WO2022076256A1

WO2022076256A1 - Engineered immune cells for immunotherapy using endoplasmic retention techniques

Info

Publication number: WO2022076256A1
Application number: PCT/US2021/053121
Authority: WO
Inventors: Yupo Ma; Kevin PINZ; Xun JIANG; Masayuki Wada
Original assignee: Icell Gene Therapeutics Llc
Priority date: 2020-10-05
Filing date: 2021-10-01
Publication date: 2022-04-14

Abstract

The present disclosure relates to engineered immune cells including, but not limited to, T or NK cells, that are engineered to downregulate a surface protein, which is the result of endoplasmic reticulum- associated retention of the surface protein. The invention also provides the methodology to co-express chimeric receptor antigens (CARs) with an agent of endoplasmic reticulum retention to prevent CAR fratricide.

Description

ENGINEERED IMMUNE CELLS FOR IMMUNOTHERAPY USING ENDOPLASMIC RETENTION TECHNIQUES

BACKGROUND OF THE INVENTION

T cells, a type of lymphocyte, play a central role in cell-mediated immunity. They are distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. T helper cells, also called CD4+ T or CD4 T cells, express CD4 glycoprotein their surface. Helper T cells are activated when exposed to peptide antigens presented by MHC (major histocompatibility complex) class II molecules. Once activated, these cells proliferate rapidly and secrete cytokines that regulate immune response. Cytotoxic T cells, also known as CD8+ T cells or CD8, express CD8 glycoprotein on their cell surface. CD8+ T cells are activated when exposed to peptide antigens presented by MHC class I molecules. Memory T cells, a subset of T cells, persist long term and respond to their cognate antigen, thus providing the immune system with "memory" against past and/or tumor cells.

T cells can be genetically engineered to produce special receptors on their surface called chimeric antigen receptors (CARs). CARs are proteins in which T cells recognize a specific protein (antigen) on tumor cells. These engineered CAR T cells are then grown in the laboratory until they expand to numbers in the billions. The expanded population of CAR T cells is then administered to a subject in need thereof.

The prior art teaches gene editing of T-cells to eliminate endogenous TCR a0 and y5 expression, which causes unwanted allogeneic immune reaction (so called GVHD - graft versus host disease). To achieve this using CAR, it commonly involves the following steps:

1) assembling gene editing construct(s) in a mRNA form or viral form to generate non-functional T-cell receptors;

2) introducing said construct to a T cell using either electroporation or viral infection;

3) selecting the absence of the targeted protein(s) on T cells; and

4) introducing CAR in a mRNA form or viral form to the T cells derived from step 3.

The use of this multistep approach has drawbacks that substantially reduce the efficiency of CAR expression, lowers viability of cells due to increased handling, and results in increased costs and time. These approaches are also risky, as current gene-editing techniques are not completely understood, have potential off-target effects, and lack complete penetrance which results in trace amounts of molecules remaining in the subject. Thus, there is a need for safer, more specific and efficient methods for genetically modifying immune cells.

The present invention is directed to a solution for the ongoing problems with gene editing of immune cells, specifically by preventing the offending molecules from being presented on the surface of the cell.

SUMMARY OF THE INVENTION

The present disclosure relates to engineered immune cells including, but not limited to, T or NK cells, that are engineered to downregulate a surface protein, which is the result of endoplasmic reticulum- associated retention of a surface protein(s). The invention also provides the methodology to co-express chimeric receptor antigens (CARs) with an agent of endoplasmic reticulum retention to prevent CAR fratricide.

The present disclosure also includes methods of engineering a T cell by inactivation of TCR or TCR signaling as a result of endoplasmic reticulum-associated retention. An engineered T cell having reduction or loss of TCR or TCR signaling useful as an "off the shelf’ therapeutic product is also disclosed.

In one embodiment, a one-step approach of introducing an expression cassette to generate, for example, an anti-surface protein CAR using non-gene editing is disclosed. This anti-surface protein CAR construct is designed to target a selected antigen, such as for example, CD7. The expression cassette encodes an anti-surface protein and an anti-surface protein scFv fused to an ER retention signal peptide, KDEL, which entraps the recognized protein within the secretion pathway, and results in the prevention of its surface location in a cell.

In a further embodiment, methods of using T cells reducing or losing the TCR signaling by the inactivation of CD45 or CD3 using a non-gene editing approach used as an "off the shelf " therapeutic product is disclosed.

The present disclosure also includes methods of engineering a T cell by inactivation of a surface antigen selected from a group of antigens including CD2, CD3, CD4, CD5, CD7 and CD52 as a result of endoplasmic reticulum-associated retention. Use of the reduction or loss of CD2, CD3, CD4, CD5, CD7, CD8, CD4 and CD52 to prevent CARs from fratricide is also disclosed.

The present disclosure also includes methods of engineering a NK cell by inactivation of a surface antigen selected from a group of antigens including CD2, CD7, CD45 and CD52 as a result of endoplasmic reticulum-associated retention. Use of the reduction or loss of CD2, CD7, CD45 and CD52 to prevent CARs from fratricide is disclosed.

In one embodiment, the present disclosure provides an engineered cell having the expression cassette encoding a polypeptide comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising a first antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a co- stimulatory domain, and a signaling domain; and a second polypeptide comprising a second antigen recognition domain fused to an ER(endoplasmic reticulum) retention signal peptide, wherein 1) the second polypeptide does not comprise a hinge region, transmembrane domain, co-stimulatory domain or a signaling domain; 2) the second antigen recognition domain entraps the recognized protein within the secretion pathway, which results in the prevention of its surface location in a cell. The two polypeptides in an expression cassette are separated by a self-cleavage site.

The disclosed invention provides methods of using a one-step approach by introducing an expression cassette in a cell, wherein the expression cassette encodes a first polypeptide comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising a first antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; and a second polypeptide comprising a second antigen recognition domain fused to an ER (endoplasmic reticulum) retention signal peptide, wherein 1) the second polypeptide does not comprise a hinge region, transmembrane domain, co-stimulatory domain or a signaling domain; 2) the second antigen recognition domain entraps the recognized protein within the secretion pathway, which results in the prevention of its surface location in a cell; wherein the first and second polypeptide comprise a single polypeptide molecule and comprise a cleavage site disposed between the first polypeptide and second polypeptide. The first and second antigen recognition domain includes at least one of CD2, CD3, CD4, CD5, CD7, CD45 or CD8; and immune cells include at least one of CD2, CD3, CD4, CD5, CD7, CD45 or CD8 surface antigens and are recruited to cancer cells. The two polypeptides in an expression cassette are separated by a self-cleavage site.

In some embodiments, the present disclosure provides a method of identifying a substance specific to at least one antigen including CD2, CD3, CD4, CD5, CD7, CD45 and CD52, that recognizes the extracellular port of these antigens in a cell.

The disclosed invention provides methods of a one-step approach by introducing an expression cassette in a cell, wherein the expression cassette encodes a first polypeptide (CAR) comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a co- stimulatory domain, and a signaling domain; and a second polypeptide comprising a second antigen recognition domain fused to an ER retention signal peptide, KDEL, wherein 1) the second polypeptide does not comprise a hinge region, transmembrane domain and costimulatory domain or a signaling domain; 2) the second antigen recognition domain entraps the recognized protein within the secretion pathway, which results in the prevention of its surface location in a cell. The second antigen recognition domain includes at least one of endogenous a and/or 0 chains or the gamma and/or delta chains of the TCR. In some embodiments, the T cell may express a CAR and/or have been modified to block TCR expression on the cell surface or inactivate TCR functions. The two polypeptides in an expression cassette are separated by a selfcleavage site

In further embodiments, the disclosed invention also relates to a one-step approach by introducing an expression cassette in a cell, wherein the expression cassette encoding a chimeric antigen receptor polypeptide (CAR); said chimeric antigen receptor polypeptide comprising an antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a costimulatory domain, and a signaling domain; and a second polypeptide and third polypeptide comprising a second and third antigen recognition domain fused to an ER retention signal peptide, KDEL, wherein 1) the second and third polypeptide does not comprise a hinge region, transmembrane domain and co-stimulatory domain or a signaling domain; 2) the second and/ or third antigen recognition domain entraps the recognized protein(s), PD1-1 and CTLA-4 within the secretion pathway, which results in the prevention of its surface location in a cell. The second and/or third antigen recognition domain includes PD-1 and CTLA-4. In a preferred embodiment, anti-PD-1 and/or CTLA-4 scFv is fused to an ER (endoplasmic reticulum) retention sequence, KDEL. When it is expressed intracellularly and retained to the ER or Golgi, the anti-PD-1 and/or anti- CTLA4 scFv entraps PD-1 and/or CLTA4 within the secretion pathway, which results in the prevention of PD-1 and CLTA4 proper cell surface location in a T cell. The polypeptides in an expression cassette are separated by a self-cleavage site(s).

The disclosed invention provides methods of one-step approach by introducing an expression cassette in a cell, wherein the expression cassette encodes a polypeptide (CAR) comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a co- stimulatory domain, and a signaling domain; and a second polypeptide comprising a second antigen recognition domain fused to an ER retention signal peptide, KDEL wherein 1) the second polypeptide does not comprise a hinge region, transmembrane domain and costimulatory domain or a signaling domain; 2) the second antigen recognition domain entraps the recognized protein within the secretion pathway, which results in blocking of its release from a cell. In a preferred embodiment, an anti-IL-6 scFv is fused to an ER retention sequence, KDEL. When it is expressed intracellularly and retained to the ER or Golgi, the anti- IL-6 scFv entraps IL-6 protein within the secretion pathway, which results in the blocking of IL-6 release from a T cell. In some embodiments, the T cell may express a CAR and/or have been modified to block or reduce release of IL-6 (Figure 43).

BRIEF DESCRIPTION OF DRAWINGS

Figures 1A-1C. CD4CAR expression. (1A), Schematic representation of recombinant lentiviral vectors encoding CD4CAR. CD4CAR expression is driven by a SFFV (spleen focusforming virus) promoter. The third generation of CD4 CAR contains a leader sequence, the anti- CD4scFv, a hinge domain (H), a transmembrane domain (TM) and intracellular signaling domains as follows: CD28, 4-1BB (both co-stimulators), and CD3 zeta. (IB), 293FT cells were transfected with lentiviral plasmids for GFP (lane 1) and CD4CAR (lane 2) for Western blot analysis at 48h post transfection and probed with mouse anti-human CD3z antibody. (1C).

Figures 2A-2D. CD4CAR T cells eliminate T-cell leukemic cells in co-culture assays. (3A), CD4CAR T cells eliminate KARPAS 299 T-cell leukemic cells in co-culture. Activated human CB buffy coat cells transduced with either GFP (middle) or CD4CAR (right) lentiviral supernatant were incubated with KARPAS 299 cells at a ratio of 2:1. After 24 hours co-culture, cells were stained with mouse- anti-human CD4 (APC) and CD8 (PerCp) antibodies and analyzed by flow cytometry for T cell subsets (N=3). (3B) and (3C), CD4CAR T cells eliminate primary T- cell leukemic cells in co-culture. Activated human CB buffy coat cells transduced with either GFP (middle) or CD4CAR (right) lentiviral supernatant were incubated with primary T-cell leukemia cells from Sezary syndrome (3B) and PTCLs (3C) at a ratio of 2:1. After 24 hours of co-culture, cells were analyzed by flow cytometry with mouse- anti-human CD4 (FITC) and CD8 (APC) antibodies (N=3). Human primary cells alone are also labeled (left). (3D) CD4CAR T cells were unable to lyse CD4-negative lymphoma cells (SP53, a B-cell lymphoma cell line). Activated human CB buffy coat cells transduced with either GFP (middle) or CD4CAR (right) lentiviral supernatant were incubated with SP53 mantle cell lymphoma cells which were prestained with the membrane dye CMTMR, at a ratio of 2:1. After 24 hours co-culture, cells were stained with mouse-anti-human CD3 (PerCp) and then analyzed by flow cytometry (N=2). SP53 cells alone, pre-stained with CMTMR were also labeled (left).

Figures 3A-3D. CD4CAR T cells efficiently mediate anti-leukemic effects in vivo with different modes. NSG mice received 2.5 Gy for sub-lethal irradiation. Twenty-four hours after irradiation, mice were injected subcutaneously with either 1 xlO⁶ (in 3A) or 0.5 xlO⁶ (in 5B and 5C) KARPAS 299 cells. Injected mice were treated with different courses and schedules of CD4CAR T cells or control T cells (injections indicated by arrows). N=5 for each group of injected mice. (3 A), a low dose of 2xl0⁶ of CD4CAR T cells was injected on day 3 followed by a large dose, 8xl0⁶, of CD4CAR T cells on day 22 after upon observed acceleration of tumor growth. (3B), two large doses of CD4CAR T cells, 8 xlO⁶ and 5.5 xlO⁶ were injected on day 3 and 10 respectively. (3C), a repeat low dose (2.5 xlO⁶) of CD4CAR T cells was injected every 5 days for a total of four administrations. (3D), overall survival of mice treated with the indicated CD4CAR T cells or control GFP T cells. N=10

Figures 4A-4D. CD4CAR NK cells demonstrate anti-leukemic effects in vivo. NSG mice were sub lethally irradiated and intradermally injected with luciferase-expressing Karpas 299 cells (Day 0) to induce measurable tumor formation. On day 1 and every 5 days for a total of 6 courses, mice were intravenously injected with 5 x 10⁶ CD4CAR NK cells or vector control NK control cells. (4 A) On days 7, 14, and 21, mice were injected subcutaneously with RediJect D- Luciferin and subjected to IVIS imaging. (4B) Average light intensity measured for the CD4CAR NK injected mice was compared to that of vector control NK injected mice. (4C) On day 1, and every other day after, tumor size area was measured and the average tumor size between the two groups was compared. (4D) Percent survival of mice was measured and compared between the two groups.

Figures 5A-5D. Generation of CD5CAR. (5A and 5B.The DNA gene construct and the translated protein construct for CD5CAR, and anchored CD5 scFv antibody and a cartoon demonstrating the creation and function of CD5CAR. The DNA construct of the third generation CD5CAR construct from 5’ to 3’ reads: Leader sequence, the anti-CD5 extracellular single chain variable fragment (Anti-CD5 ScFv), the hinge region, the trans-membrane region, and the three intracellular signaling domains that define this construct as a 3rd generation car; CD28, 4- 1BB and CD3(^. The DNA construct of the anchored CD5 scFv antibody is the same as the CD5CAR construct without the intracellular signaling domains, as is the translated protein product for anchored CD5 scFv antibody. The translated protein constructs contain the anti-CD5 ScFv that will bind to the CD5 target, the hinge region that allows for appropriate positioning of the anti-CD5 ScFv to allow for optimal binding position, and the trans-membrane region. The complete CD5CAR protein also contains the two co- stimulatory domains and an intracellular domain of CD3 zeta chain. This construct is considered as a 3rd generation CAR: CD28, 4-1BB, and CD3(^. (5C) Western blot analysis demonstrates the CD5CAR expression in HEK293 cells. HEK293 cells which had been transduced with GFP (as negative control) or CD5CAR lentiviruses for 48 h were used for Western blot analysis using CD3^ antibody to determine the expression of CD5CAR. Left lane, the GFP control HEK293 cells, with no band as expected. The right lane showing a band at about 50kDa, the molecular weight that we expected based on the CD5CAR construct. (5D) Flow cytometry analysis for CD5CAR expression on T cells surface for lentiviral transduced CD5CAR T cells. This analysis was performed on the double transduced CD5CAR T cells at day 8 after the second lentiviral transduction. Left: isotype control T cell population (negative control) ; right, transduced T cells expressing CD5 CAR showing 20.53% on T cells by flow cytometry using goat anti-mouse F(AB’)2-PE. Figures 6A-6B. Study Schema of the transduction of CD5CAR T-cells. (6A) Steps for generation of CD5 CAR T cells by single transduction. (6B) Steps for generation of CD5 CAR T cells by double transduction.

Figure 7. Comparisons of single and double transductions with CD5 CAR lentviruses in the down-regulation of surface CD5 expression on the T cells. The down-regulation of extracellular CD5 protein versus GFP T-cell control over 8 days following lentiviral transduction is analyzed. The single transduced CD5CAR T-cells do not show complete downregulation of CD5 from cell surface by day 8, with a maximum decrease in CD5 protein expression on day 6. In the double transduced population, we note the decrease in the absolute number of CD5+, CD3+ double positive CD5CAR T-cells over time, from 24.44% on day 0 to a near complete reduction of CD5 expression on day 4. In contrast, the GFP T-cell control maintains a CD5+, CD3+ double positive population above 95% from day 2 through day 8.

Figures 8A-8B. CD5CAR T cells demonstrate profound anti-leukemic effects in vivo. NSG mice were sub lethally irradiated and, after 24 hours, intravenously injected with 1 x 10⁶ luciferase-expressing CCRF-CEM cells (Day 0) to induce measurable tumor formation. On day 3 and 4, mice were intravenously injected with 5 x 10⁶ CD5CAR T cells or vector control T cells. These injections were repeated on Days 6 and 7, for a total of 2.0 x 10⁷ cells per mouse. (8A) On days 5, 8, 10 and 13, mice were injected subcutaneously with RediJect D-Luciferin and subjected to IVIS imaging. (8B) Average light intensity measured for the CD5CAR T injected mice was compared to that of vector control T injected mice.

Figure 9. The CD5 CAR NK cells (NK-92) effectively eliminate CCRF-CEM T-ALL cell line in vitro. T-lymphoblast cell line CCRF-CEM expressing CD5 was co-cultured with CD5 CAR NK cells in the indicated E:T (effector:target) cell ratios for 24 hours. Target populations were quantified with flow cytometry using CD56 and CD5 to separate the NK-CAR and target cell population respectively. Cell survival is expressed relative to transduced vector control NK cells and each bar graph represents the average statistics for duplicate samples with N=2. C, CD5CAR NK cells eliminate CCRF-CEM cells in a dose-dependent manner. T-lymphoblast cell line, CCRF-CEM expressing CD5 was co-cultured with CD5CAR NK cells in the indicated E:T (effector: target) cell ratios with the lower bound of the E:T ratio reduced. Saturation is achieved with an E:T ratio of 2:1 and co-culturing under reduced ratios results in a dosage-dependent manner of CD5 elimination. Complete elimination of CCRF-CEM was achieved at 5:1.

Figure 10. CD5CAR NK cells demonstrate potent anti-leukemic effects in vivo. NSG mice were sub lethally irradiated and, after 24 hours, intravenously injected with 1 x 10⁶ luciferase-expressing CCRF-CEM cells (Day 0) to induce measurable tumor formation. On day 3 and 4, mice were intravenously injected with 5 x 10⁶ CD5CAR NK cells or vector control NK cells. These injections were repeated on Days 6 and 7, for a total of 2.0 x 10⁷ cells per mouse. On day 5, mice were injected subcutaneously with RediJect D-Luciferin and subjected to IVIS imaging.

Figure 11A and 11B. Generation of the CD3CAR. (HAf Schematic representation of recombinant lentiviral vectors encoding CD3CAR. . (1 IB) Western blot analysis of transfected 293FT cells at 48h post transfection and probed with mouse anti-human CD3z antibody. Lane 1, GFP; Lane 2, CD3CAR.

Figures 12-13. CD3CAR NK cells demonstrate profound anti-leukemic effects in vivo. (12) NSG mice were sub lethally irradiated and, after 24 hours, intravenously injected with 1 x 10⁶ luciferase-expressing Jurkat cells (Day 0) to induce measurable tumor formation. On day 3 and 4 mice were intravenously injected with 5 x 10⁶ CD3CAR NK cells or vector control NK cells each day. These injections were repeated on Days 6 and 7, and again on Day 10, for a total of 2.5 x 10⁷ cells per mouse. (12) On days 4, 7, 9, and 13, mice were injected subcutaneously with RediJect D-Luciferin and subjected to IVIS imaging. (13) Average light intensity measured for the CD3CAR NK injected mice was compared to that of vector control NK cell injected mice.

Figure 14. Steps for generation of CAR T or NK cell targeting T-cell lymphomas or T- cell leukemias. Figure 15. Three pairs of sgRNA per gene are designed with CHOPCHOP to target CD2, CD3, CD5 and CD7. Three pairs of sgRNA were designed with CHOPCHOP to target the gene of interest. Gene-specific sgRNAs were then cloned into the lentiviral vector (Lenti U6- sgRNA-SFFV-Cas9-puro-wpre) expressing a human Cas9 and puromycin resistance genes linked with an E2A self-cleaving linker. The U6-sgRNA cassette is in front of the Cas9 element. The expression of sgRNA and Cas9puro is driven by the U6 promoter and SFFV promoter, respectively.

Figures 16A-16D. Generation of stable CD5-deficient CCRF-CEM and MOLT-4 T cells using CRISPR/Cas9 lentivirus system. (16A) Flow cytometry analysis demonstrating the loss of CD5 expression in CCRF-CEM T-cells with CRISPR/Cas9 KD using two different sgRNAs, Lenti-U6-sgCD5a-SFFV-Cas9puro (sgCD5A) and Lenti-U6-sgCD5b-SFFV-Cas9puro (sgCD5B) after puromycin selection. Wild type control is seen in the left most scatter plot. Because the CRISPR/Cas9 KD technique with sgRNA CD5A was more successful at CD5 protein downregulation, this population (denoted by the blue circle and arrow) was selected for sorting, purification and analysis in figure 16B. (16B) Flow cytometry analysis data indicating the percentage of purely sorted stable CD5 negative CCRF-CEM cells transduced using the scCD5A CRISPR/Cas9 technique. We note the >99% purity of CD45 positive, CD5 negative CCRF sgCD5A T-cells. (16C) Flow cytometry analysis demonstrating the loss of CD5 expression in MOLT-4 T-cells with CRISPR/Cas9 KD using two different sgRNA sequences (sequence CD5A and CD5B, middle and right columns) after puromycin treatment. Wild type control is seen the leftmost scatter plot. Because the CRISPR/Cas9 KD technique with primer CD5A was more successful at CD5 protein downregulation, this population (denote 16) Flow cytometry analysis data indicating the percentage of purely sorted stable CD5 negative MOLT-4 cells transduced using the scCD5A CRISPR/Cas9 technique. We note the >99% purity of CD45 positive, CD5 negative MOLT-4 sgCD5A T-cells.

