancer, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia (CLL), chronic bone marrow cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer ( The cancer may be any cancer, including any of the following: non-small cell lung cancer, lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, chronic B-lymphocytic leukemia, B-precursor acute lymphoblastic leukemia (B-ALL), childhood B-precursor acute lymphoblastic leukemia (BCP-ALL), B-cell lymphoma, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer, and uterine cancer. Preferably, the cancer is a hematological malignancy (e.g., a leukemia or lymphoma including, but not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, CLL, acute lymphocytic carcinoma, acute myeloid leukemia, chronic B-lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) (also referred to as "acute lymphoblastic leukemia"), B-ALL, BCP-ALL, B-cell lymphoma, and Burkitt's lymphoma). Preferably, the cancer is a hematological malignancy characterized by expression of one or both of CD22 and CD19, more preferably by expression of one or both of CD19 and CD22.

[0172] As used herein, the terms "treatment" and "prevention," and words derived therefrom, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art recognizes as having potential benefit or therapeutic effect. In this regard, the methods of the present invention may provide any amount or level of treatment or prevention of cancer in a mammal. Moreover, the treatment or prevention provided by the methods of the present invention may include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented. Also, for purposes of this specification, "prevention" may include delaying the onset of the disease, or a symptom or condition thereof.

[0173] Another embodiment of the invention provides the use of a CAR construct, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, or a pharmaceutical composition of the invention for the treatment or prevention of cancer in a mammal.

[0174] Another embodiment of the invention provides a method of detecting the presence of cancer in a mammal, the method comprising: (a) contacting a sample comprising one or more cells derived from a mammal with a CAR construct, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition of the invention, thereby forming a complex; and (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0175] The sample may be obtained by any suitable method, e.g., biopsy or autopsy. A biopsy is the removal of tissue and / or cells from an individual. Such removal may be the recovery of tissue and / or cells from an individual in order to perform experiments on the removed tissue and / or cells. This experiment may include experiments to determine whether an individual has and / or is suffering from a particular condition or disease state. The condition or disease may be, e.g., cancer.

[0176] With regard to one embodiment of the method of the present invention for detecting the presence of cancer in a mammal, the sample comprising mammalian cells may be a sample comprising whole cells, a lysate thereof, or a fraction of a whole cell lysate, e.g., a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction. When the sample comprises whole cells, the cells may be any cell of a mammal, e.g., a cell of any organ or tissue, including a blood cell or an endothelial cell.

[0177] For purposes of the detection methods of the present invention, the contacting can be performed in vitro or in vivo with respect to a mammal. Preferably, the contacting is in vitro.

[0178] Detection of the complex can also be performed by any number of methods known in the art. For example, the CAR constructs, nucleic acids, recombinant expression vectors, host cells, or cell populations of the invention described herein can be labeled with a detectable label, such as, for example, radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0179] Methods for testing CARs for their ability to recognize target cells and for antigen specificity are known in the art. For example, Clay et al., J.Immunol., 163:507-513 (1999) teaches methods for measuring the release of cytokines (e.g., interferon-γ, granulocyte / monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor a (TNF-α) or interleukin 2 (IL-2)). In addition, the function of CARs can be evaluated by measuring the cytotoxicity of cells, as described in Zhao et al., J.Immunol., 174:4415-4423 (2005).

[0180] The following are specific aspects of the invention.

[0181] 1.(a) cleavable domain; (b) a first antigen-binding domain, a first transmembrane domain, and First Intracellular T Cell Signaling Domain A first CAR including: (c) a second antigen-binding domain, a second transmembrane domain, and Second Intracellular T Cell Signaling Domain The second CAR, including; A chimeric antigen receptor (CAR) amino acid construct comprising: the first and second CARs are linked via a cleavable domain; the first antigen-binding domain comprises the antigen-binding domain of the m971 antibody; A construct, wherein when the first CAR is cleaved from the construct, the first antigen-binding domain has antigen specificity for CD22.

[0182] 2. The CAR construct of embodiment 1, wherein the first and second CARs are detached from the CAR construct by cleaving the cleavable domain.

[0183] 3. The CAR construct according to aspect 1 or 2, wherein the first antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence of any of SEQ ID NOs: 3 to 9, and a light chain variable region comprising an amino acid sequence of any of SEQ ID NOs: 11 to 17.

[0184] 4. The CAR construct according to any one of aspects 1 to 3, wherein the first antigen-binding domain comprises an amino acid sequence of SEQ ID NO: 3 to 9 and 11 to 17.

[0185] 5. The CAR construct according to any one of aspects 1 to 4, wherein the second antigen-binding domain has antigen specificity for CD19 when the second CAR is cleaved from the construct.

[0186] 6. The CAR construct of any one of aspects 1 to 5, wherein the second antigen-binding domain comprises the antigen-binding domain of the FMC63 antibody.

[0187] 7. The CAR construct according to any one of aspects 1 to 6, wherein the second antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 31 to 37, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 23 to 29.

[0188] 8. The CAR construct according to any one of aspects 1 to 7, wherein the second antigen-binding domain comprises the amino acid sequence of SEQ ID NOs: 23 to 29 and 31 to 37.

[0189] 9.(a) Cleavable domain; (b) a first antigen-binding domain, a first transmembrane domain, and First Intracellular T Cell Signaling Domain A first CAR including: (c) a second antigen-binding domain, a second transmembrane domain, and Second Intracellular T Cell Signaling Domain The second CAR, including; A chimeric antigen receptor (CAR) amino acid construct comprising: the first and second CARs are linked via a cleavable domain; the first antigen-binding domain comprises the antigen-binding domain of the FMC63 antibody; A construct, wherein when the first CAR is cleaved from the construct, the first antigen-binding domain has antigen specificity for CD19.

[0190] 10. The CAR construct of embodiment 9, wherein the first and second CARs are detached from the CAR construct by cleaving the cleavable domain.

[0191] 11. The CAR construct of aspect 9 or 10, wherein the first antigen-binding domain comprises a heavy chain variable region comprising an amino acid sequence of any of SEQ ID NOs: 31 to 37, and a light chain variable region comprising an amino acid sequence of any of SEQ ID NOs: 23 to 29.

[0192] 12. The CAR construct according to any one of aspects 9 to 11, wherein the first antigen-binding domain comprises an amino acid sequence of SEQ ID NOs: 23 to 29 and 31 to 37.

[0193] 13. The CAR construct according to any one of aspects 9 to 12, wherein the second antigen-binding domain has antigen specificity for CD22 when the second CAR is cleaved from the construct.

[0194] 14. The CAR construct according to any one of aspects 1 to 13, wherein the first or second transmembrane domain comprises a CD8 transmembrane domain and a CD8 hinge domain.

[0195] 15. The CAR construct of embodiment 14, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 19, and the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 18.

[0196] 16. The CAR construct of any one of aspects 1-15, wherein the first or second intracellular T cell signaling domain comprises a 4-1BB intracellular T cell signaling sequence.

[0197] The CAR construct of embodiment 16, wherein the 17.4-1BB intracellular T cell signaling sequence comprises the amino acid sequence of SEQ ID NO: 20.

[0198] 18. The CAR construct of any one of aspects 1-17, wherein the first or second intracellular T cell signaling domain comprises a CD3 zeta (ζ) intracellular T cell signaling sequence.

[0199] 19. The CAR construct of embodiment 18, wherein the CD3 zeta intracellular T cell signaling sequence comprises the amino acid sequence of SEQ ID NO: 21.

[0200] 20. The CAR construct according to any one of aspects 1-19, wherein the cleavable domain is 2A or furin.

[0201] 21. The CAR construct according to any one of aspects 1 to 20, wherein the CAR construct comprises exactly two CARs, a first and a second CAR, respectively.

[0202] 22. A chimeric antigen receptor (CAR) amino acid construct comprising the amino acid sequence of SEQ ID NO: 48, 49, 50, 51, or 52.

[0203] 23. A chimeric antigen receptor (CAR) amino acid construct comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity (e.g., 100%) to any one of SEQ ID NOs: 63 to 70.

