WO2026006283A1 - Car-enhancer dosing and timing to enhance the functionality of car immune cells - Google Patents

Abstract

Disclosed are methods of enhancing activity of chimeric antigen receptor (CAR) immune cells with a CAR-enhancer containing a first moiety that binds an epitope on an extracellular domain of the CAR connected to a second moiety containing a first immune cell effector domain.

Description

CAR-ENHANCER DOSING AND TIMING

TO ENHANCE THE FUNCTIONALITY OF CAR IMMUNE CELLS

BACKGROUND OF THE DISCLOSURE

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No: 63/664,022, filed June 25, 2024, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 2, 2025, is named 046094_793001WO_ST.xml and is 114 KB bytes in size.

BACKGROUND OF THE DISCLOSURE

[0003] Chimeric antigen receptor (CAR) expressing T cells have revolutionized the treatment of blood-borne malignancies and have shown promising results in the treatment of hematopoietic cancers. Six CAR T cell therapies targeting two antigens, CD19 and BCMA, are currently FDA- approved. CD19 is a B-cell co-receptor expressed on B cells and a wide variety of blood-borne malignancies. CD 19 CAR T cells were initially approved for the treatment of acute lymphoblastic leukemia (ALL) and have subsequently been approved for Burkitt’s Lymphoma and Mantle Cell Lymphoma. BCMA is a receptor expressed on the surface of B-cell lineage cells and a major marker of multiple myeloma (MM). MM is associated with an uncontrollable expansion of plasma cells in the bone marrow, which can progress to extra-medullary lesions forming elsewhere in the body. BCMA CAR T cell therapy has shown great promise against MM with studies showing an overall response rate of 80% even in patients with extra-medullary lesions (Gagelmann et al., Eur. J. Haematol. 104(4) :318-327 (2020)).

[0004] However, challenges persist with CAR T cell therapy. A recent meta-analysis of 22 CAR T cell clinical studies highlighted its ineffectiveness in solid tumors with a poor average overall response rate of 9% (Hou etal., Dis. Markers 20/9:3425291 (2019)). The duration of response, even in hematological cancers, remains a challenge, with almost all BCMA CAR-treated MM patients ultimately relapsing (Gagelmann et al., Eur. J. Haematol. 104(4).318-327 (2020); Roex et al., J. Hematol. Oncol. 13(1)-.164 (2020); Raje et al., N. Engl. J. Med. 380(18)-.1726-1737 (2019)). In addition, treatments can have severe side effects including cytokine release syndrome (CRS) and neurotoxicity.

[0005] CAR T cells need to home to the tumor location, expand, and persist in circulation, at least until they neutralize and kill the last remaining cancer cells. Therefore, approaches to prolong and enhance the activity of CAR T cells in a controlled way are critically needed.

SUMMARY OF THE DISCLOSURE

[0006] The presently disclosed methods of enhancing activity of chimeric antigen receptor (CAR) immune cells are expected to address the above needs. The CAR-enhancers augment CAR immune cell functionality and persistence in vivo. They may also reduce the cellular dose needed for CAR immune cell therapy, which may result in reduced adverse side effects (e.g., cytokine release syndrome) caused by the larger doses typically used in the clinic and therefore, the CAR- enhancers are also referred herein as CAR-enhancers. Further, since CAR-enhancer binding to CAR is reversible, the CAR-enhancer does not induce immune synapse formation of CAR on the CAR immune cell surface. Therefore, CAR-enhancers do not block CAR-mediated killing of cancer cells. CAR immune cells often do not persist in the body during minimal residual disease (MRD), which, as known in the art, is associated with limited cancer antigens. The disclosed CAR-enhancers may support persistence, proliferation, and efficacy of CAR T cells during states of MRD.

[0007] A first aspect of the present disclosure is directed to method of enhancing activity of CAR immune cells. The method entails administering to a subject having had CAR immune cell therapy that targets an antigen present on a cancer cell, a first course of an effective amount of CAR- enhancer therapy, wherein the CAR-enhancer comprises a first proteinaceous moiety that binds an epitope on an extracellular domain (ED) of the CAR connected to a second proteinaceous moiety comprising a first immune cell effector domain, wherein administration of the first course of CAR- enhancer therapy is initiated at any time up to about 6 months after the subject received the CAR immune cell therapy. The ED includes one or more extracellular binding domains (EBDs) of the CAR and any other extracellular portions of the CAR, e.g., a linker that connects antibody fragments, or EBDs, etc.

[0008] In some embodiments, the first course of the CAR-enhancer therapy is initiated about 2 weeks after the CAR immune cell therapy. In some embodiments, the first course of the CAR- enhancer therapy is conducted over a period of time of about 1 to about 3 weeks and comprises, e.g. , from about 1 to about 3 doses of CAR-enhancer per week. In some embodiments, the administering of the first course of the CAR-enhancer therapy is conducted over a period of time of about 2 weeks. In some embodiments, the first course of the CAR-enhancer therapy comprises administering from about 1 to about 6 doses of the CAR-enhancer. The working examples demonstrate that the timing and dosage amounts of the CAR-enhancer therapy may optimize its effects on the prior CAR immune cell therapy in terms enhancing CAR immune cells that target a cancer antigen by driving them toward generation of memory immune cells, and exhaustion-preventative proliferation of the CAR immune cells.