Figures 17A-17D. Generation and cell sorting of stable CD7 loss in CCRF-CEM cells or NK-92 cells using CRISPR/Cas9 lentivirus system. The percentage of CD7 loss in CCRF-CEM (Figure. 17A and B) or NK-92(Figures 17C and 17D) using sgCD7A (Lenti-U6-sgCD7a-SFFV- Cas9-puro) and sgCD7B (Lenti-U6-sgCD7b-SFFV-Cas9-puro) was determined by flow cytometric analysis with CD45 and CD7 antibodies after puromycin treatment. The values of insert in figures showed percentage of positive and negative expressing CD45 or CD7 among analysis. Right panel indicates the percentage purity of sorted stable CD7 negative cells in CCRF-CEM (17B) or in NK-92 cells (17D) prepared from CD7 negative cells transduced using sgCD7A or sgCD7D CRISPR lentivirus.

Figures 18A-18B. CD7CAR NK ⁷‘ -92 cells effectively lyse T cell ALL cell line T cells that express CD7. To avoid self-killing, CD7 deficient NK-92 (NK ⁷“ -92) cells were generated and transduced with CD7CAR. Two transduced preparations of CD7CAR NK ⁷“ -92 cells, #A and #B were used to test their killing ability. (18 A) Flow cytometry analysis of CCRF-CEM cells alone (left column), in co-culture with GFP NK ⁷“ -92 cells (middle column), and in co-culture with CD7CAR-NK-92-cells, #A and B# (right columns). (18B) bar graphs based on data obtained from A.

Figure 19. CD3 multimeric protein complex. CD3 includes a protein complex and is composed of four distinct chains as described the figure above. The complex includes a CD35 chain (yellow), a CD3y chain (orange), and two CD3s chains (purple). These chains associate with the T-cell receptor (TCR) composing of a0 chains (red).

Figure 20A-20B. CD2CAR NK cells eliminate T-cell leukemic cells in co-culture assays. (20A) CD2CAR NK cells eliminate leukemic cells from T-ALL patient’s cells in co-culture. NK-92 cells transduced with either GFP (top) or CD2CAR (bottom) lentiviral supernatant were incubated with primary human T-ALL cells, SAMPL1 (PT1) at a ratio of 5: 1 (1 for 100,000 cells). After 24 hours co-culture, cells were stained with mouse- anti-human CD2 (APC) antibodies and analyzed by flow cytometry (N=2). (20B) CD2CAR NK cells eliminate a T-ALL cell line, CCRF leukemic cells in co-culture. NK-92 cells transduced with either GFP (top) or CD2CAR (bottom) lentiviral supernatant were incubated with CCRF cells at a ratio of 5:1 (1 for 100,000 cells). CCRF cells were pre-stained with cell tracker dye (CMTMR). After 24 hours coculture, cells were stained with mouse-anti-human CD2 (APC) antibodies and analyzed by flow cytometry (N=2). (Percentage of target cells (CCRF or PT1) lysed compared to GFP NK experimental control.

Figure 21. Percentage of target cells (CCRF or PT1 ) lysed compared to GFP NK experimental control. At 5:1 ratio and 24 hours co-culture, CD2CAR NK cells were able to eliminate about 60% of CD2(+) CCRF and PT1 cells in co-culture assays.

Figure 22A. Schematic diagram to elucidate the one-step approach by introducing an expression cassette to generate anti-CD7-RTX-ER-CAR using non-gene editing. A CAR, anti- CD7-RTX-ER (also called CD7-ER CAR or CD7-RTX-ER CAR) construct was designed to target the CD7 antigen. The expression cassette encodes an anti-CD7 CAR and an anti-CD7 scFv fused to an ER retention signal peptide, KDEL, which entraps the recognized protein, CD7, within the secretion pathway, h and results in the prevention of its surface location in a cell.

Figure 22B. Schematic diagram to elucidate the one-step approach by introducing an expression cassette to generate anti-CD2-RTX-ER-CAR using non-gene editing. A CAR, anti- CD2-RTX-ER (also called CD2-ER CAR or CD2-RTX-ER CAR) construct was designed to target the CD2 antigen. The expression cassette encodes an anti-CD2 CAR and an anti-CD2 scFv fused to an ER retention signal peptide, KDEL which can entrap the recognized protein, CD2 within the secretion pathway, which results in the prevention of its surface location in a cell.

Figure 22C. Schematic diagram to elucidate the one-step approach by introducing an expression cassette to generate anti-CD3-RTX-ER-CAR using non-gene editing. A CAR, anti- CD3-RTX-ER (also called CD3-ER CAR or CD3-RTX-ER CAR) construct was designed to target the CD3 antigen. The expression cassette encodes an anti-CD3 CAR and an anti-CD3 scFv fused to an ER retention signal peptide, KDEL which can entrap the recognized protein, CD3 within the secretion pathway, which results in the prevention of its surface location in a cell.

Figure 22D. Schematic diagram to elucidate the one-step approach by introducing an expression cassette to generate anti-CD45-RTX-ER-CAR using non-gene editing. A CAR, anti- CD45-RTX-ER (also called CD45-ER CAR or CD45-RTX-ER CAR) construct was designed to target the CD45 antigen. The expression cassette encodes an anti-CD45 CAR and an anti-CD45 scFv fused to an ER retention signal peptide, KDEL which can entrap the recognized protein, CD45 within the secretion pathway, which results in the prevention of its surface location in a cell.

Figure 23A. Transduction of U937 Cells with CD7-RTX-ER CAR. Wild-type U937 cells were transduced with either control (left) or CD7-RTX-ER (right) viral supernatant from transfected HEK-293FT cells. After 24 hours, cells were harvested, cells were stained with goat- anti-mouse F(Ab’). Cells were washed and stained with streptavidin-PE conjugate, and mouse anti-human CD45 and CD20 antibodies (Tonbo), and analyzed by flow cytometry.

Figure 23B. Transduction of T Cells with CD7-RTX-ER CAR. Activated T cells from healthy donor peripheral blood were transduced with either control (left) or CD7ER (right) viral supernatant from transfected HEK-293FT cells. After 48 hours, cells were harvested, washed and moved to tissue culture plates with fresh media and IE-2. After 2 days incubation, cells were stained with goat-anti-mouse F(Ab’)2. Cells were washed and stained with streptavidin-PE conjugate, and mouse anti-human CD3 and CD7 antibodies (Tonbo), and analyzed by flow cytometry. Upper panels show CAR expression, lower panels show CD7 expression.

Figure 23C. CD7-RTX-ER CAR T cells eliminate CD7-expressing Jurkat tumor cells in co-culture. Activated human T cells transduced with either control (top panels), or CD7-RTX- ER CAR (bottom panels) lentiviral supernatant were incubated with CCRF-CEM cells at E:T ratios of 0.25:1, 0.5:1, or 1:1. The tumor cells were pre-labeled with CellTracker (CMTMR) to better distinguish them from T cells. After 18 hours co-culture, cells were stained with mouse- anti-human CD3 and CD5 antibodies and analyzed by flow cytometry (N=2). The left panel shows pre-labeled Jurkat cells alone. The CD7+ targeted cells (blue dots) are circled in each panel.

Figure 23D. CD7-RTX-ER CAR T cells eliminate CD7-expressing MOET4 tumor cells in co-culture. Activated human T cells transduced with either control (top panels), or CD7Q-7ER CAR (bottom panels) lentiviral supernatant were incubated with CCRF-CEM cells at E:T ratios of 0.25:1, 0.5:1, or 1:1. The tumor cells were pre-labeled with CellTracker (CMTMR) to better distinguish them from T cells. After 18 hours co-culture, cells were stained with mouse-anti- human CD3 and CD5 antibodies and analyzed by flow cytometry (N=2). The left panel shows pre-labeled MOLT4 cells alone. The CD7+ targeted cells (green dots) are circled in each panel.

Figure 24. Graphical summary of CD7-RTX-ER CAR T cells in vitro assays against various CD7-expressing cell lines. Each cell line was incubated for 18 hours with either control T or CD7-RTX-ER CAR T cells at 0.25:1, 0.5: 1, and 1:1 effector:target cell ratios. Each bar represents the average percent cell lysis, compared to control T cells, for duplicate samples; N = 2.

Figure 25. Activated T cells from healthy donor peripheral blood were transduced with CAR viruses. After 24 hours, cells were harvested, washed and moved to tissue culture plates with fresh media and IL-2 at IxlO⁶ cells per mL. Cells were then counted every 2-3 days, starting with 3 days after transduction (Day 5), and fresh media with IL-2 was added to maintain I x lO⁶ cells per mL.

Figure 26. 2^nd experiment as above. Activated T cells from two healthy donors were transduced with CAR viruses. After 48 hours, cells were harvested, washed and moved to tissue culture plates with fresh media and IL-2 at IxlO⁶ cells per mL. Cells were then counted every 2 days, starting with 4 days after transduction (Day 6), and fresh media with IL-2 was added to maintain I x lO⁶ cells per mL.

Figure 27. CD4-IL15/IL15SUSHI construct and in vitro validation. Schematic representation of recombinant lentiviral vector encoding third generation CD4 CAR linked with the P2A self-cleaving sequence to IL-15/IL-15sushi domain of the IL- 15 alpha receptor. Expression is driven by the spleen focus-forming virus (SFFV) promoter. The IL-15/IL-15sushi portion is composed of IL-2 signal peptide fused to IL- 15 and linked to sushi domain via a 26- amino acid poly-proline linker.

Figure 28.CD4 CAR and CD4-IL/IL15sushi CAR T cells reduce tumor burden in M0LM13 mouse model. (28A.) NSG mice were sub-lethally irradiated and intravenously injected with luciferase-expressing M0LM13 cells, an acute myeloid leukemia cell line that is 100% CD4⁺ to induce measurable tumor formation. Three days following tumor cell injection, 6 mice per group were intravenously injected with a course of either 8xl0⁶ vector control, CD4 CAR, or CD4-IL15/IL15sushi CAR T cells. On days 3, 6, 9, and 11, mice were injected subcutaneously with RediJect D-luciferin and subjected to IVIS imaging to measure tumor burden. (28B.) Average light intensity measured for CD4 CAR and CD4-IL15/IL15sushi was compared to that of control to determine the percentage of tumor cells in treated versus control mice. By Day 6, CD4 CAR-treated mice had 52% lower tumor burden relative to control while CD4- IL15/IL15sushi-treated mice had 74% lower. By day 11, nearly all tumor cells had been lysed in both CD4 CAR and CD4-IL15/IL15sushi CAR groups. (28C.) Mouse survival was compared across the groups. Treatment with either CD4 CAR or CD4-IL15/IL 15 sushi CAR led to significantly significant improvement in survival compared to control mice. Additionally, all leukemic mice injected with CD4-IL15/IL15sushi CAR survived longer than the mice injected with CD4 CAR.

Figure 29. CD4-IL/IL15 sushi CAR NK cells reduce tumor burden in Jurkat mouse model and allow growth in the absence of IL-2. (A.) To create a stressful condition, we utilized CAR transduced into NK cells and Jurkat tumor cells. NK cells bear a short half-life, and Jurkat cells express less than 60% CD4⁺ phenotype. NSG mice were sub-lethally irradiated and intravenously injected with luciferase-expressing Jurkat cells to induce measurable tumor formation. Three days following tumor cell injection, 5 mice were intravenously injected with a course of 10 x 10⁶ vector control, CD4 CAR, or CD4-IL15/IL15sushi CAR T cells. On days 3, 7, 10, and 14, mice were injected subcutaneously with RediJect D-luciferin and subjected to IVIS imaging to measure tumor burden. (B.) Average light intensity measured for CD4 CAR and CD4-IL15/IL15sushi was compared to that of control to determine the percentage of tumor cells in treated versus control mice. Although both conditions showed significant tumor cell lysis by Day 7, lysis percentage for CD4 CAR NK cells stayed the same to Day 14, while CD4- IL15/IL15sushi CAR NK cells increased to over 97%. (C.) Average light intensity for the three groups used to measure the data in (B.).

Figure 30. Efficiency of CD4-IL15/IL15 sushi CAR T cells in Patient 1 with Sezary syndrome. (A.) Chest skin appearance with marked erythema and swelling before treatment with CD4-IL15/IL15sushi CAR T cells. (B.) Chest skin appearance on day 28 after infusion. (C.) Leg skin appearance before treatment with CD4-IL15/IL 15 sushi CAR T cells. (D.) Leg skin appearance on day 28 after infusion. (E.) H&E staining of skin biopsy before treatment with CD4-IL15/IL15sushi CAR T cells, showing extensive lymphocytic infiltration. (F.) H&E staining of skin biopsy 28 days after treatment, showing significantly diminished lymphocyte levels. (G.) CD4 expression of leukemic Sezary cells in skin biopsy before treatment. (H.) Diminished CD4 expression in skin biopsy 28 days after treatment. (I.) CD3-CD4+ leukemic Sezary cells dramatically diminished within 20 days after infusion with CD4-IL15/IL15sushi CAR T cells. (J.) Expansion of CD3+CD8+ cells within 8 days after infusion with CD4- IL15/IL15sushi CAR T cells. (K.) Expansion of NK cells within 22 days after infusion. (L.) Decreased Treg cells within 19 days after infusion. (M.) Long-term expansion of CD3+CD8+ cells 1 year after infusion. (N.) Decreased CD3-CD4+ cell counts in peripheral blood after infusion with CD4-IL15/IL15sushi CAR T cells that remain undetectable for the next year after treatment (orange curve), whereas CD3+CD4+ cells were depressed during the first month posttreatment followed by recovery in the next few months (blue curve) after treatment, which indicates that immune reconstitution can be observed post-treatment. (O.) Transient expansion of NK cells to 84.41% during the first month post-treatment following by a gradual decrease until 6 months and maintenance of that level for 6 more months. (P.) Initial suppression of B cells for 2 months followed by expansion.

Figure 31. Measurement ofIL-15 cytokine release. Measurement of IL- 15 in all three patients demonstrate low levels of IL- 15 despite secretion of IL15/IL15sushi complex from CD4-IL15/IL15sushi CAR T cells. The level of IL-15 was low and only picogram quantities (2- 20pg/ml).

Figure 32. CD4-IL15/IL15 sushi CAR T cells improve symptoms in Patient 2 with immunoblastic T cell lymphoma. (A.) Leg skin appearance with erythema and swelling before treatment with CD4-IL15/IL15sushi CAR T cells. (B.) Leg skin appearance 2 weeks after treatment shows some improvement. (C.) Leg skin appearance after 4 weeks after treatment show even further improvement. (D.) H&E staining of skin biopsy before treatment show numerous inflammatory lymphocytes. (E.) H&E staining of skin biopsy days after infusion of CD4-IL15/IL15sushi CAR T cells show less lymphocytes in the skin. (F.) CD4 expression of malignant T cells in skin biopsy before treatment. (G.) Diminished CD4 expression in skin biopsy days after infusion. (H.) CD3+CD4+, including the malignant cells, reduced to undetectable levels by 24 days after CD4-IL15/IL15sushi CAR T cell therapy. (I.) Therapy led to rapid suppression of B cells to undetectable levels by 6 days after treatment. (J.) Expansion of NK cells 1 month after therapy.

Figure 33. CD5-RTX-IL15/IL15sushi CAR causes significant improvement in patient’s T-ALL that has spread to left eye. (A.) Structure of CD5-RTX-IL15/IL15sushi CAR. CD5- RTX-IL15/IL15sushi is a CAR linked to IL-15/IL15sushi via the P2A self-cleaving sequence. Two rituximab (RTX)-binding epitopes are located in the hinge region. Two rituximab (RTX) epitope sequences are added to the hinge region to create anti-CD5-RTX CAR (also called CD5 CAR).

Figure 34. CSF findings after CD5-RTX-IL15/IL15sushi CAR infusion. (A) WBC counts; (B) % of blast cells; (C) CD5-RTX-IL15/IL 15 sushi CAR T cells eradicates the lymphoma cells. (B) CSF pressure; (D) protein (g/L); (E) CD5+CD34+ blasts before infusion; (F) CD5+CD34+ blasts after infusion;(G) CD3+CD8+ T cells; (H) Ferrin levels; (I)IL-6 levels in peripheral blood; (J) IL- 15 levels in the peripheral blood. The level of IL- 15 was low and only picogram quantities (about 10-50pg/ml).

Figure 35. Schematic diagram to elucidate the one -step approach by introducing an expression cassette to generate anti-CD5-RTX-ER-CAR using non-gene editing. A CAR, anti- CD5-RTX-ER (also called CD5-ER CAR or CD5-RTX-ER CAR) construct was designed to target the CD5 antigen. The expression cassette encodes an anti-CD5 CAR and an anti-CD5 scFv fused to an ER retention signal peptide, KDEL which can entrap the recognized protein, CD5 within the secretion pathway, which results in the prevention of its surface location in a cell. The CD5 CAR and scFv are separated by a self-cleavage site.

Figure 36. Schematic diagram to elucidate the anti-CD7-RTX-ER-CAR co-expressing secreting IL15/IL15sushi.

Figure 37. Schematic diagram to elucidate the one -step approach by introducing an expression cassette to generate a CAR using non-gene editing. The expression cassette encodes a CAR and a scFv against one of TCR components (TCR complex shown in bottom) fused to an ER retention signal peptide, KDEL that can entrap the recognized protein, within the secretion pathway, which results in the prevention of its surface location in a cell. A CAR and scFv are separated by a self-cleavage site.

Figure 38. Schematic diagram to elucidate the one -step approach by introducing an expression cassette to generate a CAR using non-gene editing. The expression cassette encodes a CAR and a scFv against one of HLA class 1 fused to an ER retention signal peptide, KDEL which can entrap the recognized protein, within the secretion pathway, which results in the prevention of its surface location in a cell. A CAR and scFv are separated by a self-cleavage site.

Figure 39. Schematic diagram to elucidate the one -step approach by introducing an expression cassette to generate a CAR using non-gene editing. The expression cassette encodes a CAR and a scFv against one of anti-immunopressors selected from at least one of group including, but not limited to, PD-1, TGF beta, CTLA-4, LAG3, TIGIT, VISTA. The scFv is fused to an ER retention signal peptide, KDEL which can entrap the recognized protein within the secretion pathway, which results in the prevention of its surface location in a cell. A CAR and scFv are separated by a self-cleavage site. Figure 40. Schematic diagram to elucidate the one -step approach by introducing an expression cassette to generate a CAR using non-gene editing. The expression cassette encodes a complete CAR and a scFv against one of HLA class 1 as well as a scFv against one of TCR components. Each scFv is fused to an ER retention signal peptide, KDEL which can entrap the recognized protein within the secretion pathway, which results in the prevention of its surface location in a cell. A complete CAR and each scFv are separated by a self-cleavage site.

Figure 41. Schematic diagram to elucidate the one-step approach by introducing an expression cassette to generate a CAR using non-gene editing. The expression cassette encodes a CAR and a scFv against PD-1 fused to an ER retention signal peptide, KDEL which can entrap the recognized protein, PD-1 within the secretion pathway, which results in the prevention of its surface location in a cell. A CAR and scFv are separated by a self-cleavage site.

Figure 42. Schematic diagram to elucidate the one-step approach by introducing an expression cassette to generate a CAR using non-gene editing. The expression cassette encodes a complete CAR and a scFv against PD-1 as well as a scFv against CTLA4. Each scFv is fused to an ER retention signal peptide, KDEL which can entrap the recognized protein within the secretion pathway, which results in the prevention of its surface location in a cell. A complete CAR and each scFv are separated by a self-cleavage site.

Figure 43. Schematic diagram to elucidate the one -step approach by introducing an expression cassette to generate a CAR using non-gene editing. The expression cassette encodes a CAR and a scFv against IL-6 fused to an ER retention signal peptide, KDEL which can entrap the recognized protein, IL-6, within the secretion pathway, which results in the prevention of its secretion from a cell. A CAR and scFv are separated by a self-cleavage site. DETAILED DESCRIPTION

A chimeric antigen receptor (CAR) polypeptide includes a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co- stimulatory domain, and a signaling domain.

First-generation CARs include CD3z as an intracellular signaling domain, whereas second-generation CARs include at least one single co-stimulatory domain derived from various proteins. Examples of co-stimulatory domains include, but are not limited to, CD28, CD2, 4- IBB (CD137, also referred to as “4-BB”), and OX-40 (CD124). Third generation CARs include two co-stimulatory domains, such as, without limiting, CD28, 4-1BB, CD134 (OX-40), CD2, CD27, CD30, CD40, ICIS, ICAM-1, LFA-l(CDl la/CD18), CD7, B7-H3, NKG2C, and/or CD137 (4- 1BB).