[0204] 24.(a) Two or more cleavable domains; (b) a first antigen-binding domain, a first transmembrane domain, and First Intracellular T Cell Signaling Domain A first CAR including: (c) a second antigen-binding domain, a second transmembrane domain, and Second Intracellular T Cell Signaling Domain The second CAR, including; A chimeric antigen receptor (CAR) amino acid construct comprising: A construct, wherein the first and second CARs are linked via two or more cleavable domains.

[0205] 25. The CAR construct of embodiment 24, wherein the two or more cleavable domains are directly adjacent or have at least one linker between at least two of the cleavable domains.

[0206] 26. The CAR construct of embodiment 25 or 24, wherein exactly two cleavable domains are present.

[0207] 27. A nucleic acid comprising a nucleotide sequence encoding the CAR amino acid construct of any one of embodiments 1 to 26.

[0208] 28. The nucleic acid according to aspect 27, comprising any one of the nucleotide sequences of SEQ ID NOs: 53 to 57 and 71 to 78.

[0209] 29. A recombinant expression vector comprising the nucleic acid of embodiment 27 or 28.

[0210] 30. An isolated host cell comprising the recombinant expression vector of embodiment 29.

[0211] 31. A cell population comprising at least one host cell of embodiment 30.

[0212] 32. A pharmaceutical composition comprising the CAR construct of any one of aspects 1 to 26, the nucleic acid of aspect 27 or 28, the recombinant expression vector of aspect 29, the host cell of aspect 30, or the cell population of aspect 31, and a pharma- ceutical acceptable carrier.

[0213] 33. A method for detecting the presence of cancer in a mammal, comprising: (a) contacting a sample comprising one or more cells from a mammal with a CAR construct of any one of embodiments 1 to 26, a nucleic acid of embodiment 27 or 28, a recombinant expression vector of embodiment 29, a host cell of embodiment 30, a cell population of embodiment 31, or a pharmaceutical composition of embodiment 32, thereby forming a complex; and (b) detecting a complex, wherein detection of the complex is indicative of the presence of cancer in the mammal. A method comprising:

[0214] 34. A CAR construct according to any one of aspects 1 to 26, a nucleic acid according to aspect 27 or 28, a recombinant expression vector according to aspect 29, a host cell according to aspect 30, a cell population according to aspect 31, or a pharmaceutical composition according to aspect 32 for use in the treatment or prevention of cancer in a mammal.

[0215] 35. The CAR construct for use in embodiment 34, wherein the cancer is a hematological malignancy.

[0216] 36. A method for producing a chimeric antigen receptor (CAR) amino acid construct, comprising: (a) a first antigen-binding domain, a first transmembrane domain, and First Intracellular T Cell Signaling Domain A first CAR including: (b) a second antigen-binding domain, a second transmembrane domain, and Second Intracellular T Cell Signaling Domain The second CAR containing wherein the first and second CARs are linked via the two or more cleavable domains; and cloning a sequence comprising, from the N-terminus to the C-terminus, a first CAR, two or more cleavable domains, and a second CAR into a plasmid; A method comprising:

[0217] 37. The method of embodiment 36, wherein the two or more cleavable domains are directly adjacent or have at least one linker between at least two of the cleavable domains.

[0218] 38. The method of embodiment 36 or 37, wherein exactly two cleavable domains are present.

[0219] The following examples further illustrate the invention but should not be construed as in any way limiting its scope.

[0220] Example 1 This example demonstrates the generation of a CAR construct, a lentiviral vector encoding the CAR construct, and CAR-expressing T cells according to embodiments of the invention, as well as the generation of other CARs for comparison.

[0221] CAR constructs were synthesized by GENEWIZ (South Plainfield, NJ, USA) and then subcloned into the lentiviral plasmid backbone between the NhE1 and HincII sites.

[0222] Lentiviral vectors encoding CAR constructs were produced by transient transfection of the 293T cell line. Briefly, 293T cells were seeded on poly-D lysine-coated 15 cm plates (BD Biosciences, San Jose, CA, USA). The next day, 293T cells were transfected with plasmids encoding the CAR constructs together with packaging and envelope vectors (pMDLg / pRRE, pMD-2G, and pRSV-Rev) using Lipofectamine 3000 (Life Technologies, Calrsbad, CA, USA). Lentiviral supernatants were harvested 48-72 hours after transfection, centrifuged at 3000 RPM for 10 minutes to remove cellular debris, and then stored at -80°C. Human PBMCs from normal donors were activated with CD3 / CD28 microbeads (Life Technologies) at a 1:1 ratio in AIM-V medium containing 40 IU / mL recombinant IL-2 (rhIL-2; Roche, Basel, Switzerland) for 24 h. Activated T cells were resuspended at 2 million cells per mL of fresh AIM-V medium containing 3 mL of lentiviral supernatant and 10 μg / mL protamine sulfate and 100 IU / mL IL-2 and cultured in 6-well plates. Plates were centrifuged at 1000 × g for 2 h at 32 °C and then incubated overnight at 37 °C. A second transduction was performed the following day. On day 3, CD3 / CD28 beads were removed and cells were cultured at 300,000 cells / mL in AIM-V containing 100 IU / mL IL-2, and fresh IL2-containing medium was added every 2-3 days until harvest on day 8 or 9.

[0223] Vectors for the single anti-CD19 CAR, single anti-CD22 CAR, and the bispecific LoopCAR6 were generated by transient transfection of the 293T lentipackaging cell line.

[0224] The sequence of the anti-CD19 CAR is:

[0225] [ka]

[0226] It is.

[0227] The sequence of the anti-CD22 CAR is:

[0228] [ka] It is.

[0229] LoopCAR6 is described in International Patent Publication No. WO2016 / 149578 and has the following sequence:

[0230] [ka]

[0231] has.

[0232] Example 2 This example demonstrates the surface expression on human T cells of a CAR cleaved from a CAR construct according to an embodiment of the invention compared to other CARs.

[0233] Surface expression of anti-CD19 CAR and anti-CD22 CAR on V1-transduced T cells is approximately 15%, while expression of anti-CD19 CAR from a vector encoding only anti-CD19 CAR alone is 61% and expression of anti-CD22 CAR from a vector encoding only anti-CD22 CAR alone is 56% (Figures 2A-2C).

[0234] Human PBMCs from healthy donors were activated with CD3 / CD28 microbeads for 24 hours. Activated T cells were then transduced with vectors individually or co-transduced with both single anti-CD19 CAR vector and single anti-CD22 CAR vector together. Surface expression of anti-CD19 CAR and anti-CD22 CAR was analyzed on day 8. Co-transduced T cells had much lower expression of both anti-CD19 and anti-CD22 CAR compared to bispecific LoopCAR6. Expression of anti-CD19 and anti-CD22 CAR on co-transduced T cells is not in equal molar ratio. In contrast, LoopCAR6 showed a nearly 1:1 ratio in expression of anti-CD19 and anti-CD22 CAR displayed as a diagonal plot. See Figure 3.

[0235] Vectors for the bispecific LoopCAR6 and V1 and V5 CARs were produced by transient transfection of the 293T lentipackaging cell line. Human PBMCs from healthy donors were activated with CD3 / CD28 microbeads for 24 hours. Activated T cells were then transduced with the vectors. Surface expression of anti-CD19 and anti-CD22 CARs was analyzed on day 7 using flow cytometry. T cells transduced with the vector encoding the V5 CAR showed higher cell surface expression of both the isolated anti-CD19 CAR and the isolated anti-CD22 CAR provided by CAR cleavage than T cells transduced with the vector encoding the V1 CAR (Figure 4).

[0236] Example 3 This example demonstrates the cytokine production-based in vitro activity of a CAR construct according to an embodiment of the invention compared to other CARs.

[0237] CAR-transduced T cells (1E5) were washed three times with 1XPBS and then co-incubated with an equal number of target cells in 200 ml of RPMI medium in a 96-well plate in a 37°C incubator for 15–20 h. Target cells were K562 expressing CD19 or CD22, or both CD19 and CD22. K562 cells served as negative controls. Cytokine levels of IL2 and IFNγ in the culture supernatant were measured with an ELISA kit (R&D Systems, Minneapolis, MN, USA). All tests were set up in triplicate. V1 CAR T cells produced large amounts of IL2 and IFNg when co-cultured with CD22-expressing target cells, but only low levels of IL2 and IFNg when co-cultured with CD19-expressing target cells (Figures 5A and 5B).