As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound having amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can include a protein's or peptide's sequence. Polypeptides include any peptide or protein having two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides, and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

A “signal peptide” includes a peptide sequence that directs the transport and localization of the peptide and any attached polypeptide within a cell, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and/or the cell surface. The signal peptide is a peptide of any secreted or transmembrane protein that directs the transport of the polypeptide of the disclosure to the cell membrane and cell surface, and provides correct localization of the polypeptide of the present disclosure. In particular, the signal peptide of the present disclosure directs the polypeptide of the present disclosure to the cellular membrane, wherein the extracellular portion of the polypeptide is displayed on the cell surface, the transmembrane portion spans the plasma membrane, and the active domain is in the cytoplasmic portion, or interior of the cell.

In one embodiment, the signal peptide is cleaved after passage through the endoplasmic reticulum (ER), i.e. is a cleavable signal peptide. In an embodiment, the signal peptide is human protein of type I, II, III, or IV. In an embodiment, the signal peptide includes an immunoglobulin heavy chain signal peptide.

The “antigen recognition domain” includes a polypeptide that is selective for or targets an antigen, receptor, peptide ligand, or protein ligand of the target; or a polypeptide of the target.

The antigen recognition domain may be obtained from any of the wide variety of extracellular domains or secreted proteins associated with ligand binding and/or signal transduction. The antigen recognition domain may include a portion of Ig heavy chain linked with a portion of Ig light chain, constituting a single chain fragment variable (scFv) that binds specifically to a target antigen. The antibody may be monoclonal or polyclonal antibody or may be of any type that binds specifically to the target antigen. In another embodiment, the antigen recognition domain can be a receptor or ligand. In particular embodiments, the target antigen is specific for a specific disease condition and the disease condition may be of any kind as long as it has a cell surface antigen, which may be recognized by at least one of the chimeric receptor constructs present in the compound CAR architecture. In a specific embodiment, the chimeric receptor may be for any cancer for which a specific monoclonal or polyclonal antibody exists or is capable of being generated. In particular, cancers such as neuroblastoma, small cell lung cancer, melanoma, ovarian cancer, renal cell carcinoma, colon cancer, Hodgkin's lymphoma, and childhood acute lymphoblastic leukemia have antigens specific for the chimeric receptors.

In some embodiments, antigen recognition domain can be non-antibody protein scaffolds, such as but not limited to, centyrins, non-antibody protein scaffolds that can be engineered to bind a variety of specific targets with high affinity. Centyrins are scaffold proteins based on human consensus tenascin FN3 domain, and are usually smaller than scFv molecules.

The target specific antigen recognition domain preferably includes an antigen binding domain derived from an antibody against an antigen of the target, or a peptide binding an antigen of the target, or a peptide or protein binding an antibody that binds an antigen of the target, or a peptide or protein ligand (including but not limited to a growth factor, a cytokine, or a hormone) binding a receptor on the target, or a domain derived from a receptor (including but not limited to a growth factor receptor, a cytokine receptor or a hormone receptor) binding a peptide or protein ligand on the target.

In one embodiment, the antigen recognition domain includes the binding portion or variable region of a monoclonal or polyclonal antibody directed against (selective for) the target.

In another embodiment, the antigen recognition domain includes Camelid single domain antibody, or portions thereof. In one embodiment, Camelid single-domain antibodies include heavy-chain antibodies found in camelids, or VHH antibody. A VHH antibody of camelid (for example camel, dromedary, llama, and alpaca) refers to a variable fragment of a camelid singlechain antibody (See Nguyen et al, 2001; Muyldermans, 2001), and also includes an isolated VHH antibody of camelid, a recombinant VHH antibody of camelid, or a synthetic VHH antibody of camelid.

In one embodiment, the signal peptide is cleaved after passage through the endoplasmic reticulum (ER), i.e. is a cleavable signal peptide. In an embodiment, the signal peptide is human protein of type I, II, III, or IV. In another embodiment, the signal peptide includes an immunoglobulin heavy chain signal peptide.

The intracellular portion of a cell contains several organelles with various roles in the development of the cell. Many of these are involved in the transport of proteins to the extracellular surface of the cell. Once these proteins reach the surface, they can be embedded in the plasma membrane of the cell and can have portions of the peptide located variably in the intracellular cytosol, the transmembrane region, or the extracellular region. Oftentimes these proteins function as signal molecules, where they are contacted by specific molecules on the extracellular portion which leads to changes or signals being generated on the intracellular side. The proteins may also be released from the cell through secretion, either in vesicles or small bags formed from the plasma membrane or as naked proteins.

In addition to transport and retention, some peptide sequences target proteins for degradation, by lysosomes, peroxisomes, and the proteasome. Proteins tagged with peptide sequences related to Lys-Phe-Glu-Arg-Gln (KFERQ) are targeted to the lysosome( 1990. 11(1- 3): p. 291-296). Proteins bound to ubiquitin, and often a chain of four ubiquitin molecules traffick to the proteasome for degradation (2009. 5(11): p. 815-822). In another embodiment, the targeting sequence will target the antigen to the peroxisome, lysosome, or the proteasome for sequestration or degradation.

ER retention refers to a protein(s) that is retained in the endoplasmic reticulum. The protein localization to the ER is commonly dependent on a signal peptide sequence located at the N-terminus or C-terminus. A common ER retention signal is the C-terminal KDEL (Lys-Asp- Glu-Leu) peptide sequence for lumen bound proteins and KKXX for transmembrane location. ER retention receptors proteins also include, for example, e KDELR1, KDELR2 and KDELR3 (Molecular Biology of the Cell. 14 (3): 889-90). The KDEL-bearing form is restricted mainly to the ER, whereas the KKMP-bearing form is distributed mainly to the intermediate compartment and Golgi complex. (Mol Biol Cell. 2003 Mar; 14(3): 889-902).

CD2, CD3, CD4, CD5, CD7, CD45 or CD8 are expressed in CAR T or NK cells, which offset their ability of target these antigens on tumor cells. Self-killing might occur in T or NK cells armed with CARs targeting one of these antigens. Therefore, it may be necessary to inactivate an endogenous targeted antigen in a T or NK cell when used as a target to arm CARs.

The herein ER retention approach is used to block endogenous antigen surface locations, for example, generation of CD2, CD3, CD5 and CD7 CAR. A multiple-step approach, wherein a target gene is first deleted or inactivated and then a targeted CAR is introduced to a cell, is considered standard procedure for the skilled person, especially when being provided with specific sgRNAs or scFv sequences of the present application (Figure 14 and 15).

According to the herein ER retention approach, the same scFv sequence is used for both the ER retention targeted recognition domain and CAR. Evidence from the prior art documents, teaches away from further proceeding with generation of CD2, CD3, CD4, CD5 and CD7 CARs. Therefore, the herein invention is patentable over the prior art for at least the reasons set forth below:

(i) a person of ordinary skill in the art would not think to, or be motivated to use an engineered T cell genetically engineered to inactivate expression of CD2, CD3, CD5, CD4 or CD7 because CD2, CD3, CD5, CD4 and CD7 play important roles in T cell-based cell killing mechanisms.

(ii) much evidence from the prior to art has been documented with reference to two antibodies recognizing the same epitope competing with each other in terms of binding (mAbs 7:1, 110—119; January/February 2015; Cytometry 40:316-326 , 2000)

(iii) In addition, the multistep step process described above would be used to engineer a cell having a CAR with inactivation of the targeted antigen because the ordinary person skilled in the art would not consider transducing cells simultaneously with two same antibodies, due to the well-documented phenomenon of antibody competition (mAbs 7:1, 110—119; January/February 2015; Cytometry 40:316-326 , 2000). This competition would be expected to result in either a decrease in the CAR efficiency, or a lack of retention of the targeted antigen at the localized site. However, an unexpected finding by the instant inventors that expression of both scFv antibodies binding to the same epitope yielded surprising and amazing results. When transduction was performed using the two-scFv expression cassette (Figure 23-26), the CAR expression efficiency was over 90% with virtually undetectable surface targeted protein by flow cytometry analysis (Figure 23B).

(iv) The ordinary person skilled in the art would also not consider designing a longer expression cassette that incorporates both antibody domains because it is known that the longer the polypeptide, the less effective it becomes for making viruses for CARs. This would be due to the lower level of protein expression expected. Research has shown the level of expression drops with increasing insert size (Int J Biochem Mol Biol. 2013; 4(4): 201-208). Our unexpected findings shows that, in fact, a surprisingly high level of CAR expression (90%) and virtually undetectable surface targeted protein with our longer expression cassette can be achieved by the herein invention.

CD2, CD3, CD5 and CD4 or CD7 play an important role in T cell-based cell killing mechanisms. In particular, T Helper cells and T Cytotoxic cells subsets are directly responsible for T cell mediated target cell killing. Therefore, CAR T cell based therapies targeting CD2, CD3, CD5, and CD7 require genetic editing of the host T cells in order to prevent CAR mediated cell killing from destroying the CAR T cells required for target cell killing. Furthermore, in addition to being found on all T cells, CD2, CD3, CD5, and CD7 play an important role in T cell based target cell killing.

In particular, CD2 interacts with lymphocyte function-associated antigen CD58 and CD48/BCM1 to mediate adhesion between T-cells and other cell types. Moreover, CD2 has been shown to bind to CD59 on APCs and facilitate TCR binding.

CD3, in conjunction with T-cell receptor (TCR) makes up the TCR complex. The essential function of the TCR complex is to identify specific bound antigens and elicit a distinct and critical response. The signal transduction mechanism by which a T cell elicits this response upon contact with its unique antigen is termed T-cell activation. The TCR polypeptides themselves have very short cytoplasmic tails, and all proximal signalling events are mediated through the CD3 molecules.

CD5 plays an important role in TCR signalling.

CD7 is a cell surface costimulatory molecule expressed on human T and natural killer cells and on cells in the early stages of T-, B-, and myeloid cell differentiation

The disclosed invention provides methods utilizing a one-step approach by introducing an expression cassette into a cell, wherein the expression cassette encodes a polypeptide comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a co- stimulatory domain, and a signaling domain; and a second polypeptide comprising an antigen recognition domain fused to an ER retention signal peptide, such as for example, KDEL, wherein:

1) the second polypeptide does not comprise a hinge region, transmembrane domain and co-stimulatory domain or a signaling domain; 2) the second antigen recognition domain entraps the recognized protein with the secretion pathway, which results in either the prevention of its surface location in a cell, or its secretion;

3) two polypeptides in an expression cassette are separated by a self-cleavage site.

In a preferred embodiment, each engineered scFv polynucleotide has different nucleotide sequences in order to avoid homologous recombination if their targets are the same.

The present disclosure provides a method of reducing cancer cell proliferation or increasing cancer cell death by administering an engineered cell having a first polypeptide comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a co- stimulatory domain, and a signaling domain; and a second polypeptide comprising a second antigen recognition domain fused to an ER retention signal peptide, such as for example, KDEL, wherein 1) the second polypeptide does not comprise a hinge region, transmembrane domain and co-stimulatory domain or a signaling domain; and 2) the second antigen recognition domain entraps the recognized protein with the secretion pathway, which results in the prevention of its surface location in a cell. The first and second antigen recognition domain includes at least one of CD2, CD3, CD4, CD5, CD7, CD45 or CD8.

In another embodiment, the disclosed invention provides methods utilizing a one-step approach by introducing an expression cassette in a vector into a cell, wherein the expression cassette encodes a polypeptide comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain for CD2, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; and the second antigen recognition domain described above is referred to a scFv (single-chain antibody) against CD2 is derived from a monoclonal or polyclonal antibody binding to intracellular CD2 and blocks the transport of CD2 protein to the cell surface. In a preferred embodiment, anti-CD2 scFv is fused to an ER (endoplasmic reticulum) retention sequence, such as for example, KDEE. When expressed intracellularly and retained to the ER or Golgi, the anti-CD2 scFv entraps CD2 within the secretion pathway, which results in the prevention of CD2 proper cell surface location in a T or NK cell (Figure 22B). In one embodiment, the disclosure provides a CD7-RTX-ER CAR engineered cell that includes a polypeptide of CD7-RTX-ER CAR (SEQ ID NO. 1 and SEQ ID NO. 3) and corresponding polynucleotide (SEQ ID NO. 2 and SEQ ID NO. 4).

In some embodiments the disclosed invention provides methods of a one-step approach by introducing an expression cassette in a vector to a cell, wherein the expression cassette encodes a polypeptide comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain for CD3, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; and the second antigen recognition domain described above is referred to a scFv (single-chain antibody) against CD3 is derived from a monoclonal or polyclonal antibody binding to intracellular CD3 and blocks the transport of CD3 protein to the cell surface. In a preferred embodiment, anti-CD3 scFv is fused to an ER (endoplasmic reticulum) retention sequence, such as for example, KDEL. When it is expressed intracellularly and retained to the ER or Golgi, the anti-CD3 scFv entraps CD2 within the secretion pathway, which results in the prevention of CD3 proper cell surface location in a T cell (Figure 22C).

In one embodiment, the disclosure provides a CD3-RTX-ER CAR engineered cell that includes a polypeptide of CD3-RTX-ER CAR (SEQ ID NO. 5) and corresponding polynucleotide (SEQ ID NO. 6). In another embodiment, the disclosed invention provides methods of a one-step approach by introducing an expression cassette in a vector to a cell, wherein the expression cassette encodes a polypeptide comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain for CD4, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; and the second antigen recognition domain described above is referred to a scFv (single-chain antibody) against CD4 is derived from a monoclonal or polyclonal antibody binding to intracellular CD4 and blocks the transport of CD4 protein to the cell surface. In a preferred embodiment, anti-CD4 scFv is fused to an ER (endoplasmic reticulum) retention sequence, such as for example, KDEL. When it is expressed intracellularly and retained to the ER or Golgi, the anti-CD4 scFv entraps CD4 within the secretion pathway, which results in the prevention of CD4 proper cell surface location in a T cell. In another embodiment, the disclosed invention provides methods of a one-step approach by introducing an expression cassette in a vector to a cell, wherein the expression cassette encodes a polypeptide comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain for CD5, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; and the second antigen recognition domain described above is referred to a scFv (single-chain antibody) against CD5 is derived from a monoclonal or polyclonal antibody binding to intracellular CD5 and blocks the transport of CD5 protein to the cell surface. In a preferred embodiment, anti-CD5 scFv is fused to an ER (endoplasmic reticulum) retention sequence, such as for example, KDEL. When it is expressed intracellularly and retained to the ER or Golgi, the anti-CD5 scFv entraps CD5 within the secretion pathway, which results in the prevention of CD5 proper cell surface location in a T cell (Figure 35).

In one embodiment, the disclosure provides a CD5CAR engineered cell that includes secreting IL-15/IL-15sushi (SEQ ID NO.11 and SEQ ID NO.13) and corresponding polynucleotide (SEQ ID 12 and SEQ ID NO.14).

In another embodiment, the disclosed invention provides methods of one-step approach by introducing an expression cassette in a vector to a cell, wherein the expression cassette encodes a polypeptide comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain for CD7, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; and the second antigen recognition domain described above is referred to a scFv (single-chain antibody) against CD7 is derived from a monoclonal or polyclonal antibody binding to intracellular CD7 and blocks the transport of CD7 protein to the cell surface. In a preferred embodiment, anti-CD7 scFv is fused an ER (endoplasmic reticulum) retention sequence, KDEL. When it is expressed intracellularly and retained to the ER or Golgi, the anti-CD7 scFv entraps CD7 within the secretion pathway, which results in the prevention of CD7 proper cell surface location in a T or NK cell (Figure 22 A).

In one embodiment, the disclosure provides a CD7-RTX-ER CAR engineered cell that includes a polypeptide of CD7-RTX-ER CAR (SEQ ID NO. 7) and corresponding polynucleotide (SEQ ID NO. 8). In one embodiment, the disclosure provides a CD7-RTX-E-IL15/IL15sushi (also called CD7-RTX-ER-VAC) CAR engineered cell that includes secreting IL-15/IL-15sushi (SEQ ID NO. 9) and corresponding polynucleotide (SEQ ID NO. 10).

In another embodiment, the disclosed invention provides methods of one-step approach by introducing an expression cassette in a vector to a cell, wherein the expression cassette encodes a polypeptide comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain for CD45, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; and the second antigen recognition domain described above is referred to a scFv (single-chain antibody) against CD45 is derived fro45 protein to the cell surface. In a preferred embodiment, anti-CD45 scFv is fused an ER (endoplasmic reticulum) retention sequence, such as for example, KDEL. When it is expressed intracellularly and retained to the ER or Golgi, the anti-C45 proper cell surface location in a NK cell.

In one embodiment, the disclosure provides a CD45-RTX-ER CAR engineered cell that includes a polypeptide of CD45-RTX-ER CAR (SEQ ID NO. 15) and corresponding polynucleotide (SEQ ID NO. 16).

In another embodiment, the disclosed invention provides methods for utilizing a one-step approach by introducing an expression cassette in a vector to a cell, wherein the expression cassette encodes a polypeptide comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain for CD52, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; and the second antigen recognition domain described above is referred to a scFv (single-chain antibody) against CD52 is derived from a monoclonal or polyclonal antibody binding to intracellular CD52 and blocks the transport of CD52 protein to the cell surface. In a preferred embodiment, anti-CD52 scFv is fused to an ER (endoplasmic reticulum) retention sequence, such as for example, KDEL. When it is expressed intracellularly and retained to the ER or Golgi, the anti-CD52 scFv entraps CD52 within the secretion pathway, which results in the prevention of CD52 proper cell surface location in a T or NK cell.

T-antigen deficient or inactivated T and NK cells T cell lymphomas or T cell leukemias express specific antigens, which may represent useful targets for these diseases. For instance, T cell lymphomas or leukemias express CD7, CD2, CD3 and CD5. However, CD7 and CD2 are also expressed in CAR T or NK cells, which offset their ability to target these antigens. The self-killing might occur in T cells or NK cells armed with CARs targeting any one of these antigens. This makes generation of CARs targeting these antigens difficult. Therefore, it may be necessary to inactivate an endogenous antigen in a T or NK cell when it is used as a target to arm CARs.

In another embodiment, the engineered cell is further modified to inactivate a cell surface polypeptide to prevent engineered cells from acting on other engineered cells. For example, one or more of the endogenous CD2, CD3, CD4, CD5, and CD7 genes of the engineered cells may be knocked out or inactivated. In a preferred embodiment, the engineered cell is a natural killer cell having at least one of the endogenous CD2 and CD7 genes knocked out or inactivated.

In another preferred embodiment, the engineered cell is a T-cell having at least one of the endogenous CD2, CD3, CD4, CD5, CD7, and CD8 genes knocked out or inactivated. In another preferred embodiment, the engineered cell is a NK cell having at least one of the endogenous CD2 and CD7 genes knocked out or inactivated.

In one embodiment, the engineered cell expressing a CAR having a particular antigen recognition domain will have the gene expressing that antigen inactivated or knocked out. For example, a T-cell having a CD2 CAR will have an inactivated or knocked out CD2 antigen gene. In another embodiment, an engineered cell (e.g. NK cell or T-cell) having a CAR with a CD4 antigen recognition domain will be modified so that the CD4 antigen is not expressed on its cell surface. In another embodiment, an engineered cell (e.g. NK cell or T-cell) having one CAR with a CD2 antigen recognition domain and another CAR with a CD7 antigen recognition domain may have both the CD2 antigen gene and the CD7 antigen gene knocked out or inactivated.

CD2, CD4, CD3, CD5 and CD7 are present on the surface of T cells and are involved in the T cell response to targeted cells or cancers. Therefore, a person ordinary sill in the art would not use an engineering T cell genetically engineered to delete or inactivate surface expression of CD2, CD3, CD5 or CD7 because they play an important role in T cell based killing mechanisms.

With regards to CD4, CD4+ T cells are important in CD8+ T cell function and the ratio of CD4+ T cells and CD8+ T cells is also essential. The inventor surprisingly discovered that CD4 CAR T cells, which depleted CD4+ cell T cells, did not interfere with the expansion and manufacturing of CD8+ T cells and did not affect cytotoxicity in vitro or in vivo. In a pilot clinical trial, infusion of CD4-CAR T cells led to the remission of aggressive T lymphomas/leukemias. Additionally, infusion of CD4 CAR T cells showed marked expansion of CD3⁺CD8⁺ and NK cells.

In another embodiment, the disclosed invention provides methods utilizing a one-step approach by introducing an expression cassette to a cell, wherein the expression cassette encodes a polypeptide (complete CAR) comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; and the second antigen recognition domain referred to as scFv (single-chain antibody) against at least one of “immune checkpoints”, a group of molecules expressed by T or NK cells. In a preferred embodiment, anti-“immune checkpoint” scFv is fused to an ER (endoplasmic reticulum) retention sequence, such as for example, KDEL. When expressed intracellularly, the anti-immune checkpoint scFv entraps “immune checkpoints” within the secretion pathway, which results in the prevention of immune checkpoints to a proper location for their functions. These “immune checkpoints” serve as “brakes” to effectively inhibit an immune response. Immune checkpoint molecules include, but are not limited to, Programmed Death 1 (PD- 1), Cytotoxic T-Lymphocyte Antigen (CTLA-4), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7 , SIGLEC9, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1 , M ORA, IL10RB, HM0X2, IL6R, IL6ST, EIF2AK4, CSK, PAG1 , SDT , F0XP3, PRDM1 , BATF, GUCY1A2, GUCY1A3, GUCY1 B2, LAG3, HAVCR2, BY55, 2B4 , TIGIT and SIGLEC10.