[0238] CML cell line K562 was artificially transduced with CD19 or CD22 or both to express target antigen. K562 cells served as a negative control. 1E5 CAR T cells were washed three times and then co-incubated with 1E5 target cells in RPMI medium at 37°C. After 14 hours, culture supernatants were collected and cytokine production was measured by ELISA kit. V5 produces the highest levels of both IL2 and IFNg when co-incubated with target antigen expressed on K562, when compared with bispecific LoopCAR6 and anti-CD19 and anti-CD22 single CARs. V1 produces both IL2 and IFNg well when co-incubated with target antigen expressed on K562, when compared with bispecific LoopCAR6 and anti-CD19 and anti-CD22 single CARs. See Figures 6A and 6B.

[0239] B-cell leukemia cell line NALM6 expresses both CD19 and CD22 surface antigens. CD19 or CD22 was knocked out by CRISPR / Cas9 technology to eliminate the expression of these target antigens. NALM6 cells served as a positive control. 1E5 CAR T cells were washed three times and then co-incubated with 1E5 target cells in RPMI medium at 37°C. After 14 hours, culture supernatants were collected and cytokine production was measured by ELISA kit. V5 produces the highest levels of both IL2 and IFNg when co-incubated with CD19 expressed on NALM6 compared to bispecific LoopCAR6 and anti-CD19 and anti-CD22 single CARs. V5 produces less amount of IL2 and IFNg when co-incubated with CD22 expressed on NALM6 compared to anti-CD22 single CARs. V1 produces low amounts of IL2 and IFNg when co-incubated with target antigen expressed on NALM6 when compared to V5 CAR. V1 produces comparable amounts of IL2 and IFNg to bispecific LoopCAR6 and anti-CD19 and anti-CD22 single CARs. See Figures 7A and 7B.

[0240] CAR T cells were co-incubated with NALM6 tumor cells for 18 hours, and IL2 production levels in the culture supernatant were measured by ELISA. As can be seen in Figure 8, Bicis-V5 and Bicis-V6 may have a synergistic effect.

[0241] Example 4 This example demonstrates treatment of a relapsed leukemia model with a CAR construct according to an embodiment of the invention compared to other CARs.

[0242] Bioluminescence imaging was used to track leukemia progression in vivo. 500,000 CD19KO NALM6 cells were mixed with an equal number of CD22KO NALM6 cells and injected into NSG mice on day 0. Three days later, the mice were treated with 3 million CAR T cells or mock T cells.

[0243] Leukemia appeared to be completely eradicated by the use of T cells transduced with a vector encoding the V1 CAR, but not by the use of T cells transduced with vectors encoding either a single anti-CD19 CAR or a single anti-CD22 CAR (Figure 9).

[0244] Mice were euthanized on day 14. Bone marrow (BM) cells were stained with anti-CD19 or anti-CD22 antibodies for detection of leukemia, and also with CD22 Fc or CD19 anti-idiotype for detection of anti-CD22 CAR or anti-CD19 CAR, respectively.

[0245] There were no detectable levels of leukemia in mice treated with T cells transduced with vectors encoding the V1 CAR, but there was a high tumor burden in mice treated with T cells transduced with vectors encoding either a single anti-CD19 CAR or a single anti-CD22 CAR, and there was a high tumor burden in mice treated with mock T cells. V1 CAR T cells persisted in the BM compartment until day 14.

[0246] On day 0, NSG mice were inoculated with mixed leukemia cells (0.1E6 NALM6 and 0.1E6 NALM6-CD19 - and 0.1E6 of NALM6-CD22 - On the third day, the mice were injected with 6E6 CAR. + In all groups except group 2 (G2), which had T cells, mice were administered the CAR + Mice in group 5 (G5) were administered CD19 CAR and CD22 CAR co-transduced T cells. Mice in group 6 (G6) were administered 3E6 CD19 CAR and 3E6 CD22 CAR co-transduced T cells. Mice in group 9 (G9) were administered Lenti-GFP + T cells were administered and served as a negative control. Bioluminescence intensity represents tumor burden. The data suggest that at the same dose level of CAR T cells (3E6), the bicistronic-V1 CAR may be the most potent in reducing leukemia in this relapse model (Figure 10).

[0247] Example 5 This example shows the efficacy of CAR constructs according to embodiments of the invention in comparison to other CARs. - and CD22 - Demonstrating the treatment of leukemia.

[0248] Bioluminescence imaging was used to track leukemia progression in vivo. On day 0, 500,000 CD19KO NALM6 cells were mixed with an equal number of CD22KO NALM6 cells and injected into NSG mice. Three days later, these mice were treated with 3 million CAR T cells.

[0249] The use of T cells transduced with a vector encoding the V1 CAR eliminated nearly all of the leukemia, but not with T cells transduced with vectors encoding either a single anti-CD19 CAR or a single anti-CD22 CAR (Figure 11).

[0250] NSG mice were administered 1E5 NALM6 cells, 1E5 NALM6-CD19KO cells, and 1E5 CD22KO leukemia cells on day 0, followed by 3E6 CAR + T cells were administered. Bioluminescence intensity represents tumor burden. Images show that V1, V5, V6, and V7 CARs express CD19 + CD22 + Figure 11 shows that CARs are effective in reducing leukemia and both CD19- and CD22-negative leukemia cells, whereas anti-CD19 or anti-CD22 CARs alone were unable to do so.

[0251] Example 6 This example demonstrates the treatment of leukemia with a CAR construct according to an embodiment of the invention compared to other CARs.

[0252] NSG mice were administered 1E6 NALM6 leukemia cells on day 0. Mice in groups 1 and 2 (see FIG. 13 for groups of mice) received sequential treatment with 1E6 T cells transduced with vectors encoding a single anti-CD19 CAR or a single anti-CD22 on day 3, and 3E6 T cells transduced with vectors encoding other single CARs on day 7. Mice in group 3 were co-administered a total of 6E6 CAR+ T cells, 3E6 T cells transduced with vectors encoding a single anti-CD19 CAR and 3E6 T cells transduced with vectors encoding a single anti-CD22 CAR on day 3. Mice in groups 4-7 were administered 3E6 T cells transduced with vectors encoding CARs displayed in FIG. 13 on day 3. Mice in group 4 were administered approximately 10E6 total CAR+ T cells due to low expression on co-transduced T cells. FIG. 13 demonstrates that targeting both CD19 and CD22 simultaneously is superior to sequential treatment with single-targeted CARs.

[0253] On day 0, NSG mice were administered 0.25E6 NALM6-CD19KO and 0.25E6 CD22KO leukemia cells. On day 3, NSG mice were injected with 3E6 CAR+ T cells. The use of T cells transduced with a vector encoding the V1 CAR and the use of T cells transduced with a vector encoding the V5 CAR resulted in the expression of CD19 - and CD22 - Both leukemic cells appear to be completely eradicated (Figure 14).

[0254] Example 7 This example demonstrates a bispecific CAR according to an embodiment of the invention.

[0255] Human leukemia samples Patient samples were screened for antigen expression by National Cancer Institute (NCI) Institutional Review Board (IRB) approved screening protocols. Human ALL samples for xenograft cell generation were collected and stored after informed consent to a National Cancer Institute (NCI)-IRB approved tissue acquisition protocol. All study specimens from human subjects were obtained with informed consent in accordance with the Declaration of Helsinki.

[0256] Cell lines and culture conditions The following leukemia cell lines were used: erythroleukemia K562-CD22 (transduced with human CD22, GeneCopoeia, Cat: EX-Z9364-Lv151), K562-CD19 (transduced with human CD19), K562-CD19CD22 (transduced with both human CD19 and CD22), non-transduced K562 as a negative control; B-cell acute lymphoblastic leukemia lines NALM6, NALM6-GL (transduced with GFP and luciferase) and REH-TSLPR-GL (Qin et al., Blood, 126:629-39 (2015), incorporated by reference). Cell lines were cultured in medium supplemented with 10% heat inactivated FBS, 10 mM HEPES, 100 U / mL penicillin, 100 ug / mL streptomycin, and 2 mM L-glutamine (Invitrogen). Lenti-X 293T lentipackaging cell line (Clontech. Cat# 632180) was cultured in DMEM (Invitrogen) medium.