In another embodiment, the disclosed invention provides methods utilizing a one-step approach by introducing an expression cassette to a cell, wherein the expression cassette encodes a polypeptide (complete CAR) comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; and a second polypeptide and/or third polypeptide comprising a second and/or third antigen recognition domain fused to an ER retention signal peptide, such as for example, KDEL, wherein l)the second and/or third polypeptide does not comprise a hinge region, transmembrane domain and co-stimulatory domain or a signaling domain; 2) the second and/ or third antigen recognition domain entraps the recognized protein(s) with the secretion pathway, which results in the prevention of its appropriate location or surface location in a cell. The second and/or third antigen recognition domain includes at least one of PD-1, CTLA-4, PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7 , SIGLEC9, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1 , M ORA, IL10RB, HM0X2, IL6R, IL6ST, EIF2AK4, CSK, PAG1 , SIT1 , F0XP3, PRDM1 , BATF, GUCY1A2, GUCY1A3, GUCY1 B2, LAG3, HAVCR2, BY55, 2B4 , TIGIT and SIGLEC10.

In further embodiments, the disclosed invention also relates to a methods of using an engineering T cell, having a first polypeptide comprising a chimeric antigen receptor polypeptide (CAR); said chimeric antigen receptor polypeptide comprising an antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; and a second polypeptide and third polypeptide comprising a second and third antigen recognition domain each fused to an ER retention signal peptide, such as for example, KDEL, wherein 1) the second and third polypeptide does not comprise a hinge region, transmembrane domain and co-stimulatory domain or a signaling domain; 2) the second and/ or third antigen recognition domain entraps the recognized protein(s), PD1-1 and CTLA-4 with the secretion pathway, which results in the prevention of its surface location in a cell. The second and/or third antigen recognition domain includes PD-1 and CTLA-4. In a preferred embodiment, anti-PD-1 and/or CTLA-4 scFv is fused an ER (endoplasmic reticulum) retention sequence, such as for example, KDEL. When expressed intracellularly and retained to the ER or Golgi, the anti-PD-1 and/or anti- CTLA4 scFv entraps PD-1 and/or CLTA4 within the secretion pathway, which results in the prevention of PD-1 and/or proper cell surface location in a T cell.

Gene editing chimeric antigen receptors (CARs) from the prior art are introduced to T- cells, which typically involve the following several steps:

1) assembling a gene editing construct(s) in a mRNA form or viral form to eliminate a gene;

2) introducing to a T cell using either electroporation or viral infection;

3) selecting the absence of targeted protein on T cells; and

4) introducing CAR in a mRNA form or viral form to the T cells from step 3.

CAR with ER “entrappers” can be introduced to a cell using this similar strategy. However, these multiple steps 1) prolong T cell culture time; 2) excessively manipulate T cells, which may affect T cell functions as well as increase costs for generation of T cells for immunotherapy; and 3) reduce efficiency of CAR expression.

In one embodiment of the disclosed invention a solution to these limitations is provided that efficiently introduces an expression cassette, which contains CAR (s) and ER “entrappers” to a cell. In further embodiments, CAR and ER “entrappers” in an expression cassette are expressed in a single cell simultaneously.

In some embodiments, CAR expression in a T or NK cell, includes a high efficiency cleavage site or “self-cleaving” peptide, between CAR and ER “entrapper”, targeted scFv fused to an ER signal peptide and CAR. The “self-cleaving” peptide may be, without be limited to, porcine teschovirus-1 2A (P2A), FMDV 2A (abbreviated herein as F2A); equine rhinitis A virus (ERAV) 2A (E2A); and Thoseaasigna virus 2A (T2A) or a combination thereof. Preferably, the “self-cleaving” peptide is P2A. In some embodiments, a CAR can be designed to simultaneously express with any one or more of ER “entrappers”, targeted scFvs via a self-cleaving peptide as shown in Figure 42. Each ER “entrapper”, targeted scFv is fused to an ER signal peptide, such as for example, KDEL. ER “entrapper”, targeted scFv against antigens can include at least one of immune checkpoint molecules include, but are not limited to programmed death 1 (PD-1), cytotoxic T-lymphocyte antigen (CTLA-4), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7 , SIGLEC9, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1 , M ORA, IL10RB, HM0X2, IL6R, IL6ST, EIF2AK4, CSK, PAG1 , SHT , F0XP3, PRDM1 , BATE, GUCY1A2, GUCY1A3, GUCY1 B2, LAG3, HAVCR2, BY55, 2B4 , TIGIT and SIGLEC10.

In some embodiments, a CAR can be designed to sequentially express with any one or more of ER “entrappers”. Each ER “entrapper”, targeted scFv is fused to an ER signal peptide, such as for example, KDEL. ER “entrapper”, targeted scFv against antigens can include at least one of immune checkpoint molecules include, but are not limited to programmed death 1 (PD-1), cytotoxic T-lymphocyte antigen (CTLA-4), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7 , SIGLEC9, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1 , M ORA, IL10RB, HM0X2, IL6R, IL6ST, EIF2AK4, CSK, PAG1 , SfTl , F0XP3, PRDM1 , BATE, GUCY1A2, GUCY1A3, GUCY1 B2, LAG3, HAVCR2, BY55, 2B4 , TIGIT and SIGLEC10.

In some embodiments, a CAR can be designed to simultaneously express with any one or more of ER “entrappers”, targeted scFvs via a self-cleaving peptide. Each ER “entrapper”, targeted scFv is fused to an ER signal peptide, such as for example, KDEL. ER “entrapper”, targeted scFv against antigens can include at least one of this group, but not limited to CD2, CD3, CD5, CD4, CD7, CD8, CD45 and CD52.

In some embodiments, a CAR can be designed to sequentially express with any one or more of ER “entrappers”. Each ER “entrapper”, targeted scFv is fused to an ER signal peptide, such as for example, KDEL. ER “entrapper”, targeted scFv against antigens can include at least one of this group, but not limited to CD2, CD3, CD5, CD4, CD7, CD8, CD45 and CD52. In further embodiments, the CAR target antigens can include at least one of this group, but not limited to, GD2, GD3, , ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, Glycolipid, glypican 3, F77, GD-2, WT1, CEA, HER-2/neu, MAGE-3, MAGE-4, MAGE-5, MAGE- 6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, CD30, MMG49 epitope, EGFRvIII, CD33, CD123, CLL-1, immunoglobin kappa and lambda, CD38, CD52, CD47, CD200, CD70, CD19, CD20, CD22, CD38, BCMA, CS1, NKG2D receptor, April receptor, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD7, CD2, and CD138. The target antigens can also include viral or fungal antigens, such as E6 and E7 from the human papillomavirus (HPV) or EBV (Epstein Barr virus) antigens.

In some embodiments, a CAR can be designed to simultaneously express with any one or more of ER “entrappers”, targeted scFvs via bicistronic or multicistronic expression vectors. Several strategies may be employed to construct bicistronic or multicistronic vectors including, but not limited to, (1) multiple promoters fused to the open reading frames;(2) insertion of splicing signals between different portions of CAR and ER “entrappers”, targeted scFvs and ;(3) insertion of proteolytic cleavage sites (self-cleavage peptide); and (4) insertion of internal ribosomal entry sites (IRESs). In one embodiment, one or more proteolytic cleavage sites are inserted at different portions of CAR and ER “entrappers”, targeted scFvs.

Exemplary methods and compositions for disrupting or eliminating endogenous TCR «P and y6 expression (Figure 37 and Figure 22C) using a non-gene editing approach

Gene editing from the prior art is introduced to T-cells to eliminate endogenous TCR a0 and y5 expression, which causes unwanted allogeneic immune reaction (so called GVHD, graft versus host disease). To achieve this with CAR, it commonly involves in the following steps to eliminate endogenous TCR a0 and y5 expression:

1) assembling a gene editing construct(s) in a mRNA form or viral form to generate nonfunctional T-cell receptor

2) introducing to a T cell using either electroporation or viral infection

3) selecting the absence of targeted protein on T cells

4) introducing CAR in a mRNA form or viral form to the T cells derived from step 3. The use of these multistep approaches has drawbacks that substantially reduce the efficiency of CAR expression, lower viability of cells due to increased handling, and increase costs and time as well. In addition, the multistep step process described above would be used to engineer a cell having a CAR with inactivation of the targeted antigen because the ordinary person skilled in the art would not consider transducing cells simultaneously with two identical antibodies, due to the well-documented phenomenon of antibody competition (mAbs 7:1, 110— 119; January/February 2015; Cytometry 40:316-326 , 2000). This competition would be expected to result in either decrease in the CAR efficiency, or a lack of retention of the targeted antigen at the localized site. However, unexpected findings by the inventors that expression of two scFv antibodies binding to the same epitope yielded surprising and amazing results when transduction was performed using the two-scFv expression cassette (Figure 23-26).

The disclosed invention provides methods of one-step approach by introducing an expression cassette in a vector to a cell, wherein the expression cassette encodes a polypeptide comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a co- stimulatory domain, and a signaling domain; and a second polypeptide comprising an antigen recognition domain fused to an ER retention signal peptide, such as for example, KDEL, wherein: l)the second polypeptide does not comprise a hinge region, transmembrane domain and co-stimulatory domain or a signaling domain;

2) the second antigen recognition domain entraps the recognized protein with the secretion pathway, which results in either the prevention of its surface location in a cell, or its secretion;

The disclosed invention provides methods of a one-step approach by introducing an expression cassette in a vector to a cell, wherein the expression cassette encodes a polypeptide (CAR) comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; the second antigen recognition domain includes at least one of endogenous a and/or 0 chains or the gamma and/or delta chains of the TCR. In a preferred embodiment, a scFv against at least one of a, 0, gamma and delta chains of TCR is fused to an ER retention sequence, such as for example, KDEL. When it is expressed intracellularly and retained to the ER or Golgi, the anti- a, or 0 or gamma or delta chain scFv entraps one of these proteins within the secretion pathway, which results in the prevention of the protein to the proper cell surface location in a T cell. In some embodiments, the T cell may express a CAR and/or have been modified to block TCR expression on the cell surface or inactivate TCR functions.

The disclosed invention provides methods of a one-step approach by introducing an expression cassette in a vector to a cell, wherein the expression cassette encodes a polypeptide (CAR) comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; and a second polypeptide comprising a second antigen recognition domain fused to an ER retention signal peptide, such as for example, KDEL, wherein 1) the second polypeptide does not comprise a hinge region, transmembrane domain and co-stimulatory domain or a signaling domain; and 2) the second antigen recognition domain entraps the recognized protein within the secretion pathway, which results in the prevention of its surface location in a cell. The second antigen recognition domain is endogenous CD3. In some embodiments, a T cell may express a CAR and/or have been modified to block TCR expression on the cell surface or inactivate TCR functions.

The disclosed invention provides methods of a one-step approach by introducing an expression cassette in a vector to a cell, wherein the expression cassette encodes a polypeptide (CAR) comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; and a second polypeptide comprising a second antigen recognition domain fused to an ER retention signal peptide, such as for example, KDEL, wherein 1) the second polypeptide does not comprise a hinge region, transmembrane domain and co-stimulatory domain or a signaling domain; and 2) the second antigen recognition domain entraps the recognized protein within the secretion pathway, which results in the prevention of its surface location in a cell. The second antigen recognition domain is endogenous CD45. In some embodiments, a T or NK cell may express a CAR and/or have been modified to inactivate TCR signaling or functions.

In some embodiments, expression cassette in a T or NK cell, includes a “self-cleaving” peptide, between the first polypeptide (CAR) and the second polypeptide fused to the ER signal peptide. The “self-cleaving” peptide may be, without limiting, porcine teschovirus-1 2A (P2A), FMDV 2A (abbreviated herein as F2A); equine rhinitis A virus (ERAV) 2A (E2A); and thosea asigna virus (T2A).

In some embodiments, a cell described above is an immune cell including, but not limited to, a T cell, which is provided from an umbilical cord blood bank or a peripheral blood bank or an induced pluripotent stem cell or a human embryonic stem cell. In some cases, a T cell is allogeneic in reference to one or more recipients.

Exemplary methods and compositions for disrupting or eliminating endogenous components of HLA (Figure 38) using a non-gene editing approach

In some embodiments, the ER signal peptide can be used to engineer or modify a cell. It is desirable to generate universal T cells that have lost both T-cell receptor and HLA surface expression and thus will be less susceptible to immune-mediated recognition and destruction from the allogeneic recipient cells.

In some embodiments, there are more than one recognition domain, each fused to the ER signal peptide, that can be used to modify a cell. For instance, an ER signal peptide-fused polypeptide targeting at least one selected from endogenous TCR a0 and y5 can be used to block its surface expression. In another instance, the ER signal peptide-fused polypeptide can be used to block one or more human leukocyte antigens (HLA).

Exemplary methods and compositions for disrupting or eliminating endogenous IL-6 release (Figure 43) using a non-gene editing approach

Engineering T cells having a CAR (s) can be used to treat a disease effectively but are associated with complications such as cytokines release syndrome (CRS) and CAR T cells related encephalopathy (CRES). These complications are associated with IL-6 secretion. The disclosed invention provides methods of one-step approach by introducing an expression cassette in a vector to a cell, wherein the expression cassette encodes a polypeptide (CAR) comprising a chimeric antigen receptor polypeptide; said chimeric antigen receptor polypeptide comprising an antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain; and a second polypeptide comprising a second antigen recognition domain fused to an ER retention signal peptide, KDEL, wherein 1) the second polypeptide does not comprise a hinge region, transmembrane domain and co-stimulatory domain or a signaling domain; and 2) the second antigen recognition domain entraps the recognized protein within the secretion pathway, which results in the blocking of its release from a cell. In a preferred embodiment, an anti-IL-6 scFv is fused to an ER retention sequence, such as for example, KDEL. When it is expressed intracellularly and retained to the ER or Golgi, the anti- IL-6 scFv entraps IL-6 protein within the secretion pathway, which results in the blocking of IL-6 release from a T cell. In some embodiments, the T cell may express a CAR and/or have been modified to block or reduce IL-6 release.

Immunomodulator(s)

The present disclosure includes methods of improving CAR T/NK cell expansion, persistency and anti-target activity by co-expressing secretory IL-15/IL-15sushi complexes in an expression cassette. In a further embodiment, engineered CAR T/NK cells comprise a secretory IL-15/IL-15sushi (also called IL15/IL15sushi) complex, which can promote expansion of specific CAR T/NK cells, and promote infiltrate of innate immune cells to the target sites resulting in greater destruction.

Objects to be solved by the invention relevant to IL-15/IL-15sush complex

IL- 15 is a pleiotropic cytokine that is associated with a huge range of immunology and plays an important role in both adaptive and innate immunity.

A 65 amino acid sequence of the extracellular portion of IL-15Ra, called sushi domain, is involved in the binding of IL- 15.

According to the prior art,, it is known that IL- 15 has a short biological half-life. The instant inventors have discovered that when the sushi domain (IL-15Ra) is incorporated it results in an increased IL-15 half-life up to ten-fold by forming an IL-15/IL-15sushi complex, leading to longer persistency.

According to the prior art, constitutive expression of a high level of IL- 15 in mice could cause leukemia (Fehniger et al, J Exp Med. 2001 Jan 15; 193(2):219-31). Therefore, this leukemia matter with IL- 15 teaches away from generating a more powerful version of IL- 15 with a longer biological half-life and longer persistency to arm a CAR.

The herein inventors surprisingly discovered that only picogram quantities of IL-15/IL- 15sushi (Figure 31 and 34) in patients were produced by infused CAR T cells co-expressing IL- 15/IL- 15sushi and there was no evidence of abnormal T cell growth detected. In addition, a clinical trial with CD19 CAR co-expressing IL-15/IL-15sush (CD19 IL- 15/IL- 15 sushi) has been undertaken to treat patients with B-ALL (B cell acute lymphoblastic leukemia). CD19 IL-15/IL- 15sushi exhibited an expectedly superior result with a higher rate of complete remission (CR) and there was no evidence of autonomous growth or leukemic transformation in a human clinical trial after an over 2.5-year observation (see Table 2 below).

B-ALL: B-cell acute lymphoblastic leukemia; NK: normal karyotype; NA: none available, LFS: leukemia free survive. None of Patients after treating with CD19 CAR co-expressing IL15/I L15sushi developed abnormal T cell growth detected.

Table 2

SUBSTITUTE SHEET (RULE 26) In accordance with the present disclosure, the inventors have also found that immune cells transduced with secreted IL-15/IL-15sushi are superior in persistency and immunityinducing effect as compared with conventional immune cells in vivo (Figures 28 and 29).

In some embodiments, the present disclosure provides an engineered polypeptide including a chimeric antigen receptor and an immunomodulator(s). In a further embodiment, an immunomodulator can be selected from at least one of the group including, but not limited, IL-2, IL-7, IL-12, IL-15, IL-15/IL-15sush, IL-15/IL-15sushi anchor, IL-15/IL-15RA, IL-18, IL-21, IL- 21 anchor, PD-1, PD-L1, CSF1R, CTAL-4, TIM-3, cytoplasmic cytoplasmic domain of IL-15 receptor alpha, 4-1BBL, IL-21, IL-21 anchor and TGFR beta, receptors.

Combination therapy

The compositions and methods of this disclosure can be used to generate a population of CAR T lymphocyte or NK cells that deliver both primary and co- stimulatory signals for use in immunotherapy in the treatment of diseases, such as for example, cancer. In further embodiments, the present invention for clinical aspects are combined with other agents effective in the treatment of hyperproliferative diseases, such as anti-cancer agents. Anti-cancer agents are capable of reduction of tumor burdens in a subject. Anti-cancer agents include chemotherapy, radiotherapy and immunotherapy.

More than 50 % of persons with cancer will undergo surgery of some type. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Engineered cells described above can be used in conjunction with other treatments in patient in need thereof.

In another embodiment, the present disclosure provides a method of treating an autoimmune disease, said method includes administering an engineered cell according to claim 1 to a patient in need thereof; wherein said autoimmune disease comprises systemic lupus erythematosus (SLE), multiple sclerosis (MS), Inflammatory bowel disease (IBD), Rheumatoid arthritis, Sjogren syndrome, dermatomyosities, autoimmune hemolytic anemia, Neuromyelitis optica (NMO), NMO Spectrum Disorder (NMOSD), idiopathic thrombocytopenic purpura (ITP), antineutorphil cytoplasmic autoantibodies (ANCAs) associated with systemic autoimmune small vessel vasculitis syndromes or microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA, Wegener’s granulomatosis), or eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss syndrome) and TTP (thrombotic thrombocytopenic purpura).

The compositions and methods described in the present disclosure may be utilized in conjunction with other types of therapy for cancer, such as chemotherapy, surgery, radiation, gene therapy, and so forth.

In accordance with the present disclosure, natural killer (NK) cells represent alternative cytotoxic effectors for CAR driven killing. Unlike T-cells, NK cells do not need pre-activation and constitutively exhibit cytolytic functions. Further expression of CARs in NK cells allow NK cells to effectively kill cancers, particularly cancer cells that are resistant to NK cell treatment.

Further, NK cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD).

The present disclosure may be better understood with reference to the examples, set forth below. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure

Administration of any of the engineered cells described herein may be supplemented with the co-administration of a CAR enhancing agent. Examples of CAR enhancing agents include immunomodulatory drugs that enhance CAR activities, such as, but not limited to agents that target immune-checkpoint pathways, inhibitors of colony stimulating factor- 1 receptor (CSF1R) for better therapeutic outcomes. Agents that target immune-checkpoint pathways include small molecules, proteins, or antibodies that bind inhibitory immune receptors CTLA-4, PD-1, and PD- Ll, and result in CTLA-4 and PD-1/PD-L1 blockades. As used herein, enhancing agent includes enhancer as described above.

As used herein, “patient” includes mammals. The mammal referred to herein can be any mammal. As used herein, the term “mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. The mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs). The mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). The mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). Preferably, the mammal is a human. A patient includes subject.

In certain embodiments, the patient is a human 0 to 6 months old, 6 to 12 months old, 1 to 5 years old, 5 to 10 years old, 5 to 12 years old, 10 to 15 years old, 15 to 20 years old, 13 to 19 years old, 20 to 25 years old, 25 to 30 years old, 20 to 65 years old, 30 to 35 years old, 35 to 40 years old, 40 to 45 years old, 45 to 50 years old, 50 to 55 years old, 55 to 60 years old, 60 to 65 years old, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 years old, 85 to 90 years old, 90 to 95 years old or 95 to 100 years old.

The terms “effective amount” and “therapeutically effective amount” of an engineered cell as used herein mean a sufficient amount of the engineered cell to provide the desired therapeutic or physiological or effect or outcome. Such, an effect or outcome includes reduction or amelioration of the symptoms of cellular disease. Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what an appropriate “effective amount” is. The exact amount required will vary from patient to patient, depending on the species, age and general condition of the patient, mode of administration and the like. Thus, it may not be possible to specify an exact “effective amount”. However, an appropriate “effective amount” in any individual case may be determined by one of ordinary skill in the art using only routine experimentation. Generally, the engineered cell or engineered cells is/are given in an amount and under conditions sufficient to reduce proliferation of target cells.

Following administration of the delivery system for treating, inhibiting, or preventing a cancer, the efficacy of the therapeutic engineered cell can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the art will understand that a therapeutic engineered cell delivered in conjunction with the chemo-adjuvant is efficacious in treating or inhibiting a cancer in a patient by observing that the therapeutic engineered cell reduces the cancer cell load or prevents a further increase in cancer cell load. Cancer cell loads can be measured by methods that are known in the art, for example, using polymerase chain reaction assays to detect the presence of certain cancer cell nucleic acids or identification of certain cancer cell markers in the blood using, for example, an antibody assay to detect the presence of the markers in a sample (e.g., but not limited to, blood) from a subject or patient, or by measuring the level of circulating cancer cell antibody levels in the patient.