[0257] Generation of CD19neg and CD22neg leukemia relapse models Nalm6 was edited using CRISPR Cas9 technology to generate NALM6-CD19neg-GL (CRISPR CD19 (in exon 3)), NALM6-CD22neg-GL (CRISPR CD22 (in exon 6)). Lentiviral vectors for CRISPR / Cas9 gene editing of CD19 or CD22 on NALM6 were previously described (Fry et al., Nat. Med., 24:20-28 (2018), incorporated by reference). Briefly, guide RNAs were optimized and cloned into the LentiCRISPR v2 plasmid (Addgene Plasmid 52,961) by http: / / crispr.mit.edu / . The plasmids were then co-transfected with packaging plasmids and transformed into HEK293T cells. After 2 days, the supernatants were harvested, filtered, and concentrated. For viral transduction, 10 5 NALM6 cells were incubated with 10 ml of concentrated viral supernatant for 2 days and then expanded in RPMI medium. Cell phenotype was assessed by flow cytometry, followed by sorting and single-cell cloning of cells with phenotypic changes. Single-cell clones were sequenced to confirm genotypic changes.

[0258] Production of CAR lentiviral vectors and transduction of T cells Bivalent CAR constructs were designed and synthesized and then cloned into lentiviral transfer plasmids. Lentiviral vectors encoding bivalent CARs were produced by transient transfection of the Lenti-X 293T lentipackaging cell line. Briefly, Lenti-X 293T cells were seeded on poly-D lysine-coated 15 cm plates (BD Biosciences). The next day, Lenti-X 293T cells were transfected with the plasmids encoding the CAR constructs together with packaging and envelope vectors (pMDLg / pRRE, pMD-2G, and pRSV-Rev) using Lipofectamine 3000 (Thermo Fisher Scientific). Lentiviral supernatants were harvested 24 and 48 hours after transfection, centrifuged at 3000 RPM for 10 minutes to remove cellular debris, frozen on dry ice, and stored at -80°C. Human PBMCs from normal donors were obtained by NIH approved protocol and activated with CD3 / CD28 microbeads (Dynabeads Human T-Expander CD3 / CD28, Thermo Fisher Scientific, Cat#11141D) at a 1:3 ratio in AIM-V medium containing 40 IU / mL recombinant IL-2 and 5% FBS for 24 hours. Activated T cells were resuspended in 6-well plates at 2 million cells per mL of fresh AIM-V medium containing 2 mL of lentiviral supernatant and a final concentration of 10 ug / mL protamine sulfate and 100 IU / mL IL-2. Plates were centrifuged at 1000×g for 2 hours at 32°C and incubated overnight at 37°C. A second transduction was performed the next day by repeating the same transduction procedure as above. Three days after transduction, CD3 / CD28 beads were removed and cells were cultured at 300,000 cells / mL in AIM-V containing 100 IU / mL IL2, and fresh IL2-containing medium was added every 2-3 days until harvest on day 8 or 9.

[0259] Flow cytometry analysis Surface expression of CD22 CAR-transduced T cells was determined by flow cytometry using CD22-Fc (R&D Systems) followed by incubation with PE-F(ab)2 or APC-F(ab)2 specific for human IgG-Fc (Jackson ImmunoResearch Laboratories). Surface expression of CD19 CAR-transduced T cells was detected by anti-CD19 idiotype or recombinant human CD19 Fc chimeric protein (R&D Systems) conjugated to APC by using the Lightning-Link APC Antibody Labeling Kit (Novus Biologicals). A combination of both detection reagents was used to assess the expression of bivalent CARs as indicated for the individual figures. Expression of CD19 and CD22 on B-ALL lines was detected using the following anti-human antibodies: CD45-PerCP-Cy5.5 (eBioscience), CD19-Pacific Blue, CD19-APC-Cy7, CD10-PE-Cy7, and CD22-PE (Biolegend). T cells were characterized with the following antibodies: CD3-APC-Cy7, CD4-Pacific Blue, and CD8a-PE-Cy7 (BioLegend).

[0260] Incucyte Cytotoxicity Assay 5E4 target tumor cells in 100 ul of RPMI medium were plated onto a 96-well plate (Corning® BioCoat TM Poly-L-Lysine 96-Well Clear TC-Treated Flat Bottom Assay Plates). The next day, equal amounts of CAR T cells were added to the designated wells. Apoptosis markers (Essen BioScience) were diluted in 100ul PBS and 1ul of the dilution was added to each well. Plates were scanned for GFP fluorescence expression to monitor apoptosis (GFP-positive cell loss) every 30 minutes for a duration of 40 hours using the IncuCyte ZOOM® system. The percentage of cell death at each time point was determined relative to baseline.

[0261] Analysis of cytokine production Target tumor cells and transduced CAR-positive T cells were washed three times with PBS and resuspended in RPMI at 1E6 / ml. 100ul of tumor cell suspension and 100ul of CAR T cell suspension were loaded into each well of a 96-well plate in duplicate or triplicate, along with T cell only and tumor cell only controls. After 18 hours in a 37°C incubator, culture supernatants were collected for cytokine detection using either ELISA (R&D Biosciences) or multiplex assays (Meso Scale Discovery).

[0262] In vivo studies Animal studies were performed under protocols approved by the NCI Bethesda Animal Care and Use Committee. B-ALL cell lines and xenografted human B-ALL specimens were injected IV into NSG mice (NOD.Cg-PrkdscscidIl2rgtm1Wjl / SzJ, Jackson Laboratories). For luciferase-expressing lines, leukemia was detected using a Xenogen IVIS Lumina (Caliper Life Sciences). Mice were injected intraperitoneally with 3 mg D-luciferin (Caliper Life Sciences) and imaged 4 min later with an exposure time of 30 s for NALM6 and 2 min for PDX. Bioluminescence signal flux was measured for each mouse in photons / sec / cm using Living Image Version 4.1 software (Caliper Life Sciences). 2 Leukemic burden in xenografted cells that do not express luciferase was measured by flow cytometry in peripheral blood, bone marrow, and spleen.

[0263] Patient-derived xenograft cells Primary sample: CD19 - ALL and CD19 + CD22 dim(De novo relapse specimens ALL_H0113_post22_r (CAR3), ALL_H0090_post19_pd (HMB15) were used to generate PDX models. PDXs were generated by intravenous injection of 1E6–10E6 patient ALL cells into NSG mice (NOD scid gamma, NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ; Jackson ImmunoResearch Laboratories). After two successful passages, PDX lines were transduced with lenti-GFP-Luc virus and selected for high expression of GFP and luciferase after the first and second passages. The in vivo leukemia burden of GFP-transduced PDXs was assessed weekly by fluorescent imaging, and once human ALL was detectable by fluorescent imaging, animals were transduced with CAR 3-positive cells by tail vein injection. T cells were treated. Elutriated human lymphocytes from healthy donors were obtained from the Department of Transfusion Medicine at the National Institutes of Health (NIH) Clinical Center under an IRB-approved protocol. Human lymphocytes were cultured in AIM-V medium.

[0264] Genomic analysis of PDX models Nucleic acid extraction was performed on viably cryopreserved samples using the Qiagen AllPrep micro kit according to the manufacturer's protocol (Qiagen). DNA and RNA were quantified and assessed for quality using an Agilent 2100 BioAnalyzer. Polyadenylated RNA libraries were generated and sequenced using TruSeq 4.0 chemistry on a Hiseq2500 (Illumina) platform. Whole-exome data were generated using Agilent SureSelect XT Human All Exon V5 and TruSeq V4 chemistry and sequenced to a median coverage of 100x using a HiSeq 2500 (Illumina).

[0265] Whole-exome and RNA-sequencing data were mapped and analyzed using the CCR Collaborative Bioinformatics (CCBR) pipeline (https: / / bioinformatics.cancer.gov). Reads were aligned to the reference genome Hg19. MuTect 20 Somatic variants were called using the Sigma-Aldrich ...

[0266] statistical analysis Statistical analysis was performed using Prism 7.0 software. For patient CD19 and CD22 analyses, statistical significance was calculated using the Mann-Whitney test.