Throughout this specification, quantities are defined by ranges, and by lower and upper boundaries of ranges. Each lower boundary can be combined with each upper boundary to define a range. The lower and upper boundaries should each be taken as a separate element.

Reference throughout this specification to “one embodiment,” “an embodiment,” “one example,” or “an example” means that a particular feature, structure or characteristic described in connection with the embodiment or example is included in at least one embodiment of the present embodiments. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” “one example,” or “an example” in various places throughout this specification are not necessarily all referring to the same embodiment or example. Furthermore, the particular features, structures or characteristics may be combined in any suitable combinations and/or subcombinations in one or more embodiments or examples. In addition, it is appreciated that the figures provided herewith are for explanation purposes to persons ordinarily skilled in the art and that the drawings are not necessarily drawn to scale.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, article, or apparatus.

Further, unless expressly stated to the contrary, “or” refers to an inclusive “or” and not to an exclusive “or”. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

Additionally, any examples or illustrations given herein are not to be regarded in any way as restrictions on, limits to, or express definitions of any term or terms with which they are utilized. Instead, these examples or illustrations are to be regarded as being described with respect to one particular embodiment and as being illustrative only. Those of ordinary skill in the art will appreciate that any term or terms with which these examples or illustrations are utilized will encompass other embodiments which may or may not be given therewith or elsewhere in the specification and all such embodiments are intended to be included within the scope of that term or terms. Language designating such nonlimiting examples and illustrations includes, but is not limited to: “for example,” “for instance,” “e.g.,” and “in one embodiment.”

Within a group of parameters, each member may be combined with any one or more of the other members to make additional sub-groups. For example, if the members of a group are a, b, c, d, and e, additional sub-groups specifically contemplated include any one, two, three, or four of the members, e.g., a and c; a, d, and e; b, c, d, and e; etc.

As used herein, a XXXX antigen recognition domain is a polypeptide that is selective for XXXX. “XXXX” denotes the target as discussed herein and above. For example, a CD5 antigen recognition domain is a polypeptide that is specific for CD5.

As used herein, CDXCAR refers to a chimeric antigen receptor having a CDX antigen recognition domain.

EXAMPLES

Targeting of human T cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells

Blood donors, primary tumor cells and cell lines

Human lymphoma cells and peripheral blood mononuclear cells were obtained from residual samples. Umbilical cord blood cells were obtained from donors at Stony Brook University Hospital. SP53 and KARPAS 299 lymphoma cell lines were obtained from ATCC (Manassas, VA).

Lentivirus production and transduction of T cells

To produce viral supernatant, 293FT cells were co-transfected with pMD2G and pSPAX viral packaging plasmids, and with either pRSC.CD4.3G or GFP Lentiviral vector, using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) per manufacturer’s protocol. Prior to lentiviral transduction, umbilical cord or peripheral blood mononuclear buffy coat cells were activated for two days in the presence of 300 lU/mL IL-2 and 1 pg/mL anti-human CD3 (Miltenyi Bio tec, Germany).

T cell expansion CAR-transduced T cells were expanded for 7 days in T cell media (50% AIM-V, 40% RPMI 1640, 10% FBS and lx penicillin/streptomycin; all Gibco) supplemented with IL-2. Cells were counted every day and media was added every 2-3 days in order to maintain T cell counts below 2 x 10⁶cells/mL.

CAR immunophenotype

For the analysis of CAR cell immunophenotype, following 7 days of expansion, CD4CAR T cells and GFP control cells were stained with CD45RO, CD45RA, CD62L and CD8 (all from BD Biosciences) for flow cytometry analysis.

Co-Culture target cell ablation assays

CD4CAR T cells or GFP T cells (control) were incubated with target cells at ratios of 2:1, 5:1 and 10:1 (200,000, 500,000 or 1 million effector cells to 100,000 target cells, respectively) in 1 mL T cell culture media, without IL-2 for 24h. Target cells were KARPAS 299 cells (anaplastic large T cell lymphoma expressing CD4), leukemia cells from a patient with CD4+ T cell leukemia - Sezary syndrome - and from a patient with CD4+ PTCL lymphoma. As a negative control, CD4CAR T cells and GFP T cells were also incubated with SP53 (mantle cell lymphoma) cells, which do not express CD4, in the same ratios in 1 mL separate reactions. After 24 hours of co-culture, cells were stained with mouse anti-human CD8 and CD4 antibodies. In the experiments with SP53 cells, SP53 cells were labeled with CMTMR (Life Technologies) prior to co-culture with T cells, and T cells were labeled with mouse anti-human CD3 (PerCp) after co-culture incubation.

In vivo mouse xenogenic model

NSG mice (NOD. Cg-Prkdc^scld Il2rg^tmlWji 7SzJ) from the Jackson Laboratory were used under a Stony Brook University lACUC-approved protocol. Mice were all male and between 8 and 12 weeks old. Three sets of in vivo experiments were performed with no blinding. For each set, 10 mice were irradiated with a sub lethal (2.5 Gy) dose of gamma irradiation and assigned randomly to the treatment or control group. 24h later, mice were given one intradermal injection of 0.5 xlO⁶ or 1.0 xlO⁶ KARPAS 299 cells in order to form a measurable subcutaneous tumor within 7 days. Tumor size area was measured every other day. In the first set, three days after the injection of 1 million KARPAS 299 cells, 2 million CD4CAR T (5 mice) or 2 million GFP T control cells (5 mice) were administered to the mice intravenously (by tail vein injection). A second dose of 8 million cells was injected intravenously on Day 22. In the second set, 10 NSG mice was irradiated and injected with 0.5 x 10⁶ KARPAS 299 cells. On day 2, mice were injected intravenously with one course of 8 million CD4CAR T cells (5 mice) and 8 million GFP T control cells (5 mice). A second dose of 5.5 million cells was injected intravenously on Day 10. In the third set, 10 NSG mice were irradiated and injected with 0.5 xlO⁶ KARPAS 299 cells. On day 1, mice were intravenously injected with 2.5 xlO⁶CD4CAR T cells or with GFP T control cells (5 mice per group). Intravenous injections were repeated every 5 days for a total of four courses.

Generation of the third generation of CD4CAR

The scFv (single-chain variable fragment) nucleotide sequence of the anti-CD4 molecule was derived from humanized monoclonal ibalizumab (also known as Hu5A8 or TNX-355). This monoclonal antibody has been used in a variety of Phase I or II clinical trials. To improve signal transduction through the CD4CAR, the intracellular domains of CD28 and 4- IBB co- stimulators were fused to the CD3 zeta signaling domain. Additionally, the leader sequence of CD8 was introduced for efficient expression of the CD4CAR molecule on the cell surface. Indeed, the anti-CD4 scFv is linked to the intracellular signaling domains by a CD8-derived hinge (H) and transmembrane (TM) regions (Figure 1A and C). The CD4CAR DNA molecule was sub-cloned into a lentiviral plasmid. Because of the presence of two co-stimulatory domains (CD28 and 4- 1BB), CD4CAR is considered to be a third generation CAR. CD4CAR expression is controlled under a strong SFFV (spleen focus-forming virus) promoter and is well suited for hematological applications.

Characterization of CD4CAR

In order to verify the CD4CAR construct, transfected 293-FT cells were subjected to Western blot analysis. Immunoblotting with an anti-CD3zeta monoclonal antibody showed bands of predicted size for the CD4CAR CD3zeta fusion protein (Figure IB). As expected, no CD3zeta expression was observed for the GFP control vector (Figure IB).

CD4CAR T cells highly enriched for CD8+ T cells were generated. The cells were then tested in vitro for anti-leukemic functions using the KARPAS 299 cell line. The KARPAS 299 cell line was initially established from the peripheral blood of a patient with anaplastic large T cell lymphoma expressing CD4. Cytogenetic analysis has previously shown that KARPAS 299 cells have many cytogenetic abnormalities. During co-culture experiments, CD4CAR cells exhibited profound leukemic cell killing abilities (Figure 2A).

Studies were also conducted using patient samples. Patient 1 presented with an aggressive form of CD4+ T cell leukemia, Sezary syndrome, which did not respond to standard chemotherapy. Patient 2 presented with an unspecified CD4+ PTCL lymphoma. Flow cytometry analysis of both patient samples revealed strong and uniform CD4 expression, with almost all leukemic cells expressing CD4 (Figure 2B and 2C).

As visualized by flow cytometry analysis, co-culture of patient samples with CD4CAR for 24 hours resulted in rapid and definitive ablation of CD4+ malignancies, with, once again, approximately 98% ablation observed for both Sezary syndrome and PTCL co-cultures, consistent with the ablation of KARPAS previously shown (Figure 2B and 2C). Therefore, we show that, in a co-culture assay, CD4CAR T cells efficiently eliminate two different types of aggressive CD4+ lymphoma/leukemia cells directly from patient samples even at the low E:T ratio of 2:1 (Figure 2B and 2C). These data support that CD4 is a promising therapeutic target for CD4 positive T-cell leukemias and lymphomas, analogous to the role of CD19 in the targeting of B-cell malignancies via anti-CD19 CAR. Therefore, our patient sample and CD4CAR co-culture assay extends the notion of using CAR to target CD4 positive malignancies.

CD4CAR T cells exhibit significant anti-tumor activity in vivo.

In order to evaluate in vivo anti-tumor activities, we developed a xenogeneic mouse model using the KARPAS 299 cell line. Multiple different settings were used to test CD4CAR T cell efficacy in vivo. We first tested ability of the CD4CAR T cells to delay the appearance of leukemia in the NSG mice with a single low dose. Prior to the injection, modified T cells displayed ~40 to 50% of cells expressing CD4CAR as demonstrated by flow cytometry analysis. Mice received intradermal injections of KARPAS 299 cells and then a low dose (2 million) of single systemic injection (intravenous administration) of CD4CAR T cells was given. A single low dose of systemic CD4CART cells administration to leukemia-bearing mice caused only transient regression or delayed the appearance of leukemic mass (Figure 3A). When leukemia growth started to accelerate, an additional course of administration of 8 xlO⁶CD4CAR T cells remarkably arrested the leukemic growth (Figure 3A). To further test the efficacy of CD4CAR anti-leukemia activity, we administered two courses of relatively large doses of CD4CAR T cells. Similarly, two injections totaling 13.5 xlO⁶ CD4CAR T cells caused more pronounced leukemia growth arrest as compared to a lower CD4CAR dose but eventually the leukemic cell population recovered (Figure 3B). Finally, we investigated the efficacy of multiple course injections of a low dose of CD4CAR T cells (each 2.5 x 10⁶ cells). We treated the mice bearing subcutaneous leukemia with repeat intravenous injections of CD4CAR T cells, once every 4 or 5 days for total of 4 injections. After four courses of CD4CAR T cell administration, one of four treated mice was tumor free and exhibited no toxic appearance. Multiple dose CD4CAR T cell-treated mice displayed more significant antileukemic effect compared to single dose (Figure 3C and 3A). Moreover, treatment with CD4CAR T cells significantly prolonged the survival of mice bearing KARPAS 299 lymphoma as compared to treatment with the GFP-transduced control T cells (Figure 3D).

CD4CAR NK cells exhibit significant anti-tumor activity in vivo

Generation of CD4CAR NK92 cells (CD4CAR NK cells)

CD4CAR NK transduction efficiency was determined to be 15.9%, as determined by flow cytometry. Next, fluorescence-activated cell sorting (FACS) was used in order to further enrich for CD4CAR⁺ NK cells. Following sorting, collected CD4CAR^hlgh NK cells were confirmed to be more than 85% CD4CAR positive. After FACS collection of CD4CAR^hlgh cells, CD4CAR expression levels remained consistently stable at 75-90% on NK cells during expansion of up to 10 passages and following cryopreservation. Indeed, at the onset of co-culture experiments, expanded CD4CAR^hlgh NK cells expressed CAR at 85% .

In order to evaluate the in vivo anti-tumor activity of CD4CAR NK cells, we developed a xenogeneic mouse model using NSG mice sub lethally irradiated and intradermally injected with luciferase-expressing Karpas 299 cells to induce measurable tumor formation. On day 1, 24 hours following Karpas 299 cell injection, and every 5 days afterwards for a total of 6 courses, mice were intravenously injected with 5 x 10⁶ CD4CAR NK cells or vector control NK control cells per administration. On days 7, 14, and 21, mice were injected subcutaneously with RediJect D-Luciferin and underwent IVIS imaging to measure tumor burden (Figure 4A). Average light intensity measured for the CD4CAR NK injected mice was compared to that of vector control NK injected mice (Figure 4B). By Day 21, the CD4CAR NK injected mice had significantly less light intensity and therefore thus less tumor burden compared to vector control (p <0.01). On day 1, and every other day afterwards, tumor size area was measured and the average tumor size between the two groups was compared (Figure 4C). Unpaired student T test analysis revealed that the average tumor size of CD4CAR NK injected mice was significantly smaller than that of vector control NK injected mice starting on day 17 (p <0.05) and continuing on days 19-25 (p <0.01). Next, we compared mouse survival across the two groups (Figure 4D). All of the CD4CAR NK injected mice survived past day 30. However, percent survival of vector control NK injected mice started to decrease on day 17 with no survival by day 23. In summary, these in vivo data indicate that CD4CAR NK cells significantly reduce tumor burden and prolong survival in Karpas 299-injected NSG mice.

Generation of the third generation of CD5CAR

The construct for CD5CAR, as well as anchored CD5 scFv antibody were designed to test the function and mechanism of CD5CAR T cells in terms of both the targeting and lysis of CD5 expressing cells and the ability of CD5CAR T cells to down-regulate CD5 expression within their own CD5CAR T-cell population (Figure 5A). To confirm the CD5CAR construct, the generated CD5CAR lentiviruses were transduced into HEK293 cells. After 48h treatment with CD5CAR or GFP-lentiviruses, the expression of CD5CAR in HEK293 cells was verified by Western blot analysis using CD3zeta antibody, which recognize C-terminal region of CD5CAR protein (Figure. 5B). The resulting band was the predicted size of CD5CAR protein in CD5CAR transduced HEK293 cells, but GFP transduced HEK293 cells did not exhibit any specific band by Western blot analysis. In order to evaluate the function of CD5CAR protein for future experiments, CD5CAR lentiviruses were transduced into activated human T cells. The expression of CD5CAR on surface of T cells was evaluated by flow cytometry analysis using goat anti-mouse F(ab’) antibody, which recognizes scFv region of CD5CAR protein. Flow cytometric analysis showed that about 20% of CD5CAR expression was observed on CD5CAR transduced T-cells compared to isotype control (Figure 5C). These results indicated that we successfully generated CD5CAR expression T cell for following experiments. Down-regulation of CD5 expression for CAR therapy

Prior to CD5CAR T cell co-culture and animal assays, the expression of CD5 on the surface of CD5CAR T cells is down regulated to avoid self-killing within the CD5CAR T population. The down-regulation of CD5 will prevent the self-killing of CAR T cells within the CAR T cell population, and the down-regulation of CD5 is associated with an increased killing ability of T-cells. A CAR that is produced within T-cells that has no CD5 expression could be a super-functional CAR, no matter the construct of the CAR itself. The steps for generation of CD5 CAR T cells and the comparison of CD5 down-regulation using single or double transduction of CD5 CAR lentiviuses are shown in Figure 6A and B. The single transduced CD5CAR T cells with unconcentrated lent-CD5 CAR viruses did not show complete downregulation of CD5 protein from cell surface by day 8, with a maximum CD5 negative population up to 46% on day 6 (Figure 7). In the double transduced population, about 90% of transduced T cells became CD5 negative on day 4-day incubation. In contrast, the GFP T-cell control maintains a CD5+, CD3+ double positive population above 95% from day 2 through day 8 (Figure 7).

CD5CAR T cells exhibit profound anti-tumor activity in vivo.

In order to evaluate the in vivo anti-tumor activity of CD5CAR T cells as a predictor of their therapeutic efficacy in patients, we developed a xenograft mouse model using NSG mice sub lethally (2.0 Gy) irradiated and intravenously injected with 1.0 x 10⁶ firefly luciferaseexpressing CCRF-CEM cells (CD5+) to induce measurable tumor formation. On day 3 days following CCRF-CEM-Luc+ cell injection, mice were intravenously injected with 5 x 10⁶ CD5CAR T cells or vector control T cells. These injections were repeated on Day 4, Day 6, and Day 7, for a total of 20 x 10⁶ T cells per mouse. On days 5, 8, 10 and 13, mice were injected subcutaneously with RediJect D-Luciferin (Perkin-Elmer) and subjected to IVIS imaging (Caliper LifeSciences) to measure tumor burden (Figure 8A). Average light intensity measured for the CD5CAR T cell injected mice was compared to that of vector control T injected mice (Figure 8B). Paired T test analysis revealed a very highly significant difference between the two groups by day 13 with less light intensity and thus less tumor burden in the CD5CAR T injected group compared to control (p <0.0012). Anti-CD5 Chimeric Antigen Receptor (CD5CAR) NK cells efficiently eliminate CD5 positive Hematologic Malignancies.

CD5CAR NK cells effectively eliminate human T-cell acute lymphomblastic leukemia (T-ALL) cell lines

CD5CAR NK cells were tested for anti-T-ALL activity in vitro using CCRF-CEM, MOLT-4 and Jurkat cell lines. All these T-ALL cell lines highly expressed CD5.

During co-culture experiments, CD5CAR NK cells demonstrated profound killing of CCRF-CEM at the low effector cell to target cell ratio (E:T) of 2:1 and 5:1. At these ratios, CD5CAR NK cells virtually eliminated CCRF-CEM cells . CD5CAR NK cells lysed CCRF- CEM leukemic cells in vitro in a dose-dependent manner at effector: target ratios of 0.25:1, 0.5:1, 1:1, 2:1 and 5:1 (Figure 9).

CD5CAR NK cells demonstrate a potent anti-leukemic activity in vivo.

A similar strategy for CD5CAR T cells, animal studies were employed to determine the in vivo anti-tumor activity of CD5CAR NK cells. Sub-lethally irradiated NSG mice were intravenously injected with 1.0 x 10⁶ firefly luciferase-expressing CCRF-CEM cells to induce measurable tumor formation. 3 days following CCRF-CEM-Luc+ cell injection, mice were intravenously injected with 5 x 10⁶ CD5CAR NK cells or vector control T cells. These injections were repeated on Day 4 for a total of 10 x 10⁶ T cells per mouse. On day 5, mice were injected subcutaneously with RediJect D-Luciferin and subjected to IVIS imaging to measure tumor burden. Average light intensity measured for the CD5CAR NK cell injected mice was compared to that of vector control NK cell injected mice (Figure. 10). Tumor burden was two thirds lower for treated mice on day 5 after tumor injection. Paired T test analysis revealed a very highly significant difference (P=0.0302) between the two groups. These in vivo data indicate that CD5CAR NK cells significantly reduce tumor burden in CCRF-CEM-injected NSG mice in a rapid manner when compared to vector control NK cells.

Anti-CD3 Chimeric Antigen Receptor (CD3CAR) NK cells efficiently lyse CD3 positive Hematologic Malignancies Generation of the CD3CAR

The anti-CD3 molecule is a modular design, comprising of a single-chain variable fragment (scFv) in conjunction with CD28 and 4- IBB domains fused to the CD3zeta signaling domain to improve signal transduction making it a third generation CAR. A strong spleen focus forming virus promoter (SFFV) was used for efficient expression of the CD3CAR molecule on the NK cell (NK-92) surface and the CD8 leader sequence was incorporated into the construct. The anti-CD3 scFv is attached to the intracellular signaling domains via a CD8-derived hinge (H) and transmembrane (TM) regions (Figure. 11 A). This CD3CAR construct was then cloned into a lentiviral plasmid.

Characterization of CD3CAR

Western blot analysis was performed on HEK293-FT cells transfected with CD3CAR lentiviral plasmid and vector control plasmid. Immunoblots with anti-CD3zeta monoclonal antibody show bands of predicted size for the CD3CAR-CD3zeta fusion protein (Figure. 1 IB) versus no bands for the vector control protein.

Generation of CD3CAR NK cells using NK-92 cells

The transduction efficiency of the CD3CAR was determined by flow cytometry analysis. To enrich for CD3CAR NK cells, the highest expressing NK cells were harvested using fluorescence-activated cell sorting (FACS). Following sorting, NK cells with relatively high expression of CD3CAR was obtained. Expression of CD3CAR following flow cytometry sorting was stable around 30% of CAR expression for subsequent NK cell expansion and cryopreservation.

CD3CAR NK cells exhibit profound anti-leukemic activity in vivo

To determine the in vivo anti-tumor efficacy of CD3CAR NK cells, sub lethally irradiated NSG mice were intravenously injected with 1.0 x 10⁶ firefly luciferase-expressing Jurkat cells, which are CD3 positive (-80%), and measurable tumor formation was detected by Day 3 or 4. Three days following Jurkat-Luc+ cell injection, mice were intravenously injected with 5 x 10⁶ CD3CAR NK cells or vector control NK cells per mouse, 6 per group. These injections were repeated on Day 3, 6, 7 and 10 for a total of 25 x 10⁶ T cells per mouse. On days 4, 7, 9 and 13 mice were subjected to IVIS imaging to measure tumor burden (Figure. 12A). Two treated mice died due to injection procedure on day 13. Average light intensity measured for the CD3CAR NK cell injected mice was compared to that of vector control NK injected mice (Figure. 12B). After an initial lag period, tumor burden then dropped to approximately two-thirds lower for treated mice by Day 9 and just 13% on Day 13. Paired T test analysis revealed a highly significant difference (P=0.0137) between the two groups. We conclude that these in vivo data demonstrate that CD3CAR NK cells significantly reduce tumor burden and prolong survival in Jurkat-injected NSG mice when compared to vector control NK cells.

CRISPR/Cas nucleases target to CD2, CD3, CD5 and CD7 expressed on T or NK cells.

T or NK cells appear to share some of surface antigens, such as CD2, CD3, CD5 and CD7 with leukemia or lymphoma. CD2, CD3, CD5, and CD7 could be good targets for T and NK cells as they are expressed in most of T cell leukemia/lymphoma.

Therefore, when one of surface antigens, CD2, CD3, CD5, and CD7 is selected as a target, this antigen is needed to delete or down-regulate in T or NK cells used to generate CAR if they share this antigen, to avoid self-killing within the CAR T or NK cell population.

Steps for generation of CAR T or NK cell targeting T-cell lymphomas or T-cell leukemia are described in Figure 14. Three pairs of sgRNA were designed with CHOPCHOP to target CD2, CD3, CD5, and CD7. Gene-specific sgRNAs (Figure. 15) were then cloned into the lentiviral vector (Lenti U6-sgRNA-SFFV-Cas9-puro-wpre) expressing a human Cas9 and puromycin resistance genes linked with an E2A self-cleaving linker. The U6-sgRNA cassette is in front of the Cas9 element. The expression of sgRNA and Cas9puro is driven by the U6 promoter and SFFV promoter, respectively.

CRISPR/Cas nucleases target to CD5 on T cell lines.

Lentiviruses carried gene-specific sgRNAs were used to transduce CCRF-CEM and MOLT cells. Initially, the loss of CD5 expression was observed in both of these T cell lines using two different two CDISPR/Cas9 sgRNA sequences (Figure.16A and 16C). The most successful population in terms of the loss of CD5 expression was chosen for each cell line, and these cells were sorted, expanded normally and found to be of >99% purity CD45+ and CD5- (Figure. 16B and 16D). CRISPR/Cas nucleases target to CD7 on T cell lines and NK cells.

Lentiviruses carried gene-specific sgRNAs were used to transduce CCRF-CEM, MOLT cells and NK cells (Figure. 17). Flow cytometry analysis demonstrated the loss of CD7 expression in CCRF-CEM and NK-92 cells with CRISPR/Cas9 approach using two different sgRNAs (Figure. 17A and 17B). The population (denoted by the blue circle and arrow) was selected for sorting, expansion and analysis in figure 17B. The loss of CD5 expression by flow cytometry analysis was also seen in NK-92 cells using a similar approach described above with CRISPR/Cas nucleases targeting to CD7 (Figure. 17C and 17D) The sorted CD7 negative NK-92 cells (Figure. 17D) were expanded and used to generate CD7CAR NK cells to eliminate CD7 positive leukemic cells.

CD7CAR NK ⁷‘ -92 cells have a robust anti-leukemic activity

CD7 is expressed in both NK and T-ALL leukemic cells. To avoid self-killing within the CD7CAR NK-92 population, CD7 expression first needs to be inactivated. CD7 deficient NK- 92 cells (NK^7- -92 cells) were generated as described in (Figure. 7D) and expanded. The expanded NK ⁷“ -92 cells were transduced with lentivirus expressing a CD7CAR. CD7CAR includes an anti-CD7 scFV in conjunction with CD28 and 4-BB domains fused to CD3zeta signaling domain making it a third generation CAR. CD7CAR NK ⁷“ -92 cells were used to test their lysis ability of leukemic cells expressing CD7. As shown in Figure. 18, CD7CAR NK ⁷“ -92 cells displayed a potent anti-leukemic activity against a T-ALL cell line, CCRF-CEM. As analyzed by flow cytometry, co-culture of CCRF-CEM cells effectively resulted in the lysis of approximately 50% of leukemic cells at E:T ratio of 5:1 (Figure. 18A andl8B).

CD3 multimeric protein complex is elucidated in Figure. 19. The complex includes a CD35 chain, a CD3y chain, and two CD3s chains. These chains associate with the T-cell receptor (TCR) composing of a0 chains.

CD3CAR is used for graft-versus-host disease (GvHD).

CD3CAR is administered to a patient prior to or after a stem cell transplant. The patient is tested for elevated levels of white blood cells. CD3CAR is administered to a patient prior to or after a bone marrow transplant. The patient is tested for elevated levels of white blood cells.

CD3CAR is administered to a patient prior to or after a tissue graft. The patient is tested for elevated levels of white blood cells.

Organ transplant

CD3CAR is administered to an organ transplant patient before organ transplant surgery. The patient is tested for organ rejection. The following histological signs are determined: (1) infiltrating T cells, in some cases accompanied by infiltrating eosinophils, plasma cells, and neutrophils, particularly in telltale ratios, (2) structural compromise of tissue anatomy, varying by tissue type transplanted, and (3) injury to blood vessels.

CD3CAR is administered to an organ transplant patient after organ transplant surgery. The patient is tested for organ rejection. The following histological signs are determined: (1) infiltrating T cells, in some cases accompanied by infiltrating eosinophils, plasma cells, and neutrophils, particularly in telltale ratios, (2) structural compromise of tissue anatomy, varying by tissue type transplanted, and (3) injury to blood vessels.

CD2 CAR NK cells

As a proof of concept, we next investigated that CD2CAR in NK-92 cells response to the CD2 antigen in leukemic cells as NK-92 cells only bear a low number of cells expressing CD2 antigen. The NK-92 cells were transduced with lentiviruses expressing CD2CAR and resulting CD2CAR NK-92 cells were used to test their anti-leukemic activity.

CD2CAR NK cells especially lyse CD2+ T-ALL (T-acute lymphoblastic leukemia) cells

To assess CD2CAR NK92 anti-leukemic activity, we conducted co-culture assays using a T-ALL cell line, CCRF-CEM and a T-ALL primary human patient sample. We demonstrated that CD2CAR NK-92 cells consistently displayed robust lysis of leukemic cells. Following 24- hour incubation at a low effective to target cell (E:T ratio 5:1), CD2CAR NK-92 cells Effectively lysed [M 1] leukemic cells.

One-step approach by introducing an expression cassette to generate anti-CD7-RTX-ER- CAR using non-gene editing A CAR, anti-CD7-RTX-ER (also called CD7-ER CAR or CD7-RTX-ER CAR) construct was designed to target the CD7 antigen. The expression cassette encodes anti-CD7 CAR and anti- CD7 scFv fused to an ER retention signal peptide, KDEL which can entrap the recognized protein, CD7 protein within the secretion pathway, which results in the prevention of its surface location in a cell (Figure 22A). Flow cytometry analysis of the expression of T-cells and U937 cells transduced with CD7-RTX-ER CAR encoding lentivirus is necessary to validate the expression of CAR molecules on the surface of a cell. Typically, in CAR applications, FAB fragment antibodies are used to detect the antibody expressing portions of the CAR on the T-cell and NK cell surface. In addition, CD7 expression in transduced T cells needs to be followed to determine if CD7 antigen expression can be shut down. Finally, the expansion of transduced T cells needs to be tracked to indicate the health of CD7- cells.

CAR T-cells and U937 cells were generated by transduction of primary peripheral blood T-cells and wild-type U937 cells with the lentiviral construct shown in Figure 23A. The translated CAR proteins were then expressed on the surface of the T-cell and U937 cells, where they can recognize and bind the target proteins on the surface of tumor cells. The pharmacologic effect and mechanism of the CARs are mediated by CD7 CAR recognition of the antigen, which triggers cytotoxic T-cell and NK cell activity, further enhanced by the incorporation of CD28 coactivation domains in the construct. In addition, normal CD7 surface antigen expression in T cells needs to be shut down.

Eentiviral CD7-RTX-ER CAR was used to transduce U937 cells and human peripheral blood T cells. Flow cytometry results showed that CD7 CAR was expressed on roughly 36% of U937 cells (Figure 23A) and 93% of T cells (Figure 23B). In addition, CD7+ expression was completely shut down in the CAR T cells. The experiment was repeated, with peripheral blood from two different donors, and transduced with CD7 CAR lentiviral vector with and without concentration. In each case, greater than 40% of the transduced cells expressed CAR, while CD7 expression was again eliminated.

CD7-RTX-ER CAR T cells expanded at roughly the same rate as non-transduced control cells following transduction and recovery (Figure 25 and 26)). A second experiment based on the cells from Figure 26 confirmed this, with expansion through Day 16. This indicates that the transduced cells have recovered from the shutting down of CD7 expression. Evaluation of the efficacy of CD7-RTX-ER CAR (CD7-ER)

We evaluated the efficacy of CD7-RTX-ER CAR T- cells in vitro. Co-culture killing assays, in which three T-ALL target tumor cell lines that express the CD7+ cell surface phenotype (CEM-CCRF, Jurkat, and MOLT4) are incubated with CD7-RTX-ER CAR T cells to determine the anti-tumor function of CAR T cells in vitro against bulk CD7+ disease.

To assay the ability of the CD7-RTX-ER CAR T cells to target CD7⁺ cells, 18 h cocultures with either control or CD7-RTX-ER CAR T cells versus three different CD7-expressing tumor cell lines were performed in an E:T ratio of 0.2:1, 0.5:1, and 1:1. After 18 hours coculture, CCRF-CEM target cells were ablated by CD7-RTX-ER CAR T cells at each ratio at 58, 88 and 95%, respectively (Figure 23C). Jurkat cells were ablated at 87, 88 and 95%, respectively at each of the three ratios (Figure 23D). M0ET4 cells, were lysed at 44, 45 and 65%, respectively at 18 hours (Figure 23D), but this increased to approximately 80-85% after 48 hours (data not shown). The results of these three experiments are summarized in Figure 24. These results confirm the ability of the CD7-RTX-ER CAR T cells to effectively target and lyse CD7⁺ cells.

These results show that CD7-RTX-7ER CAR T cells can ablate T-AEE tumor cells in a robust manner.

Cytokine, IL-15/IL-15sushi enhances CAR efficacy and persistency as well as modulates immune system

Generation of the third generation CD4-IL15/IL15sushi CAR

The CD4 CAR was then linked to the IE15/IE15sushi domain by a P2A self-cleaving sequence. The IE15/IE15sushi domain consists of an IE-2 signal peptide fused to IL-15, which is linked to the soluble, sushi domain of the IL- 15 a receptor via a 26-amino acid poly-proline linker (Figure 27). The construct was transduced into both T cells and NK cells.

CD4-IL15/IL15sushi CAR T cells exhibit significant anti-tumor activity in vivo

To evaluate the in vivo anti-tumor activity of the CD4-IL15/IL15sushi CAR T cells, we developed a xenogeneic mouse model using NSG mice sub-lethally irradiated and intravenously injected with luciferase-expressing M0LM13 cells to induce measurable tumor formation. Three days following tumor cell injection, 6 mice each were intravenously injected with 8xl0⁶ vector control, CD4 CAR, or CD4-IL15/IL15sushi CAR T cells. On days 3, 6, 9, and 11, mice were injected subcutaneously with RediJect D-luciferin (Perkin Elmer) and subjected to IVIS imaging to measure tumor burden (Figure 28 A). Average light intensity determined that CD4 CAR T cell-treated mice had a 52% lower tumor burden relative to control on Day 6, whereas CD4- IL15/IL15sushi CAR T cells had a 74% lower tumor burden (Figure 28B). On Day 11, nearly all the tumor cells had been lysed in both treatment groups. Unpaired t-test analysis revealed a very significant difference (P = 0.0045) between the control and two treatment groups by Day 9. Mouse survival was also significantly improved in CD4 CAR T cell-treated mice and CD4- IE15/IE15sushi T cell-treated mice compared to control (Figure 28C). Additionally, all mice injected with CD4-IE15/IE15sushi CAR T cells survived longer than the mice injected with CD4 CAR T cells (log-rank mantel-cox test p= 0.0087). These results demonstrate that both CD4 CAR and CD4-IE15/IE15sushi were able to significantly reduce tumor burden and improve survival compared to control-treated mice.

CD4-IL15/IL15sushi CAR NK92 cells demonstrate improved outcomes in “stressed” in vivo environment

To further compare the function of the CD4-IE15/IE15sushi CAR and CD4 CAR, we created a “stressful” condition by using CD4-IE15/IE15sushi CAR NK92 cells and Jurkat tumors. The NK92 cells bear a short half-life property, and the Jurkat cells showed less than 60% CD4⁺ phenotype as assayed by flow cytometry.

We used a xenogeneic mouse model using NSG mice sub-lethally irradiated and intravenously injected with luciferase-expressing Jurkat cells to induce measurable tumor formation. Three days after tumor injection, mice were intravenously injected with a course of 10xl0⁶ vector control NK92 cells, CD4 CAR NK92 cells, or CD4-IE15/IE15sushi CAR NK92 cells. Mice were subjected to IVIS imaging to measure tumor burden on days 3, 7, 10, and 14 (Figure 29 A). Measurement of average light intensity showed that both CD4 CAR NK92-treated and CD4-IE15/IE15sushi CAR NK92-treated mice showed significant tumor lysis by Day 7 (Figure 29B, C). However, tumor lysis remained relatively constant in the CD4 CAR group to Day 14, while the lysis in the CD4-IE15/IE15sushi CAR group increased to over 97%. Unpaired t-test analysis of the radiance indicated an extremely significant difference (P < 0.0001) between the CD4 CAR and CD4-IL15/IL15sushi CAR groups by Day 14. This suggests that while CAR T cells or NK cells are unable to eliminate dimly expressed or target-negative tumor cells, the secretion of IL15/IL15sushi may be able to supplement this defect through greater expansion of cell numbers.

Clinical trials Examples

CD4-IL15/IL15sushi CAR T cells were tested in three patients in a pilot clinical trial (NCT04162340). Patient 1 is a 54-year-old male with relapsed refractory stage IVB Sezary syndrome. He had been having symptoms of erythroderma, pruritus, and scaling of the skin for over 10 years. Patient 1 received a total dose of 2.8xl0⁶ autologous CD4-IL15/IL15sushi CAR T cells. While the patient had extensive skin lesions, covering >80% of total skin area prior to CD4-IL15/IL15sushi CAR T cell treatment (Figure 30A, C), the patient’s skin showed remarkable improvement 28 days post-therapy (Figure 30B, D). Skin biopsy before treatment showed extensive lymphocytic infiltration of CD4⁺ cells (Figure 30E, G), which was cleared in a repeat skin biopsy 28 days after infusion (Figure 30F, H). Molecular testing for T cell gene rearrangement, flow cytometry of peripheral blood and virtual absence of CD3⁺ and CD4⁺ cells on skin biopsy further confirm the complete molecular remission of this patient’s Sezary syndrome.

CD4-IL15/IL15sushi CAR T cells demonstrated potent targeted lysis of CD3 CD4⁺ Sezary leukemic T cells. While the percentage of Sezary leukemic cells detected in peripheral blood prior to CD4-IL15/IL15sushi CAR T cell therapy was about 50%, the leukemic cells were undetectable by Day 10 after treatment (Figure 301). The CD4-IL15/IL15sushi CAR T cell is also expected to lyse normal CD4⁺ helper T cells, which are critical to the expansion of other immune cells. Despite this CD4⁺ aplasia, CD3⁺CD8⁺ cells markedly expanded from about 18% to 70% of lymphocytes in the first week post-infusion (Figure 30J). NK expansion followed CD8⁺ T cell expansion and reached about 65% of lymphocytes on day 22 post- infusion (Figure 30K). This may be a consequence of the CD4-IL15/IL15sushi CAR T cells ablating the immunosuppressive CD4⁺ Treg cells in the first month following infusion (Figure 30L). Additionally, the inclusion of the secreting IL15/IL15sushi from CAR T cells may have also potentiated the proliferation of CD3⁺CD8⁺ and NK cells. Importantly, the CD4⁺ aplasia was not associated with the development of any infections, which may have been due to the expansion of the CD3⁺CD8⁺ and NK cell populations.

Long-term observation of the peripheral blood populations showed elevated percentage of CD3⁺CD8⁺ cells 1 year after treatment (Figure 30M). Within a month after treatment, both CD3⁺CD4⁺ and leukemic CD3 CD4⁺ declined to undetectable levels (Figure 4N). While the CD3 CD4⁺ T cells remained low 1 year after treatment, the CD3⁺CD4⁺ T cell levels began to rise 3 months after treatment (Figure 30N), indicating the remission of leukemic cells and the regeneration of normal CD4⁺ cells. While the NK cell levels had risen immediately after therapy, they began to decline to a normal level after 2 months (Figure 40). Additionally, B cells, which had been suppressed during the first few months of treatment, expanded after 3 months (Figure 4P). Taken together, these results demonstrate the ability of CD4-IL15/IL15sushi CAR T cells to transiently alter the immune milieu, resulting in the complete remission of Sezary syndrome in this patient. Patient 1 has remained in complete remission over 15 months. Interestingly, the level of IL- 15 was low and only picogram quantities (2-20pg/ml) was seen in three patients treated with the CAR co-expressing IL15/IL15sushi (Figure 31).

Patient 2 is a 45-year-old female diagnosed with relapsed /refractory severe mycosis fungoid lymphoma (stage IVb) presenting with numerous cutaneous lesions. Patient received a total dose of about 3.2xl0⁶/kg autologous CD4-IL15/IL15sushi CAR T cells. Imaging of the patient’s skin before infusion (Figure 32A), 2 weeks after infusion (Figure 32B), and 4 weeks after infusion (Figure 32C) revealed a significant improvement due to CD4-IL15/IL15sushi CAR T cell therapy. Skin biopsy before therapy revealed intensive CD4⁺ lymphoma cell infiltrates (Figure 32D, F), which showed remarkable improvement 28 days post-infusion (Figure 32E, G). These cutaneous improvements were associated with a rapid decline of CD3⁺CD4⁺ cells to undetectable levels by Day 24 post-therapy (Figure 32H). B cells also decreased to undetectable levels by Day 24 post-infusion (Figure 321), likely due to the loss of CD4⁺ helper T cells. On the other hand, this patient had a profound expansion of NK cells during the first month (Figures 32J). Similar to Patient 1, no significant opportunistic infections were observed in Patient 2 although anti-viral drug, valaciclovir was given for safety precautions. Patient 2 remains in remission over 5 months post-treatment. CD5 CAR T cells armed with IL-15/IL15sushi (CD5-RTX-IL15/IL15sushi)

A 22-year-old male patient with relapsed and refractory T-acute lymphoblastic lymphoma (T-LBL) and later developed to a leukemic phase, acute lymphoblastic leukemia (T- ALL) with invasion of the cerebrospinal fluid (CSF) with left eye involvement. After intensive chemotherapies, residual disease was observed only in the eye and CSF. Central nervous system (CNS) involvement with T-ALL in adult patients is associated with a very poor prognosis.

This patient was enrolled in a clinical trial of CD5-RTX-IL15/IL15sushi CAR. CD5- RTX-IL15/IL15sushi CAR contains a soluble IL15/IL15sushi complex that is linked to the CAR construct via P2A (Figure 33A). Additionally, the hinge region of CD5 CAR contains two rituximab-binding epitopes (Figure 33A). The incorporation of rituximab-binding epitopes in the hinge region can be used to depletion of CAR T cells in vivo.

The source of the T cells was from the same allogeneic-HSCT donor he received nine years earlier (the patient’s sister). The patient received a pretreatment with FC regimen (fludarabine 30 mg/m²dl-d3, cyclophosphamide 300 mg/m² dl-d3) before the initiation of CD5- RTX-H-15/IL-15sushi CAR T cell infusion. The patient received a total dose of 2.0xl0⁶/kg CAR T cells (6.3xl0⁷/m² CAR T cells) with split dose in two days.