[0267] Heterogeneous and dynamic expression of CD19 and CD22 on pediatric B ALL. Patient samples, primarily from patients with multiply relapsed disease, were evaluated for CD19 and CD22 expression. There was a wide range in CD19 and CD22 expression prior to immunotherapy (Figure 42). Loss of the CD19 epitope has been well described following CD19-targeted immunotherapy. In a matched pair analysis, CD22 expression was evaluated in patients before and after CD19 loss, demonstrating a consistent decrease in CD22 expression associated with CD19 loss. This suggests that loss of a single antigen can also broadly modulate antigen expression (Figure 15). These results demonstrate the challenges associated with single antigen-targeted immunotherapy.

[0268] Simultaneous targeting of both CD19 and CD22 is superior to sequential treatment in preventing relapse or disease progression in the antigen loss-relapse model. To model the CD19 and CD22 relapse phenomenon seen in clinical trials, CRISPR / Cas9 gene editing was used to delete CD19 and / or CD22 from the pre-B ALL cell line NALM6 cells, with specific data provided in Table 17 (Figure 16).

[0269] [Table 17]

[0270] Both the CRISPR-edited NALM6 line and the parental NALM6, after single-cell cloning to ensure stability, all showed disease progression when engrafted into NSG mice, despite deletion of B cell receptor-associated genes and loss of corresponding surface protein expression (Figure 17).

[0271] One approach to apply immunotherapeutic pressure against two antigens is via sequential infusion of anti-CD19-CAR T cells followed by anti-CD22 CAR T cells, or vice versa. To test this strategy, mice were injected with a mixture of CD19neg, CD22neg, and parental NALM6 (CD19pos / CD22pos) ALL to simulate antigen-negative relapse. Administration of single antigen-specific CAR T cells resulted in relapse of leukemia that does not express the target antigen, thus validating the relapse model (Figure 18). Surprisingly, sequential infusion of therapeutic doses of anti-CD19 and anti-CD22 CAR T cells, 6 days apart, did not prevent ALL progression. Importantly, the relapse phenotype demonstrated a reduced efficacy of a second CAR infusion. Co-administration (co-infusion) of both anti-CD19 and anti-CD22 targeted CARTS was superior to sequential infusion but still led to progression of CD19neg ALL that expressed CD22, suggesting that anti-CD19 CAR may be dominant.

[0272] Given the apparent superiority of the anti-CD19 CAR when co-injected with the CD22 CAR, the next step was to introduce both anti-CD19 and anti-CD22 CARs into the same T cells through co-transduction, which generated a pool of T cells containing bispecific CAR T cells. However, the co-transduction efficiency was consistently low, yielding only a quarter of the total T cell product, which expressed both anti-CD19 and anti-CD22 CARs (Figure 19). Furthermore, the relapse phenotype (CD22+CD19neg and CD22negCD19neg, Figure 20) suggests that anti-CD19 CAR T cells may still dominate when T cells expressing both anti-CARs or the anti-CD22 CAR alone are administered. Therefore, based on the inefficiency of gene transfer with two vectors, the technical challenges and costs associated with administering two vectors, and the possibility that inclusion of T cells expressing two single CARs may compromise the expansion of the bispecific T cell population, we pursued an approach to deliver the bispecifics from the same vector.

[0273] Development of bivalent CARs with tandem sequencing of scFv Bivalent CARs were generated by combining two different scFv domains into a single CAR construct. The approach taken in constructing the anti-CD19xanti-CD22 CAR was to combine the heavy chain (V H ) and light chain (V L The goal of this study was to create a tandem CAR (TanCAR) by sequentially arranging the Vs derived from each single CAR. H and V LThe original linker between the scFvs was maintained, and the two scFvs were connected using a G(S)4x5 linker (this format could be detected on the cell surface after transduction at levels comparable to single antigen-targeted CARs). TanCAR1 is described in International Patent Publication No. WO2016 / 149578. Importantly, CAR-expressing T cells could all be detected using anti-CD22 Fc fusion and anti-FMC63 idiotype. For TanCAR2 (SEQ ID NO: 63), the order of anti-CD19 and anti-CD22 scFvs was reversed, resulting in much lower detection on the surface. Despite good surface detection and comparable IL2 production levels of TanCAR1 compared to monovalent CD19 CART against CD19pos / CD22neg ALL, IL-2 production was very low when co-incubated with CD9neg / CD22pos ALL (Figures 22A and 22B). Anti-CD22 V H and V L Given the very short linker (G4S) between the anti-CD22 scFv, TanCAR3 (SEQ ID NO: 64) was constructed by increasing the length of the linker in the CD22 scFv (this format abolished CD22 Fc and anti-idiotype binding) (Figure 21). For TanCAR4 (SEQ ID NO: 65), the parental short linker for the anti-CD22 scFv was maintained, but the linker length between the anti-CD19 scFv and the anti-CD22 scFv was decreased. This resulted in enhanced CAR surface expression (anti-CD22 Fc and anti-FMC63 idiotype binding) and function against CD22, as measured by IL2 production against CD19- / CD22+ ALL, compared to TanCAR1 (Figures 22A and 22B).

[0274] The cytotoxicity of TanCAR1 and TanCAR4 was further evaluated and demonstrated comparable activity to CD19 and CD22 monovalent CARs. Despite in vitro activity, neither TanCAR1 nor TanCAR4 completely eradicated CD19CD22ALL in vivo (Figures 23A-23D).

[0275] Development of bivalent CARs with alternative sequences of scFv resulting in loop structures A series of bivalent CAR constructs were constructed (Figure 24). LoopCAR1 (SEQ ID NO: 66) was constructed with anti-CD22 scFv (maintaining a short linker) between the VH and VL of anti-CD19 scFv (this format is only detectable at a low percentage of the cell surface). For LoopCAR2 (SEQ ID NO: 67), the length of the linker was increased between the anti-CD22 scFv to facilitate folding of the loop structure, and the amino acid structure of the linker was slightly modified between the anti-CD19 variable chain and the anti-CD22 scFv to facilitate disulfide bond formation. This improved the surface detection of the CAR. LoopCAR1 failed to induce IL-2 production against either CD19 or CD22. Despite the improved surface detection and the production of some IL-2 against CD19, LoopCAR2 did not produce detectable IL-2 against the CD22 antigen. See Figures 25A and 25B.

[0276] LoopCDAR3 (SEQ ID NO: 68) was further modified by reducing the length of the linker between the anti-CD19 heavy chain and the anti-CD22 scFv and introducing the V H and V L A slightly longer linker between the anti-CD19 scFv and the anti-CD22 scFv was maintained. This resulted in improved IL-2 production against CD19neg / CD22pos ALL. For the next series of constructs, the anti-CD19 scFv was placed in a membrane-distal position and between the variable chains of the anti-CD22 scFv. In LoopCAR4 (SEQ ID NO: 69), the linker between the anti-CD19 scFv and the anti-CD22 scFv variable chains introduced in LoopCAR3 was maintained, which resulted in higher levels of CAR detection and better IL2 production compared to either of the previous formats. This suggests that the membrane-proximal location of the anti-CD22 scFv may be optimal. Given that IL-2 production against CD19neg / CD22pos ALL was still inferior to the monovalent anti-CD22 CAR, LoopCAR5 (SEQ ID NO: 70) was modified to promote disulfide bond formation (this structure did not improve cytokine production). See Figure 25C.

[0277] Finally, in LoopCAR6 (SEQ ID NO: 61), a short linker was incorporated between the anti-CD19 scFv and the anti-CD22 variable chain, which dramatically improved both CAR detection and IL-2 production for both CD19pos / CD22neg and CD19neg / CD22pos ALL (Figure 26), as well as in vitro killing of ALL expressing a single antigen (Figures 27 and 28). Notably, the kinetics of killing CD19neg ALL by T cells expressing LoopCAR6 compared to T cells expressing anti-CD22 CAR suggested slightly lower potency against CD22. LoopCAR6 produced multiple cytokines in response to both CD19 and CD22 (Figures 29A-29F), further confirming the potency and multifunctionality of LoopCAR6-expressing T cells. Thus, loop design may be optimal for bivalent CARs incorporating CD19xCD22 specificity, likely due to the difficulty in maintaining CD22 binding. Among multiple constructs designed and tested, Loop6 was identified as the most potent format and was further tested in an in vivo model.