CD5-RTX-I1-15/IL- 15 sushi CAR T cells exhibited robust targeted lysis of CD5+CD34+ T-ALL leukemic cells. While the percentage of leukemic cells detected in CSF prior to CAR infusion was about 81% (Figure 34E), the leukemic cells were undetectable 1-week post-CAR therapy (Figure 34F). This finding was also confirmed by morphology study (Figure 34A). Additionally, the levels of protein and pressure in the CSF also returned to a normal level (Figure 34C). The CD5-RTX-Il-15/IL-15sushi CAR T cells are expected to deplete normal T cells. Interestingly CD3+CD8+ cells still expanded from about 10% to 55% of lymphocytes in the peripheral blood. The common serum biomarkers including IL15, IL-6, Ferritin and CRP (C- reactive protein) were measured (Figure 34G, H, and J). Interestingly, the level of IL15/IL15sushi was low and only picogram quantities (10-50pg/ml (Figure 34J) were detected. Taken together, these results demonstrate the ability of CD5-RTX-Il-15/IL-15sushi CAR T cells to eliminate aggressive leukemic cells, resulting in the complete remission of T-ALL in this patient. Sequences Disclosed

SEQ ID NO: 1 (CD2b-RTX-ER CAR amino acid sequence)

MALPVTALLLPLALLLHAARPDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWY

QQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPL

TFGAGTKLELRRGGGGSGGGGSGGGGSQVQLQQPGTELVRPGSSVKLSCKASGYTFTS

YWVNWVKQRPDQGLEWIGRIDPYDSETHYNQKFTDKAISTIDTSSNTAYMQLSTLTSDA

SAYYCSRSPRDSSTNLADWGQGTLVTVSSACPYSNPSLCSGGGGSELPTQGTFSNVSTN

VSPAKPTTTACPYSNPSLCSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH

TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKH

YQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP

EMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD

TYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPDIVMTQSPATLSVTPGDRVSLSCR

ASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVY

YCQNGHSFPLTFGAGTKLELRRGGGGSGGGGSGGGGSQVQLQQPGTELVRPGSSVKLS

CKASGYTFTSYWVNWVKQRPDQGLEWIGRIDPYDSETHYNQKFTDKAISTIDTSSNTAY

MQLSTLTSDASAVYYCSRSPRDSSTNLADWGQGTLVTVSSGGGGSGGGGSGGGGSGGG

GSAEKDEL

SEQ ID NO: 2 (CD2b-RTX-ER CAR nucleotide sequence)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCC AGGCCG

GACATCGTGATGACCCAGAGCCCCGCCACCCTGAGCGTGACCCCCGGCGACAGGGT

GAGCCTGAGCTGCAGGGCCAGCCAGAGCATCAGCGACTACCTGCACTGGTACCAGC

AGAAGAGCCACGAGAGCCCCAGGCTGCTGATCAAGTACGCCAGCCAGAGCATCAGC

GGCATCCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCAGCGACTTCACCCTGAGCAT

CAACAGCGTGGAGCCCGAGGACGTGGGCGTGTACTACTGCCAGAACGGCCACAGCT

TCCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAGGAGGGGCGGCGGCGGC

AGCGGTGGTGGCGGTAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGCCCG

GCACCGAGCTGGTGAGGCCCGGCAGCAGCGTGAAGCTGAGCTGCAAGGCCAGCGGC

TACACCTTCACCAGCTACTGGGTGAACTGGGTGAAGCAGAGGCCCGACCAGGGCCT

GGAGTGGATCGGCAGGATCGACCCCTACGACAGCGAGACCCACTACAACCAGAAGT

TCACCGACAAGGCCATCAGCACCATCGACACCAGCAGCAACACCGCCTACATGCAG

CTGAGCACCCTGACCAGCGACGCCAGCGCCGTGTACTACTGCAGCAGGAGCCCCAG

GGACAGCAGCACCAACCTGGCCGACTGGGGCCAGGGCACCCTGGTGACCGTGAGCA

GCGCCTGCCCCTACAGCAACCCCAGCCTGTGCAGCGGCGGCGGCGGCAGCGAGCTG

CCCACCCAGGGCACCTTCAGCAACGTGAGCACCAACGTGAGCCCCGCCAAGCCCAC

CACCACCGCCTGTCCTTATTCCAATCCTTCCCTGTGTAGCGGCGGCGGCGGCAGCAC

CACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCC

TGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGG

GGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGG

GTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAGGAGTAAGAGGAGCAGGCTC

CTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCA TTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTT

CAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACG

AGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGG

GACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGT

ACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAA

AGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAG

CCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGAAGC

GGAGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGG

CCCCGATATTGTTATGACCCAAAGTCCGGCGACCCTGAGCGTGACCCCGGGCGATCG

CGTGAGCCTGAGCTGCCGCGCGAGCCAGAGCATTAGCGATTATCTGCATTGGTATCA

GCAGAAAAGCCATGAAAGCCCGCGCCTGCTGATTAAATATGCGAGCCAGAGCATTA

GCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCAGCGATTTTACCCTGAGC

ATTAACAGCGTGGAACCGGAAGATGTGGGCGTGTATTATTGCCAGAACGGCCATAG

CTTTCCGCTGACCTTTGGCGCGGGCACCAAACTGGAACTGCGCCGCGGTGGTGGTGG

TAGCGGCGGCGGCGGCAGCGGTGGTGGTGGTAGCCAGGTGCAGCTGCAGCAGCCGG

GCACCGAACTGGTGCGCCCGGGCAGCAGCGTGAAACTGAGCTGCAAAGCGAGCGGC

TATACCTTTACCAGCTATTGGGTGAACTGGGTGAAACAGCGCCCGGATCAGGGCCTG

GAATGGATTGGCCGCATTGATCCGTATGATAGCGAAACCCATTATAACCAGAAATTT

ACCGATAAAGCGATTAGCACCATTGATACCAGCAGCAACACCGCGTATATGCAACT

GAGTACCCTGACCAGTGATGCGAGCGCGGTGTATTATTGCAGCCGCAGCCCGCGCG

ATAGCAGCACCAACCTGGCGGATTGGGGTCAGGGTACCCTGGTTACCGTGAGTAGC

GGGGGGGGCGGCAGCGGGGGAGGCGGTTCCGGGGGTGGTGGTAGTGGTGGGGGTG

GCTCCGCCGAGAAGGACGAGCTGTAAGTTTAAAC

SEQ ID NO: 3 (CD2-RTX-ER CAR amino acid sequence)

MALPVTALLLPLALLLHAARPDIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQK

NYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCH Q

YLSSHTFGGGTKLEIKRGGGGSGGGGSGGGGSQLQQPGAELVRPGSSVKLSCKASGYTF

T

RYWIHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSGTAYMQLSSLTSE

D

SAVYYCATEDLYYAMEYWGQGTSVTVSSACPYSNPSLCSGGGGSELPTQGTFSNVSTN VS

PAKPTTTACPYSNPSLCSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT

RGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHY

Q

PYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE

MGG

KPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA

LHM

QALPPRGSGATNFSLLKQAGDVEENPGPDIMMTQSPSSLAVSAGEKVTMTCKSSQSVLY S SNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAV

Y

YCHQYLSSHTFGGGTKLEIKRGGGGSGGGGSGGGGSQLQQPGAELVRPGSSVKLSCKA

SG

YTFTRYWIHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSGTAYMQLSS

L

TSEDSAVYYCATEDLYYAMEYWGQGTSVTVSSSGGGGSGGGGSGGGGSGGGGSAEKD

EL

SEQ ID NO: 4 (CD2-RTX-ER CAR nucleotide sequence)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCC

AGGCCG

GACATTATGATGACACAGTCGCCATCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTC

ACTATGACCTGTAAGTCCAGTCAAAGTGTTTTATACAGTTCAAATCAGAAGAACTAC

TTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTACTGATCTACTGGGCA

TCCACTAGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGAT

TTTACTCTTACCATCAGCAGTGTGCAACCTGAAGACCTGGCAGTTTATTACTGTCATC

AATACCTCTCCTCGCACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGGT

GGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTCAACTGCAGCAGCC

TGGGGCTGAGCTGGTGAGGCCTGGGTCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGG

CTACACCTTCACCAGGTACTGGATACATTGGGTGAAGCAGAGGCCTATACAAGGCCT

TGAATGGATTGGTAACATTGATCCTTCTGATAGTGAAACTCACTACAATCAAAAGTT

CAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCGGCACAGCCTACATGCAGC

TCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAACAGAGGATCTTT

ACTATGCTATGGAGTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCTGCCTGCC

CCTACAGCAACCCCAGCCTGTGCAGCGGCGGCGGCGGCAGCGAGCTGCCCACCCAG

GGCACCTTCAGCAACGTGAGCACCAACGTGAGCCCCGCCAAGCCCACCACCACCGC

CTGTCCTTATTCCAATCCTTCCCTGTGTAGCGGCGGCGGCGGCAGCACCACGACGCC

AGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGC

GCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGA

CTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTC

CTGTCACTGGTTATCACCCTTTACTGCAGGAGTAAGAGGAGCAGGCTCCTGCACAGT

GACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCC

CTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAG

CGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATC

TAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAG

ATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAAC

TGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCG

CCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGG

ACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGAAGCGGAGCCACC

AACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCGACAT CATGATGACCCAGAGCCCCAGCAGCCTGGCCGTGAGCGCCGGCGAGAAGGTGACCA

TGACCTGCAAGAGCAGCCAGAGCGTGCTGTACAGCAGCAACCAGAAGAACTACCTG

GCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCAG

CACCAGGGAGAGCGGCGTGCCCGACAGGTTCACCGGCAGCGGCAGCGGCACCGACT

TCACCCTGACCATCAGCAGCGTGCAGCCCGAGGACCTGGCCGTGTACTACTGCCACC

AGTACCTGAGCAGCCACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGGGC

GGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGCTGCAGCAGC

CCGGCGCCGAGCTGGTGAGGCCCGGCAGCAGCGTGAAGCTGAGCTGCAAGGCCAGC

GGCTACACCTTCACCAGGTACTGGATCCACTGGGTGAAGCAGAGGCCCATCCAGGG

CCTGGAGTGGATCGGCAACATCGACCCCAGCGACAGCGAGACCCACTACAACCAGA

AGTTCAAGGACAAGGCCACCCTGACCGTGGACAAGAGCAGCGGCACCGCCTACATG

CAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCACCGAGGA

CCTGTACTACGCCATGGAGTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCA

GCGGGGGGGGCGGCAGCGGGGGAGGCGGTTCCGGGGGTGGTGGTAGTGGTGGGGG

TGGCTCCGCCGAGAAGGACGAGCTGTAAGTTTAAAC

SEQ ID NO: 5 (CD3-RTX-ER CAR amino acid sequence)

MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQ

QTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFT

FGQGTKLQIGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTM

HW

VRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYF

C

ARYYDDHYCLDYWGQGTPVTVSSACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKP

T

TTACPYSNPSLCSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF

ACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP

P

RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP

QRR

KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ

ALPP

RGSGATNFSLLKQAGDVEENPGPDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQ

Q

TPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTF

GQGTKLQIGGGGSGGGGSGGGGSQVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTM

HWV

RQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFC

A

RYYDDHYCLDYWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSAEKDEL SEQ ID NO: 6 (CD3-RTX-ER CAR nucleotide sequence)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCC AGGCCG

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGT

GACCATCACCTGCAGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGA

CCCCCGGCAAGGCCCCCAAGAGATGGATCTACGACACCAGCAAGCTGGCCAGCGGC

GTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTACACCTTCACCATCAG

CAGCCTGCAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACC

CCTTCACCTTCGGCCAGGGCACCAAGCTGCAGATCGGCGGCGGCGGCAGCGGCGGC

GGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGGCGGCG

TGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGCAAGGCCAGCGGCTACACCTTC

ACCAGATACACCATGCACTGGGTGAGACAGGCCCCCGGCAAGGGCCTGGAGTGGAT

CGGCTACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGGTGAAGGACA

GATTCACCATCAGCAGAGACAACAGCAAGAACACCGCCTTCCTGCAGATGGACAGC

CTGAGACCCGAGGACACCGGCGTGTACTTCTGCGCCAGATACTACGACGACCACTA

CTGCCTGGACTACTGGGGCCAGGGCACCCCCGTGACCGTGAGCAGCGCCTGCCCCT

ACAGCAACCCCAGCCTGTGCAGCGGCGGCGGCGGCAGCGAGCTGCCCACCCAGGGC

ACCTTCAGCAACGTGAGCACCAACGTGAGCCCCGCCAAGCCCACCACCACCGCCTG

TCCTTATTCCAATCCTTCCCTGTGTAGCGGCGGCGGCGGCAGCACCACGACGCCAGC

GCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCC

AGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTC

GCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGT

CACTGGTTATCACCCTTTACTGCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACT

ACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATG

CCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCA

GACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGG

ACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGG

GGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA

GAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG

AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACAC

CTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGAAGCGGAGCCACCAACTT

CAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCGACATTCAAA

TGACGCAGTCTCCTTCAAGCCTGTCGGCAAGTGTAGGCGATCGTGTAACTATAACTT

GCAGCGCCTCTAGCAGCGTGAGCTATATGAACTGGTATCAACAAACACCTGGAAAG

GCTCCAAAACGTTGGATTTATGACACCTCGAAGTTGGCCTCCGGAGTTCCCAGCCGT

TTCAGCGGCTCGGGTAGTGGCACGGACTATACTTTCACAATATCTTCTCTCCAGCCG

GAGGACATTGCAACTTATTACTGCCAGCAGTGGAGCTCGAACCCCTTCACCTTCGGA

CAAGGGACCAAGCTTCAGATTGGGGGAGGCGGCTCTGGGGGTGGAGGGAGCGGGG

GAGGCGGGAGCCAAGTGCAGCTTGTTCAAAGCGGCGGAGGAGTCGTACAACCGGGT

CGATCCTTACGCCTTTCATGCAAAGCCTCCGGTTATACCTTTACCAGGTACACTATGC

ACTGGGTCCGCCAAGCACCAGGAAAAGGACTCGAGTGGATAGGGTACATCAACCCT

TCGAGGGGGTACACAAACTACAATCAGAAGGTTAAAGATCGTTTTACCATCTCGCGT

GACAATTCTAAGAACACGGCCTTCTTACAGATGGATTCCTTGAGGCCAGAAGATACA

GGTGTCTATTTTTGCGCCAGGTACTACGACGACCATTATTGTTTAGACTATTGGGGG

CAAGGGACTCCTGTAACAGTCTCAAGCGGGGGGGGCGGCAGCGGGGGAGGCGGTTC CGGGGGTGGTGGTAGTGGTGGGGGTGGCTCCGCCGAGAAGGACGAGCTGTAAGTTT

AAAC

SEQ ID NO: 7 (CD7-RTX-ER CAR amino acid sequence)

MALPVTALLLPLALLLHAARPGAQPAMAAYKDIQMTQTTSSLSASLGDRVTISCSAS

QGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATY

YCQQYSKLPYTFGGGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGG

SL

KLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNIL

YEQMSSERSEDTAMYYCARDEVRGYEDVWGAGTTVTVSSACPYSNPSECSGGGGSEEP

TQ

GTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEA

CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMT

P

RRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV

LD

KRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ

GLST

ATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLLHAA

RP

GAQPAMAAYKDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIY

Y

TSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKRGG

GGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQ

TPE

KRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVR

G

YLDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSAEKDEL*

SEQ ID NO: 8 (CD7-RTX-ER CAR nucleotide sequence)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCC

AGGCCGGGCGCCCAGCCCGCCATGGCCGCCTACAAGGACATCCAGATGACCCAGAC

CACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCAGCGCCA

GCCAGGGCATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTG

AAGCTGCTGATCTACTACACCAGCAGCCTGCACAGCGGCGTGCCCAGCAGATTCAG

CGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCCCGAGG

ACATCGCCACCTACTACTGCCAGCAGTACAGCAAGCTGCCCTACACCTTCGGCGGCG

GCACCAAGCTGGAGATCAAGAGAGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGG

CGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGC

GGCCTGGTGAAGCCCGGCGGCAGCCTGAAGCTGAGCTGCGCCGCCAGCGGCCTGAC

CTTCAGCAGCTACGCCATGAGCTGGGTGAGACAGACCCCCGAGAAGAGACTGGAGT

GGGTGGCCAGCATCAGCAGCGGCGGCTTCACCTACTACCCCGACAGCGTGAAGGGC AGATTCACCATCAGCAGAGACAACGCCAGAAACATCCTGTACCTGCAGATGAGCAG

CCTGAGAAGCGAGGACACCGCCATGTACTACTGCGCCAGAGACGAGGTGAGAGGCT

ACCTGGACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGCGCCTGCCCCTAC

AGCAACCCCAGCCTGTGCAGCGGCGGCGGCGGCAGCGAGCTGCCCACCCAGGGCAC

CTTCAGCAACGTGAGCACCAACGTGAGCCCCGCCAAGCCCACCACCACCGCCTGTC

CTTATTCCAATCCTTCCCTGTGTAGCGGCGGCGGCGGCAGCACCACGACGCCAGCGC

CGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAG

AGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCC

TGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCAC

TGGTTATCACCCTTTACTGCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACA

TGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCC

CACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGAC

GCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG

AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG

GAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAA

AGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGG

GGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTA

CGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGAAGCGGAGCCACCAACTTCA

GCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGGCCCTGCCC

GTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGGCCCGGCGCC

CAGCCGGCGATGGCAGCGTATAAAGACATCCAGATGACGCAGACCACTAGCAGCCT

TTCTGCCTCCTTGGGGGACCGCGTAACCATTTCCTGCAGCGCCTCCCAAGGTATATC

CAATTATTTGAACTGGTACCAGCAGAAACCGGATGGAACCGTGAAGTTGCTGATAT

ACTACACAAGTAGTCTTCACAGTGGGGTCCCGTCACGGTTCTCAGGTTCTGGCAGTG

GTACTGACTACTCCCTGACCATCTCTAACCTGGAACCAGAGGACATCGCGACTTATT

ATTGTCAACAATATTCTAAACTCCCGTACACTTTTGGCGGTGGCACGAAGCTCGAAA

TAAAGCGAGGTGGCGGCGGGTCAGGGGGAGGAGGATCCGGCGGCGGTGGAAGTGG

CGGTGGTGGATCAGAGGTTCAACTCGTAGAATCTGGGGGCGGGCTTGTAAAACCAG

GTGGGAGTCTGAAACTCAGCTGTGCGGCTTCTGGCCTGACGTTTAGCTCTTATGCAA

TGTCATGGGTGCGCCAAACTCCCGAAAAGCGCCTCGAGTGGGTGGCTTCCATCTCAT

CCGGTGGGTTTACGTATTATCCGGATTCAGTGAAGGGAAGATTCACGATATCACGGG

ACAACGCGAGGAACATTCTGTACCTGCAGATGTCTTCACTTCGCTCTGAAGATACAG

CTATGTATTATTGTGCTAGGGACGAAGTGAGAGGGTACTTGGATGTGTGGGGAGCA

GGAACCACTGTTACTGTCTCAAGCGGGGGGGGCGGCAGCGGGGGAGGCGGTTCCGG

GGGTGGTGGTAGTGGTGGGGGTGGCTCCGCCGAGAAGGACGAGCTGTAAGTTTAAA

C

SEQ ID NO: 9 (CD7-RTX-ER -VAC CAR amino add sequence)

MALPVTALLLPLALLLHAARPGAQPAMAAYKDIQMTQTTSSLSASLGDRVTISCSAS

QGISNYENWYQQKPDGTVKEEIYYTSSEHSGVPSRFSGSGSGTDYSETISNEEPEDIATY

YCQQYSKLPYTFGGGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGG

SL

KLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNIL YLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSSACPYSNPSLCSGGGGSELP TQ

GTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEA

CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMT

P

RRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV

LD

KRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ

GLST

ATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLLHAA

RP

GAQPAMAAYKDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIY

Y

TSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKRGG

GGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQ

TPE

KRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVR

G

YLDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSAEKDELGSGEGRGSLLTCGDVE

ENP

GPMYRMQLLSCIALSLALVTNSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVT

AMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKE

FLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVK

S

YSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

SEQ ID NO: 10(CD7-RTX-ER-VAC CAR nucleotide sequence)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCC

AGGCCGGGCGCCCAGCCCGCCATGGCCGCCTACAAGGACATCCAGATGACCCAGAC

CACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGCTGCAGCGCCA

GCCAGGGCATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTG

AAGCTGCTGATCTACTACACCAGCAGCCTGCACAGCGGCGTGCCCAGCAGATTCAG

CGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCCCGAGG

ACATCGCCACCTACTACTGCCAGCAGTACAGCAAGCTGCCCTACACCTTCGGCGGCG

GCACCAAGCTGGAGATCAAGAGAGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGG

CGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGC

GGCCTGGTGAAGCCCGGCGGCAGCCTGAAGCTGAGCTGCGCCGCCAGCGGCCTGAC

CTTCAGCAGCTACGCCATGAGCTGGGTGAGACAGACCCCCGAGAAGAGACTGGAGT

GGGTGGCCAGCATCAGCAGCGGCGGCTTCACCTACTACCCCGACAGCGTGAAGGGC

AGATTCACCATCAGCAGAGACAACGCCAGAAACATCCTGTACCTGCAGATGAGCAG

CCTGAGAAGCGAGGACACCGCCATGTACTACTGCGCCAGAGACGAGGTGAGAGGCT

ACCTGGACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGCGCCTGCCCCTAC

AGCAACCCCAGCCTGTGCAGCGGCGGCGGCGGCAGCGAGCTGCCCACCCAGGGCAC CTTCAGCAACGTGAGCACCAACGTGAGCCCCGCCAAGCCCACCACCACCGCCTGTC

CTTATTCCAATCCTTCCCTGTGTAGCGGCGGCGGCGGCAGCACCACGACGCCAGCGC

CGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAG

AGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCC

TGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCAC

TGGTTATCACCCTTTACTGCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACA

TGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCC

CACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGAC

GCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG

AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGG

GAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAA

AGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGG

GGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTA

CGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGAAGCGGAGCCACCAACTTCA

GCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGGCCCTGCCC

GTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGGCCCGGCGCC

CAGCCGGCGATGGCAGCGTATAAAGACATCCAGATGACGCAGACCACTAGCAGCCT

TTCTGCCTCCTTGGGGGACCGCGTAACCATTTCCTGCAGCGCCTCCCAAGGTATATC

CAATTATTTGAACTGGTACCAGCAGAAACCGGATGGAACCGTGAAGTTGCTGATAT

ACTACACAAGTAGTCTTCACAGTGGGGTCCCGTCACGGTTCTCAGGTTCTGGCAGTG

GTACTGACTACTCCCTGACCATCTCTAACCTGGAACCAGAGGACATCGCGACTTATT

ATTGTCAACAATATTCTAAACTCCCGTACACTTTTGGCGGTGGCACGAAGCTCGAAA

TAAAGCGAGGTGGCGGCGGGTCAGGGGGAGGAGGATCCGGCGGCGGTGGAAGTGG

CGGTGGTGGATCAGAGGTTCAACTCGTAGAATCTGGGGGCGGGCTTGTAAAACCAG

GTGGGAGTCTGAAACTCAGCTGTGCGGCTTCTGGCCTGACGTTTAGCTCTTATGCAA

TGTCATGGGTGCGCCAAACTCCCGAAAAGCGCCTCGAGTGGGTGGCTTCCATCTCAT

CCGGTGGGTTTACGTATTATCCGGATTCAGTGAAGGGAAGATTCACGATATCACGGG

ACAACGCGAGGAACATTCTGTACCTGCAGATGTCTTCACTTCGCTCTGAAGATACAG

CTATGTATTATTGTGCTAGGGACGAAGTGAGAGGGTACTTGGATGTGTGGGGAGCA

GGAACCACTGTTACTGTCTCAAGCGGGGGGGGCGGCAGCGGGGGAGGCGGTTCCGG

GGGTGGTGGTAGTGGTGGGGGTGGCTCCGCCGAGAAGGACGAGCTGGGCAGCGGCG

AAGGCCGCGGCAGCCTGCTGACCTGCGGCGATGTGGAAGAAAACCCGGGCCCCATG

TACAGAATGCAGCTGCTGAGCTGCATCGCCCTGAGCCTGGCCCTGGTGACCAACAG

CAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCA

TGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTG

ACCGCCATGAAGTGCTTCCTGCTGGAGCTGCAGGTGATCAGCCTGGAGAGCGGCGA

CGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGA

GCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGA

GAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAA

CACCAGCTCCGGCGGCGGCTCCGGCGGCGGCGGCTCCGGCGGCGGCGGCTCCGGCG

GCGGCGGCTCCGGCGGCGGCTCCCTGCAGATCACCTGCCCCCCCCCCATGAGCGTGG

AGCACGCCGACATCTGGGTGAAGAGCTACAGCCTGTACAGCAGAGAGAGATACATC

TGCAACAGCGGCTTCAAGAGAAAGGCCGGCACCAGCAGCCTGACCGAGTGCGTGCT

GAACAAGGCCACCAACGTGGCCCACTGGACCACCCCCAGCCTGAAGTGCATCAGAT

AAGTTTAAAC SEQ ID NO: 11 (CD5b-RTX-VAC CAR amino add sequence)

MALPVTALLLPLALLLHAARPDIQMTQSPSSMSASLGDRVTITCRASQDINSYLSWF

HHKRGKSPKTLIYRANRLVDGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPW

TFGGGTKLEIKGGGGSGGGGSGGGGSQIQLVQSGPGLKKPGGSVRISCAASGYTFTNYG

M

NWVKQAPGKGLRWMGWINTHTGEPTYADDFKGRFTFSLDTSKSTAYLQINSLRAEDTA

TY

FCTRRGYDWYFDVWGQGTTVTVSSACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPA

KP

TTTACPYSNPSLCSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD

FACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA

P

PRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK

PQR

RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALP

PRGSGATNFSLLKQAGDVEENPGPMYRMQLLSCIALSLALVTNSGIHVFILGCFSAGLPK

TEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDA

SIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGG

GSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRK

A

GTSSLTECVLNKATNVAHWTTPSLKCIR*

SEQ ID NO: 12 (CD5b-RTX-VAC CAR nucleotide sequence)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCC AGGCCG

GACATCCAGATGACCCAGAGCCCCAGCAGCATGAGCGCCAGCCTGGGCGACAGGGT

GACCATCACCTGCAGGGCCAGCCAGGACATCAACAGCTACCTGAGCTGGTTCCACC

ACAAGAGGGGCAAGAGCCCCAAGACCCTGATCTACAGGGCCAACAGGCTGGTGGA

CGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTACACCCTGACCA

TCAGCAGCCTGCAGTACGAGGACTTCGGCATCTACTACTGCCAGCAGTACGACGAG

AGCCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGAGGGGGGGGATC

CGGGGGAGGAGGCTCCGGCGGAGGCGGAAGCCAGATCCAGCTGGTGCAGAGCGGC

CCCGGCCTGAAGAAGCCCGGCGGCAGCGTGAGGATCAGCTGCGCCGCCAGCGGCTA

CACCTTCACCAACTACGGCATGAACTGGGTGAAGCAGGCCCCCGGCAAGGGCCTGA

GGTGGATGGGCTGGATCAACACCCACACCGGCGAGCCCACCTACGCCGACGACTTC

AAGGGCAGGTTCACCTTCAGCCTGGACACCAGCAAGAGCACCGCCTACCTGCAGAT

CAACAGCCTGAGGGCCGAGGACACCGCCACCTACTTCTGCACCAGGAGGGGC

TACGACTGGTACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGC

CTGCCCCTACAGCAACCCCAGCCTGTGCAGCGGCGGCGGCGGCAGCGAGCTGCCCA

CCCAGGGCACCTTCAGCAACGTGAGCACCAACGTGAGCCCCGCCAAGCCCACCACC

ACCGCCTGTCCTTATTCCAATCCTTCCCTGTGTAGCGGCGGCGGCGGCAGCACCACG ACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTC

CCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGG

CTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTC

CTTCTCCTGTCACTGGTTATCACCCTTTACTGCAGGAGTAAGAGGAGCAGGCTCCTG

CACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTA

CCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAG

CAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGC

TCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGAC

CCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACA

ATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGG

CGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCA

CCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGAAGCGGA

GCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCC

CATGTACAGAATGCAGCTGCTGAGCTGCATCGCCCTGAGCCTGGCCCTGGTGACCAA

CAGCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGCCTGCCCAAGACCGA

GGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGA

GCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAG

GTGACCGCCATGAAGTGCTTCCTGCTGGAGCTGCAGGTGATCAGCCTGGAGAGCGG

CGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCC

TGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGA

GGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCA

TCAACACCAGCTCCGGCGGCGGCTCCGGCGGCGGCGGCTCCGGCGGCGGCGGCTCC

GGCGGCGGCGGCTCCGGCGGCGGCTCCCTGCAGATCACCTGCCCCCCCCCCATGAG

CGTGGAGCACGCCGACATCTGGGTGAAGAGCTACAGCCTGTACAGCAGAGAGAGAT

ACATCTGCAACAGCGGCTTCAAGAGAAAGGCCGGCACCAGCAGCCTGACCGAGTGC

GTGCTGAACAAGGCCACCAACGTGGCCCACTGGACCACCCCCAGCCTGAAGTGCAT

CAGATAAGTTTAAAC

SEQ ID NO: 13 (CD5m-RTX-VAC CAR amino add sequence)

MALPVTALLLPLALLLHAARPDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWF

HHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCQQYDESP

W

TFGGGTKLEIKGGGGSGGGGSGGGGSQIQLVQSGPELKKPGETVKISCKASGYTFTNYG

M

NWVKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDT

ATY

FCTRRGYDWYFDVWGAGTTVTVSSACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPA

KP

TTTACPYSNPSLCSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD

FACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA

P

PRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK

PQR

RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALP

PRGSGATNFSLLKQAGDVEENPGPMYRMQLLSCIALSLALVTNSGIHVFILGCFSAGLPK TEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDA

SIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGG

GSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRK

A

GTSSLTECVLNKATNVAHWTTPSLKCIR

SEQ ID NO: 14 (CD5m-RTX-VAC CAR nucleotide sequence)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCC

AGGCCGGACATCAAGATGACCCAGAGCCCCAGCAGCATGTACGCCAGCCTGGGCGA

GAGGGTGACC

ATCACCTGCAAGGCCAGCCAGGACATCAACAGCTACCTGAGCTGGTTCCACCACAA

GCCCGGCAAGAGCCCCAAGACCCTGATCTACAGGGCCAACAGGCTGGTGGACGGCG

TGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCCAGGACTACAGCCTGACCATCAGC

AGCCTGGACTACGAGGACATGGGCATCTACTACTGCCAGCAGTACGACGAGAGCCC

CTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGAGGGGGGGGATCCGGG

GGAGGAGGCTCCGGCGGAGGCGGAAGCCAGATCCAGCTGGTGCAGAGCGGCCCCG

AGCTGAAGAAGCCCGGCGAGACCGTGAAGATCAGCTGCAAGGCCAGCGGCTACACC

TTCACCAACTACGGCATGAACTGGGTGAAGCAGGCCCCCGGCAAGGGCCTGAGGTG

GATGGGCTGGATCAACACCCACACCGGCGAGCCCACCTACGCCGACGACTTCAAGG

GCAGGTTCGCCTTCAGCCTGGAGACCAGCGCCAGCACCGCCTACCTGCAGATCAAC

AACCTGAAGAACGAGGACACCGCCACCTACTTCTGCACCAGGAGGGGCTACGACTG

GTACTTCGACGTGTGGGGCGCCGGCACCACCGTGACCGTGAGCAGCGCCTGCCCCT

ACAGCAACCCCAGCCTGTGCAGCGGCGGCGGCGGCAGCGAGCTGCCCACCCAGGGC

ACCTTCAGCAACGTGAGCACCAACGTGAGCCCCGCCAAGCCCACCACCACCGCCTG

TCCTTATTCCAATCCTTCCCTGTGTAGCGGCGGCGGCGGCAGCACCACGACGCCAGC

GCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCC

AGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTC

GCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGT

CACTGGTTATCACCCTTTACTGCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACT

ACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATG

CCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCA

GACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGG

ACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGG

GGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCA

GAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGG

AGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACAC

CTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGAAGCGGAGCCACCAACTT

CAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGTACAGAA

TGCAGCTGCTGAGCTGCATCGCCCTGAGCCTGGCCCTGGTGACCAACAGCGGCATCC

ACGTGTTCATCCTGGGCTGCTTCAGCGCCGGCCTGCCCAAGACCGAGGCCAACTGGG

TGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATC

GACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCAT

GAAGTGCTTCCTGCTGGAGCTGCAGGTGATCAGCCTGGAGAGCGGCGACGCCAGCA

TCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAAC

GGCAACGTGACCGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACA TCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCT CCGGCGGCGGCTCCGGCGGCGGCGGCTCCGGCGGCGGCGGCTCCGGCGGCGGCGGC TCCGGCGGCGGCTCCCTGCAGATCACCTGCCCCCCCCCCATGAGCGTGGAGCACGCC

GACATCTGGGTGAAGAGCTACAGCCTGTACAGCAGAGAGAGATACATCTGCAACAG CGGCTTCAAGAGAAAGGCCGGCACCAGCAGCCTGACCGAGTGCGTGCTGAACAAGG CCACCAACGTGGCCCACTGGACCACCCCCAGCCTGAAGTGCATCAGATAAGTTTAA

AC

SEQ ID NO: 15(CD45-RTX-ER -VAC CAR amino add sequence)

MALPVTALLLPLALLLHAARPDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSY

LHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHS R

ELPFTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLKLSCAASGFDF

S

RYWMSWVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQMSKVRSE

D

TALYYCARGNYYRYGDAMDYWGQGTSVTVSACPYSNPSLCSGGGGSELPTQGTFSNV STN

VSPAKPTTTACPYSNPSLCSGGGGSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV

HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRK H

YQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP

EM

GGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DAL

HMQALPPRGSGATNFSLLKQAGDVEENPGPDIVLTQSPASLAVSLGQRATISCRASKSVS

TSGYSYLHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATY

YCQHSRELPFTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLKLSCA A

SGFDFSRYWMSWVRQAPGKGLEWIGEINPTSSTINFTPSLKDKVFISRDNAKNTLYLQM

S

KVRSEDTALYYCARGNYYRYGDAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGG SAEK

DEL

SEQ ID NO: 16(CD45-RTX-ER-VAC CAR nucleotide sequence)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCC AGGCCG

GACATCGTGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGGGC

CACCATCAGCTGCAGGGCCAGCAAGAGCGTGAGCACCAGCGGCTACAGCTACCTGC

ACTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACCTGGCCAGC AACCTGGAGAGCGGCGTGCCCGCCAGGTTTAGCGGTAGCGGTAGCGGCACCGACTT

CACCCTGAACATCCACCCCGTGGAGGAGGAGGACGCCGCCACCTACTACTGCCAGC

ACAGCAGGGAGCTGCCCTTCACCTTCGGCAGCGGCACCAAGCTGGAGATCAAGGGC

GGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGG

TGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAAGCTGAGCTGCGCC

GCCAGCGGCTTCGACTTCAGCAGGTACTGGATGAGCTGGGTGAGGCAGGCCCCCGG

CAAGGGCCTGGAGTGGATCGGCGAGATCAACCCCACCAGCAGCACCATCAACTTCA

CCCCCAGCCTGAAGGACAAGGTGTTCATCAGCAGGGACAACGCCAAGAACACCCTG

TACCTGCAGATGAGCAAGGTGAGGAGCGAGGACACCGCCCTGTACTACTGCGCCAG

GGGCAACTACTACAGGTACGGCGACGCCATGGACTACTGGGGCCAGGGCACCAGCG

TGACCGTGAGCGCCTGCCCCTACAGCAACCCCAGCCTGTGCAGCGGCGGCGGCGGC

AGCGAGCTGCCCACCCAGGGCACCTTCAGCAACGTGAGCACCAACGTGAGCCCCGC

CAAGCCCACCACCACCGCCTGTCCTTATTCCAATCCTTCCCTGTGTAGCGGCGGCGG

CGGCAGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGT

CGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTG

CACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGG

ACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAGGAGTAAGAGG

AGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCAC

CCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAG

AGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGC

TCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGA

CGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGG

AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATT

GGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCT

CAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCG

CGGAAGCGGAGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAG

AACCCCGGCCCCGATATTGTGCTGACGCAGAGCCCGGCGAGCCTGGCGGTGAGCCT

GGGCCAGCGCGCGACCATTAGCTGCCGCGCGAGCAAAAGCGTGAGTACCAGTGGTT

ATAGCTATCTGCATTGGTATCAGCAGAAACCGGGCCAGCCGCCGAAACTGCTGATTT

ATCTGGCGAGCAACCTGGAAAGCGGCGTGCCGGCGCGCTTTAGCGGCAGCGGCAGC

GGCACCGATTTTACCCTGAACATTCATCCGGTGGAAGAAGAAGATGCGGCGACCTA

TTATTGCCAGCATAGCCGCGAACTGCCGTTTACCTTTGGCAGCGGCACCAAACTGGA

AATTAAAGGTGGTGGTGGTAGCGGCGGCGGTGGCAGCGGTGGTGGTGGTAGCCAAG

TTCAGCTGGTTGAAAGTGGTGGTGGTCTGGTTCAACCGGGCGGCAGCCTGAAACTGA

GCTGCGCGGCGAGCGGCTTTGATTTTAGCCGCTATTGGATGAGCTGGGTGCGCCAGG

CGCCGGGCAAAGGCCTGGAATGGATTGGCGAAATTAACCCGACCAGCAGCACCATT

AACTTTACCCCGAGCCTGAAAGATAAAGTGTTTATTAGCCGCGATAACGCGAAAAA

CACCCTGTATCTGCAGATGAGCAAAGTGCGCAGCGAAGATACCGCGCTGTATTATTG

CGCGCGCGGCAACTATTATCGCTATGGCGATGCGATGGATTATTGGGGTCAAGGTAC

GAGTGTTACGGTTTCCAGCGGGGGGGGCGGCAGCGGGGGAGGCGGTTCCGGGGGTG

GTGGTAGTGGTGGGGGTGGCTCCGCCGAGAAGGACGAGCTGTAAGTTTAAAC

Claims

1. An engineered T cell or NK cell co-expressing two distinct chimeric antigen receptor (CAR) units at the cell surface, wherein the engineered T cell or NK cell comprises a nucleotide sequence comprising from 5’ to 3’ a comprising a first polynucleotide encoding a first CAR, a second nucleotide encoding a second CAR, a nucleotide encoding a viral self-cleavage peptide disposed between the first CAR and second CAR, under the transcriptional control of a single promoter, wherein

(i) the first CAR comprises a signal peptide, a first antigen recognition domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signaling domain to form a first fusion protein; and

(ii) the second CAR comprises a second antigen recognition domain fused to a retention signal peptide, wherein: the second CAR does not comprise a hinge region, transmembrane domain, co-stimulatory domain or a signaling domain; and the second antigen recognition domain entraps a recognized protein within its secretion pathway, which results in either prevention of its surface location in a cell, or its secretion.

2. The engineered T cell or NK cell according to claim 1, wherein the cleavage site is selected from the group consisting of porcine teschovirus-1 2A (P2A), FMDV 2A (abbreviated herein as F2A); equine rhinitis A virus (ERAV) 2A (E2A);_and Thoseaasigna virus 2A (T2A).

3. The engineered T cell or NK cell according to claim 1, wherein the first antigen recognition domain and second antigen recognition domain are different or the same.

4. The engineered T cell or NK cell according to claim 1, wherein the engineered cell is a CD4 T-cell, CD8 T-cell, NKT cell, or NK-92 cell or macrophage.

5. The engineered T cell or Nk cell according to claim 1, wherein the target of the first antigen recognition domain is selected from the group consisting of and scFv of CD2, an scFv of CD3, an scFv of CD4, and scFv of CD5 and an scFv of CD7; and the target of the second antigen recognition domain is selected from the group consisting of an scFv of CD2, and scFv of CD3, an scFv of CD4, an scFv of CD5 and an scFv of CD7.

77

6. The engineered T cell or NK cell according to claim 1, wherein the target of the first antigen recognition domain is an scFv of CD2; and the target of the second antigen recognition domain is an scFv of CD2.

7. The engineered T cell or NK cell according to claim 1, wherein the target of the first antigen recognition domain is and scFv of CD3; and the target of the second antigen recognition domain is and scFv of CD3.

8. The engineered T cell or NK cell according to claim 1, wherein the target of the first antigen recognition domain is an scFv of CD7 ; and the target of the second antigen recognition domain is an scFv of CD7.

9. The engineered T cell or NK cell according to claim 1, wherein the target of the first antigen recognition domain is an scFv of CD45; and the target of the second antigen recognition domain is an scFv of CD45.

10. The engineered T cell or NK cell according to claim 1, wherein the second antigen recognition domain includes at least one of endogenous a and/or 0 chains or gamma and/or delta chains of the TCR.

11. The engineered T cell or NK cell according to claim 1, wherein the second antigen recognition domain is an scFv of CD3.

12. The engineered T cell or NK cell according to claim 1, wherein the target of the first antigen recognition domain is selected from the group consisting of interleukin 6 receptor, NY- ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, CLL-1, CD2, CD3, CD4, CD5, CD7, CD45 and CS1; and the target of the second antigen recognition domain is CD3.

13. The engineered T cell or NK cell according to claim 1, wherein the second antigen recognition domain includes at least one of endogenous IL-1, IL-6 and IL-1 receptor.

14. The engineered T cell or NK cell according to claim 1, wherein the target of the first antigen recognition domain is selected from the group consisting of interleukin 6 receptor, NY- ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138,

78 CD267, CD269, CD38, Flt3 receptor, CLL-1, CD2, CD3, CD4, CD5, CD7, CD45 and CS1; and the target of the second antigen recognition domain is selected from the group consisting of IL- 1 and/or IL The engineered cell according to claim 1, wherein the target of the first antigen recognition domain is selected from the group consisting of interleukin 6 receptor, NY- ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, CLL-1, CD2, CD3, CD4, CD5, CD7, CD45 and CS1; and the target of the second antigen recognition domain is IL-6.

15. The engineered T cell or NK cell according to claim 1, wherein the target of the first antigen recognition domain is selected from the group consisting of interleukin 6 receptor, NY- ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, CLL-1, CD2, CD3, CD4, CD5, CD7, CD45 and CS1; and the target of the second antigen recognition domain is selected from the group consisting of IL- 1

16. The engineered T cell or NK cell according to claim 1, wherein the target of the second antigen recognition domain is selected at least one from the group consisting of immune checkpoint molecules including Programmed Death 1 (PD-1), Cytotoxic T-Lymphocyte Antigen (CTLA-4), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7 , SIGLEC9, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1 , M ORA, IL10RB, HM0X2, IL6R, IL6ST, EIF2AK4, CSK, PAG1 , SIT1 , F0XP3, PRDM1 , BATF, GUCY1A2.

17. The engineered T cell or NK cell according to claim 1, wherein the second antigen recognition domain is Programmed Death 1 (PD-1) and/or Cytotoxic T-Lymphocyte Antigen (CTLA-4),

18. The engineered T cell or NK cell according to claim 1, wherein the target of the first antigen recognition domain is selected from the group consisting of interleukin 6 receptor, NY- ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, CLL1, CD2, CD3, CD4, CD5, CD7, CD45 and CS1; and the target of the second antigen recognition domain is selected at least one from the group

79 consisting of immune checkpoint molecules including Programmed Death 1 (PD-1), Cytotoxic T-Lymphocyte Antigen (CTLA-4), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7 , SIGLEC9, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1 , M ORA, IL10RB, HM0X2, IL6R, IL6ST, EIF2AK4, CSK, PAG1 , SDT , F0XP3,19

19. The engineered T cell or NK cell according to claim 1, wherein the target of the first antigen recognition domain is selected from the group consisting of interleukin 6 receptor, NY- ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor,CLL-l, CD2, CD3, CD4, CD5, CD7 and CS1; and the target of the second antigen recognition domain is Programmed Death 1 (PD-1) and/or Cytotoxic T- Lymphocyte Antigen (CTLA-4).

20. The engineered T cell or NK cell according to claim 1, wherein the engineered cell further comprises an immunomodulatory selected from IL15, IL-15/IL-15sushi, IL-2, IL-7, IL- 12 IL- 18, IL-21, functional fragments thereof, or combinations thereof.

21. The engineered T cell or NK cell according to claim 1, wherein the immunomodulator comprises IL-15/IL-15sushi.

22. The engineered T cell or NK cell according to claim 1, wherein the immunomodulator is IL-15/IL-15.

22. The engineered T cell or NK cell according to claim 1, wherein the immunomodulator is IL-7.

24. The engineered T cell or NK cell according to claim 1, wherein the immunomodulator is

IL- 12.

80