[0278] LoopCAR6 efficiently eradicates CD19posCD22neg and CD19neg PDXs Next, LoopCAR6 was tested against Nalm6 xenograft cells. 6 A dose of LoopCAR6 appeared to eradicate CD19pos / CD22pos Nalm6 (Figure 30), whereas a dose of 3x10 6 LoopCAR6 retained activity up to doses of 0.01 mg / mL (Figure 31). Against CD19 / CD22 ALL, LoopCAR6 was superior to continuous infusion (Figure 32). However, at low doses, LoopCAR6 did not perform as well against CD19 / CD22 leukemia, a cell line with low expression of CD22 compared to the parental NALM6 (Figure 33A).

[0279] LoopCAR6 was further tested in a "spike in" relapse model in which the engrafted ALL inoculum contained 1% CD19neg or CD22neg ALL along with 99% CD19pos / CD22pos ALL (an assay mimicking relapse from a small pre-existing clone). In this model, LoopCAR6 was comparable to anti-CD19 CAR in clearing CD22neg ALL, confirming its efficacy against CD19 comparable to monovalent anti-CD19 CAR. However, in contrast to monovalent anti-CD22 CAR, LoopCAR6 was unable to completely clear CD19neg / CD22pos ALL at low CD22 site density (Figure 33B). Taken together, and as suggested by the kinetics of in vivo killing of CD22-single-expressing ALL (Figure 27), the in vivo experiments suggest that LoopCAR6 has comparable potency against CD19 to the monovalent anti-CD19 CAR, but is slightly less potent against CD22 than the monovalent anti-CD22 CAR.

[0280] To further explore the in vivo activity of LoopCAR6 in clinically relevant models of anti-CD19 CAR resistance, we utilized two different patient-derived xenograft cells (HMB15; CD19pos / CD22pos and HMB28; CD19neg / CD22pos) generated from de novo relapse specimens. Whole-exome and whole-transcriptome sequencing was performed to characterize the two PDX model lines. HMB15 harbors a translocation that creates an in-frame fusion oncogene between the N-terminus of MLL (exons 1-6) and the C-terminus of MLLT10. In this model, the CD19 and CD22 genomic loci are intact. The primary oncogene driver in HMB28 PDX is a point mutation in KRAS G12D. Additionally, this model has a premature stop codon in CD19 (Table 18).

[0281] [Table 18]

[0282] HMB15 appeared to be cleared by both monovalent CAR and LoopCAR6 (Figures 34A and 34B). HMB28 was resistant to monovalent anti-CD19 CAR and was therefore a good model of anti-CD19 CAR resistance. Encouragingly, LoopCAR6 prevented progression in HMB28, indicating that LoopCAR6 may be effective in preventing anti-CD19 CAR resistance.

[0283] No evidence of off-target activity of LoopCAR6 Two distinct V H and V L Considering the possibility of mispairing of NALM6, we performed a functional screen of T cells expressing LoopCAR6. LoopCAR6 T cells were co-incubated with human iPSC cell lines representing multiple normal tissues and IFNγ production was measured in the culture supernatant. As all active CAR constructs developed induce IFNγ, IFNγ production was used to measure reactivity. Two separate ALL cell lines expressing both NALM6 and REH-TSLPR, CD19 and CD22 were used as positive controls.

[0284] In this assay, LoopCAR6 induced IFNγ in both NALM6 and REH-TSLPR T cells. IFNγ production was not detected in the supernatants of LoopCAR6-expressing T cells in the presence of either iPS cell line (Figures 35A and 35B).

[0285] Table 19 presents a summary of the results.

[0286] [Table 19]

[0287] Example 8 This example demonstrates a cleavable CAR according to an embodiment of the present invention.

[0288] Cell lines and culture conditions The following leukemia cell lines: erythroleukemia K562-CD22 (transduced with human CD22, GeneCopoeia, Cat: EX-Z9364-Lv151), K562-CD19 (transduced with human CD19), K562-CD19CD22 (transduced with both human CD19 and CD22), non-transduced K562 as negative control; B-cell acute lymphoblastic leukemia lines NALM6, NALM6-GL (transduced with GFP and luciferase), NALM6-CD19 - -GL (Crisper KO of CD19 in exon 3), NALM6-CD22 - -GL (Crisper KO of CD22 in exon 6) was used. Cell lines were cultured in medium supplemented with 10% heat-inactivated FBS, 10 mM HEPES, 100 U / mL penicillin, 100 ug / mL streptomycin, and 2 mM L-glutamine (Invitrogen). Lenti-X 293T lentipackaging cell line (Clontech. Cat# 632180) was cultured in DMEM (Invitrogen) medium.

[0289] Primary human leukemia samples and patient-derived xenograft cells Human ALL (acute lymphoblastic leukemia) samples were collected and stored after informed consent to an IRB approved tissue acquisition protocol (protocol number: 15-C-0029). All study specimens from human subjects were obtained with informed consent in accordance with the Declaration of Helsinki. The following primary samples: CD19 - ALL and CD19 + CD22 dim(De novo relapse specimens ALL_H0113_post22_r (CAR3), ALL_H0090_post19_pd (HMB15)) were used for in vivo testing of bispecific CAR constructs. PDX models were generated by intravenous injection of 1E6-10E6 patient ALL cells into NSG mice (NOD scid gamma, NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ; Jackson ImmunoResearch Laboratories). PDX lines were transduced with lenti-GFP-Luc virus and sorted for leukemic cells expressing GFP and luciferase after the first and second passages. For these studies, second and later passages of PDX were used for relapsed and de novo ALL specimens, respectively. The in vivo leukemia burden of GFP-transduced PDXs was assessed weekly by fluorescent imaging, and when human ALL was detectable by fluorescent imaging, animals were treated with CAR T cells by tail vein injection. Elutriated human lymphocytes from healthy donors were obtained from the Department of Transfusion Medicine at the National Institutes of Health (NIH) Clinical Center under an IRB-approved protocol. Human lymphocytes were cultured in AIM-V medium.

[0290] Generation of CD19-negative or CD22-negative leukemias with CRISPR We generated lentiviral vectors for CRISPR knockout of CD19 or CD22 in NALM6. Briefly, guide RNAs were optimized and cloned into the LentiCRISPR v2 plasmid (Addgene Plasmid 52,961) by http: / / crispr.mit.edu / . The plasmids were then co-transfected with packaging plasmids and transformed into HEK293T cells. After 2 days, CRISPR supernatants were harvested, filtered, and concentrated. For viral transduction, 10 5NALM6 cells were incubated with 10 ml of concentrated viral supernatant for 2 days and then expanded in RPMI medium. Cell phenotype was assessed by flow cytometry, followed by sorting and single-cell cloning of cells with phenotypic changes. Single-cell clones were sequenced to confirm genotypic changes.

[0291] Creation of lenti-viral CAR constructs CD19 / CD22 bicistronic CARs were generated with different combinations of CD28 or 4-1BB costimulatory domains in each CD19 and CD22 CAR, linked by a cleavable linker between them. Each CD19 and CD22 CAR begins with a leader sequence followed by a single chain variable fragment of CD19 or CD22, then either a CD8 transmembrane domain linked to 4-1BB and a CD3 zeta domain, or a CD28 transmembrane domain linked to CD28 and a CD3 zeta domain. These CARs were subcloned into a pELNS lentivector backbone. All restriction enzymes were purchased from New England Biolabs. The sequences of all CAR constructs were confirmed by sequencing at Macrogen.

[0292] The CARs described in this example are: 22-BB / 19-28 (also listed herein as V5), 22-28 / 19-BB (also listed herein as V6), 22-BB / 19-BB (also listed herein as V7), and 22-28 / 19-28 (also listed herein as V8).

[0293] Generation of CAR T cells Lentiviral vectors encoding bicistronic CARs were produced by transient transfection of the Lenti-X 293T lenti-packaging cell line. Briefly, Lenti-X 293T cells were seeded on poly-D lysine-coated 15 cm plates (BD Biosciences). The next day, Lenti-X 293T cells were transfected with the plasmid encoding the CAR construct along with packaging and envelope vectors (pMDLg / pRRE, pMD-2G, and pRSV-Rev) using Lipofectamine 3000 (Thermo Fisher Scientific). Lentiviral supernatants were harvested 24 and 48 hours after transfection, centrifuged at 3000 RPM for 10 minutes to remove cell debris, frozen on dry ice, and stored at -80°C. Human PBMCs from normal donors were obtained by NIH-approved protocol and activated with CD3 / CD28 microbeads (Dynabeads Human T-Expander CD3 / CD28, Thermo Fisher Scientific, Cat#11141D) at a 1:3 ratio in AIM-V medium containing 40 IU / mL recombinant IL-2 and 5% FBS for 24 hours. Activated T cells were resuspended in 6-well plates at 2 million cells per mL of lentiviral supernatant and fresh AIM-V medium containing 10 mg / mL protamine sulfate and 100 IU / mL IL-2. Plates were centrifuged at 1000×g for 2 hours at 32°C and incubated overnight at 37°C. A second transduction was performed the next day by repeating the same transduction procedure described above. Three days after transduction, CD3 / CD28 beads were removed and cells were cultured at 300,000 cells / mL in AIM-V containing 100 IU / mL IL2, and fresh IL2-containing medium was added every 2-3 days until harvest on day 8 or 9.

[0294] Flow cytometry Surface expression of T cells transduced with CD22 CAR was measured by flow cytometry using CD22-Fc (R&D Systems) followed by incubation with PE-F(ab)2 or APC-F(ab)2 specific for human IgG-Fc (Jackson ImmunoResearch Laboratories). Surface expression of T cells transduced with CD19 CAR was detected by anti-CD19 idiotype or recombinant human CD19 Fc chimeric protein (R&D Systems) conjugated to APC by using Lightning-Link APC Antibody Labeling Kit (Novus Biologicals). Expression of CD19, CD22 on B-ALL lines was detected using the following anti-human antibodies: CD45-PerCP-Cy5.5 (eBioscience), CD19-Pacific Blue, CD19-APC-Cy7, CD10-PE-Cy7, and CD22-PE (Biolegend). T cells were characterized with the following antibodies: CD3-APC-Cy7, CD4-Pacific Blue, and CD8a-PE-Cy7 (BioLegend).

[0295] Cytotoxicity assay 5E4 target tumor cells were plated in 100 ul of RPMI medium onto a 96-well plate (Corning® BioCoat TM Poly-L-Lysine 96-Well Clear TC-Treated Flat Bottom Assay Plates). The next day, the same amount of CAR T cells was added to the designated wells. The first incucyte apoptosis marker (Essen BioScience) was diluted in 100ul of PBS and 1ul of the dilution was added to each well. The plates were scanned for GFP and / or RFP fluorescence expression using the IncuCyteZOOM® system to monitor cell apoptosis every 30 minutes for a duration of 40 hours. The percentage of cell death at each time point was baseline corrected.

[0296] Analysis of cytokine production Target tumor cells and transduced CAR-positive T cells were washed three times with 1XPBS and resuspended in RPMI at 1E6 / ml. 100ul of tumor cells were loaded into each well of a 96-well plate along with 100ul of CAR-positive T cells. T cell only and tumor cell only controls were set up. All tests were performed in duplicate or triplicate. Cells were incubated at 37°C for 18 hours, and 120ul of culture supernatant was collected for detection of cytokine production. Cytokine levels in the supernatant were measured using either an ELISA kit (R&Dsystems) or a multiplex assay (Meso Scale Discovery).

[0297] RNA sequencing analysis NALM6 and CAR T cells were resuspended at 1E6 / ml. 5E5 NALM6 were co-incubated with 5E5 CAR+T cells in 10 ml of AIMV culture medium containing 40 U IL2 in a 25 ml culture flask in duplicate or triplicate for 24 hours. NALM6 with CD19 microbeads was removed by LD column. Total eluate was collected and cells were pelleted by centrifugation at 1200 rpm for 6 min. tRNA was immediately extracted with RNAeasy Plus Mini Kit. RNA samples were sent to the NIH Core Facility for analysis. RNA quality was assessed by TapeStation Analysis Software (Agilent Technologies). RNA sequencing was performed on NextSeq FASTQ with TruSeq LT assay.

[0298] Bioenergy Analysis For glycolysis stress test, CAR T cells were suspended in serum-free unbuffered DMEM medium (Sigma-Aldrich) supplemented with L-glutamine (200 mM) and NaCl (143 mM). 0.6 mL of 0.5% phenol red solution (Sigma P0290) was added at a final concentration of 3 mg / L, and the pH was adjusted to 7.35 + / - 0.05. CAR T cells were seeded (3E5 cells per well) in Seahorse cell plates and coated with Cell-Tak (Corning) to promote T cell adhesion. Briefly, the cartridges were hydrated the day before the assay. On the day of the assay, the plates were coated with Cell-Tak, and the cells were seeded on the Cell-Tak-coated plates and placed in the XF24 analyzer for the assay. The detailed procedure is as follows: The assay cartridge was first hydrated with 200ul / well of XF calibrant solution, hydro booster was added, flexed on parafilm, the sensor cartridge was placed on top of the utility plate, and incubated overnight at 37°C without CO2. The cell culture plates were then coated with Cell-Tak as follows: For one plate, 46μl of Cell-Tak was diluted in 204μl of TC water and 1ml of NaHCO3. The mixer was dispensed into each well at 50μl, and the plate was incubated at room temperature for at least 20 minutes. After removal of the Cell-Tak solution, each well was washed with 250ml of TC water. CAR T cells (3E5 / well) were seeded in 158μl of assay medium. The cell culture plate was then spun at 450 rpm for 1 second with low acceleration and no deceleration, then the plate was reversed and spun at 650 rpm for 1 second with low acceleration and no deceleration. The plate was then incubated at 37°C, 0% CO2 for 25-30 minutes. After the 25-30 minute incubation, 158ul of warmed assay medium was slowly and gently added to the top of each well along the side of the wall using a manual P200 pipettor. The cell plate was incubated for 15-25 minutes. After 15-25 minutes, the plate was placed in the XF24 Analyzer (after calibration). The XF assay was run.Solutions were injected sequentially through three ports: Port A: Glucose 80 mM (96 μl stock solution in 3 ml assay medium); Port B: Oligomycin 18 μM (10.8 μl stock solution in 3 ml assay medium); Port C: 2-deoxyglucose (2DG) was used as a stock solution. Glycolysis stress tests were performed by measuring ECAR (mpH / min) at steady state after loading 75 μl of drug solution into the cartridge ports. For mitochondrial stress tests, CAR T cells were suspended in serum-free unbuffered DMEM medium containing D-glucose (25 mM) and sodium pyruvate (1 mM). Mitochondrial stress tests were performed as above by measuring OCR (pmol / min) at steady state after sequential injection of oligomycin (0.5 μM), FCCP (0.5 μM), rotenone (1 μM) and antimycin A (1 μM) (Sigma-Aldrich). Experiments with the Seahorse system utilized the following assay conditions: 2 min mix; 2 min wait; 3 min measure.

[0299] Fluorescence microscopy imaging and analysis T cells were co-transduced to express CAR-Cerulean or CAR-mCherry fusion proteins. CAR-positive T cells were sorted and stained with lipophilic tracer-DiD membrane dye (Life Technologies) and LIVE / DEAD Fixable Blue Dead Cell (Life Technologies) in PBS. Cells were then washed and mounted. Images were acquired with a Zeiss Apotome equipped with an AxioCam MRm camera using a Zeiss plan apochromat 20x objective. Exposure settings were identical for the entire experiment. ImageJ software was used for data analysis. Dead cells were excluded. DiD membrane staining was used to identify each cell. Area and maximum intensity of Cerulean (CFP)- or mCherry-positive areas were measured. Only maximum intensities greater than 1 were measured. The threshold for DiD staining was set at 10% of maximum pixel intensity. The threshold for Cerulean and mCherry channels was set at 20% of maximum pixel intensity.

[0300] In vivo experiments Animal studies were performed under protocols approved by the NCI Bethesda Animal Care and Use Committee. B-ALL cell lines and xenografted human B-ALL specimens were injected IV into NSG mice. For luciferase-expressing lines, leukemia was detected using Xenogen IVIS Lumina (Caliper Life Sciences). NSG were injected intraperitoneally with 3 mg D-luciferin (Caliper Life Sciences) and imaged 4 min later with exposure times of 30 s for NALM6 cells and 2 min for PDX. Bioluminescence signal flux was measured for each mouse in photons / sec / cm using Living Image Version 4.1 software (Caliper Life Sciences). 2 Leukemic burden in xenografted cells that do not express luciferase was measured by flow cytometry in peripheral blood, bone marrow, and spleen.

[0301] statistical analysis Statistical analysis was performed using Prism 7.0 software. Plots are presented as mean + / - SD. For patient CD19 and CD22 analysis, statistical significance of all data was calculated using the Mann-Whitney test.

[0302] Development of bicistronic CARs When tested in a highly aggressive relapse model with low doses of CAR, bivalent LoopCAR6 inhibited CD19 neg and CD22 neg Does not completely eradicate leukemia. After testing 11 forms of bivalent CAR, it was found to be difficult to ensure CD22 activity in a bivalent form.

[0303] Bicistronic CAR expression was measured by flow cytometry to confirm the expression of the biscistronic construct. As shown in Figure 36, upon protein translation, the bicistronic CD19 CAR and CD22 CAR became two separate fragments, and were finally expressed as two CARs on the cell surface. The clustering of cells in the fourth quadrant indicated equimolar expression of CD19 and CD22 CARs on the cell surface. CAR with 22-BB / 19-28 expression system was found to have the highest percentage of double positive cells, with 70.3% positive cells. The lowest level of double expression was associated with either double BB or double 28, 40.8% and 58.1%, respectively. When the costimulatory domain was switched, approximately 10% expression was lost in the double positive population.

[0304] Bicistronic CARs with a combination of 4-1BB and CD28 have superior in vitro function compared to bivalent and other bicistronic CARs It has been previously reported that the costimulatory endodomains CD28 and 4-1BB have different effects on the immune regulation of CAR function. To clarify the sensitivity of the single target CAR constructs, each CAR-T cell was co-cultured with leukemia expressing the allogeneic antigen at various densities, and IL-2 levels were measured in the supernatant of the co-culture for 18 hours. Target antigen density caused as much as a 10-fold difference in cytokine production, with the CD22 CAR being particularly sensitive to target density (Figure 37A). The costimulatory domain also contributed to the difference in cytokine production, but the difference due to the costimulatory domain was mild compared to the effect of target antigen density.

[0305] Next, bicistronic CARs with various combinations of costimulatory domains were incubated with K562 and NALM6-derived cells to determine whether antigen density affects cytokine production. Bicistronic CARs combining both CD28 and 4-1BB costimulatory domains produce more cytokines than those with only CD28 or 4-1BB costimulatory domains or single target CARs (Figure 37B). Antigen density again had the greatest effect on CAR T cell cytokine production. 22-BB / 19-28 produces slightly more cytokines than 22-28 / 19-BB CARs. 22-BB / 19-28 CARs also produced more cytokines than CD19 / CD22 bivalent CARs (Figure 37C). All bicistronic CARs demonstrated effective killing of target cell lines (Figures 37D-37G).

[0306] RNA sequencing analysis reveals distinct gene expression associated with different combinations of costimulatory domains To investigate the biological pathways associated with the combination of different costimulatory domains, we performed RNA sequencing analysis. + CD22 + NALM6 cells were co-incubated and total RNA was extracted for RNA sequencing analysis. Principal component analysis (PCA) shows that the various combinations are associated with distinct gene expression profiles (Figure 38).

[0307] Bicistronic CARs are expressed in CD19 + CD22 + Leukemia and CD19 neg or CD22 neg Efficiently reduces both leukemic blasts CD19 + CD22 + The NALM6 line was used to test the in vivo activity of bicistronic CARs. Single CARs of CD19 and CD22 with CD28 or 4-1BB were used as controls. As shown in Figure 39, in general, bicistronic CARs are superior to single-targeted CARs. Various combinations of co-stimulation of CD28 and 4-1BB induce different rates of tumor elimination. 22-BB / 19-28 is optimal in eliminating leukemic blasts.

[0308] In relapsed patients, CD19 neg To simulate this clinical situation, we used CRISPR Cas9 technology to generate CD19 and low CD22 blasts using NALM6 cells. neg and CD22 neg A leukemia line was generated. CD19 neg , CD22 neg A mixture of 4-1BB and parental NALM6 cells was injected into immunodeficient NSG to generate a malignant xenograft model to simulate a clinical relapse situation. The three bicistronic CARs were compared with single targeting CARs of CD19 and CD22 (Figure 39B). Single targeting CARs were unable to prevent leukemia progression. The bicistronic CAR containing both CD28 and 4-1BB had very strong activity in eliminating leukemia.

[0309] CD19 neg and CD22 neg Further comparison was made with bicistronic CARs and bivalent CARs in vivo using mixed leukemia (Figure 40). neg and CD22 neg It is superior to bivalent CAR in reducing leukemia.

[0310] Strong activity of bicistronic CAR Clinically relevant CD19 neg The bicistronic CAR was tested using a PDX model (HMB28). The leukemia for the PDX model was derived from a patient who had previously been treated with CD19 CAR, relapsed with CD19-negative leukemia relapse, and subsequently treated with anti-CD22 CAR-T cells (which failed to eliminate the leukemia due to the appearance of blasts that weakly express CD22). The bicistronic CAR could completely eradicate the CD19-negative leukemia blasts (Figure 41).

[0311] All references cited herein, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and to the same extent as if each reference was set forth in its entirety herein.

[0312] With respect to describing the invention (particularly with respect to the claims which follow), use of the terms "a" and "an" and "the" and "at least one" and similar referents should be construed to cover both the singular and the plural, unless otherwise stated herein or clearly contradicted by context. Use of the term "at least one" following a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item (A or B) selected from the listed items or any combination of two or more of the listed items (A and B), unless otherwise stated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise stated herein. The recitation of ranges of values ​​herein is intended only to serve as a shorthand method of referring individually to each individual value falling within the range, unless otherwise stated herein, and each individual value is incorporated herein as if it were individually stated herein. All methods described herein can be performed in any suitable order, unless otherwise stated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary phrases (e.g., "such as") provided herein is intended only to better illustrate the invention, and does not impose limitations on the scope of the invention unless specifically claimed. No phrase in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0313] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those of skill in the art upon reading the foregoing description. The inventors anticipate that such variations will be employed by those of skill in the art, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. A chimeric antigen receptor (CAR) amino acid construct comprising the amino acid sequence of SEQ ID NO: 48, 49, 50, 51, or 52.

2. The CAR amino acid construct described in claim 1, wherein the CAR amino acid construct comprises the amino acid sequence of SEQ ID NO:

48.

3. 2. The CAR amino acid construct of claim 1, wherein the CAR amino acid construct comprises the amino acid sequence of SEQ ID NO:

49.

4. The CAR amino acid construct described in claim 1, wherein the CAR amino acid construct comprises the amino acid sequence of SEQ ID NO:

50.

5. The CAR amino acid construct described in claim 1, wherein the CAR amino acid construct comprises the amino acid sequence of SEQ ID NO:

51.

6. The CAR amino acid construct described in claim 1, wherein the CAR amino acid construct comprises the amino acid sequence of SEQ ID NO:

52.

7. A nucleic acid comprising a nucleotide sequence encoding the CAR amino acid construct of any one of claims 1 to 6.

8. 8. An isolated host cell comprising a recombinant expression vector comprising the nucleic acid of claim 7.

9. A pharmaceutical composition comprising the CAR amino acid construct of any one of claims 1 to 6, the nucleic acid of claim 7, the host cell or a cell population thereof of claim 8, and a pharma- ceutical acceptable carrier.

10. A pharmaceutical composition for treating cancer in a mammal, comprising as an active ingredient the CAR amino acid construct of any one of claims 1 to 6, the nucleic acid of claim 7, the host cell or a cell population thereof of claim 8, or the pharmaceutical composition of claim 9.

11. The pharmaceutical composition of claim 10, wherein the cancer is a hematological malignancy.

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