[0009] The working examples also present a hypothesis of a fundamental mechanism of action as between the CAR-enhancer and the CAR immune cells. More specifically, the working examples demonstrate that the two binding events, namely the binding between the immune effector domain of the CAR-enhancer and the cognate receptor on the immune cells, and the binding between the moiety of the CAR-enhancer that binds the ED of the CAR, produces a synergistic, molecular “cross-talk” between the intracellular domain (endodomain) of the cognate receptor and the endodomain stimulatory regions of the CAR, respectively, that results in production of IFN-y and TNF-a, and ultimately the generation of memory immune cells, and exhaustion-preventative proliferation of the CAR immune cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[00010] FIG. 1 schematically illustrates the domains of a CAR-enhancer according to some embodiments that contains an ectodomain of an antigen on the surface of a cancer cell (Ag), a CH3 dimerization domain, and an immune cell effector domain (ICE). The CAR-enhancer may be a monomer, a dimer, or a multimer.

[00011] FIGs. 2A - 21 are a set of illustrations and line plots that show three CAR-enhancers. FIG. 2A schematically illustrates a CAR-enhancer that contains a BCMA ectodomain and a Neo2/15 synthetic cytokine immune cell effector domain. FIG. 2B schematically illustrates a CAR-enhancer that contains a BCMA ectodomain and two weak affinity mutated IL-2 (mIL2) synthetic cytokine immune cell effector domains. FIG. 2C schematically illustrates a CAR-enhancer that contains a BCMA ectodomain and a 4-1BBL immune cell effector domain. FIG. 2D is a line plot that shows dose-dependent staining of CAR T cells that bind CD 19 or non-transduced T cells (NT T cells) with CAR-enhancer or control proteins. FIG. 2E is a line plot that shows dose-dependent staining of CAR T cells that bind BCMA or non-transduced T cells (NT T cells) with CAR-enhancer or control proteins. FIGs. 2F and 2G are a line and bar plot, respectively, that together show dose-dependent activation of CAR T cells after CAR-enhancer treatment. FIG. 2H is a line plot showing that the BCMA-muIL2 CAR-enhancer does not block the killing efficacy of the CAR T cells. FIG. 21 is a line plot that shows phosphorylation of signal transducer and activator of transcription (STAT5) in the BCMA CAR T cells.

[00012] FIGs. 3A - 3B are a set of illustrations and line plots showing the effects of CAR- enhancers on non-transduced T cells. FIG. 3A schematically illustrates the experimental design. FIG. 3B is a set of line plots that show T cell count and carboxyfluorescein succinimidyl ester (CFSE) staining of non-transduced, activated T cells treated with teceleukin, a CAR-enhancer containing an BCMA ectodomain and two mutated weak affinity IL-2 (muIL2), or CAR-enhancer containing an BCMA ectodomain and a Neoleukin domain.

[00013] FIGs. 4A - 4C are a set of illustrations and line plots showing that CAR-enhancers specifically activate CAR T cells. FIG. 4A schematically illustrates the experimental design. FIG. 4B is a bar plot that shows the percentage of CD69⁺ anti-BCMA CAR-transduced, activated T cells after treatment with BCMA CAR-enhancer, BCMA CAR-enhancer without an immune cell effector domain control, or a non-antigen-specific CAR-enhancer control. FIG. 4C is a bar plot that shows the percentage of CD69⁺ anti-CD19 CAR-transduced, activated T cells after treatment with CD19 CAR-enhancer or a non-anti gen-specific CAR-enhancer control.

[00014] FIG. 5 is a line plot showing that CAR-enhancers do not inhibit BCMA CAR T cell killing and that the percentage of 0PM2 target cell survival after incubation with CAR T cells and CAR- enhancers (red) or non-transduced T cells (blue).

[00015] FIGs. 6A - 6C are a set of illustrations and photographs showing that CAR-enhancers reduce tumor burden in vivo. FIG. 6A schematically illustrates the experimental design. FIGs. 6B - 6C are a set of photographs that show tumor burden in mice before and after CAR T cell infusion and CAR-enhancer treatment.

[00016] FIGs. 7A - 7C are a set of flow cytometry plots showing the tumor burden in mice after CAR T cell infusion and CAR-enhancer treatment. FIG. 7A is a set of flow cytometry plots that shows 0PM2 tumor burden in blood, spleen, and lymph nodes. FIG. 7B is a set of flow cytometry plots that shows OPM2 tumor burden in bone marrow and lung. FIG. 7C is a set of flow cytometry plots that shows 0PM2 tumor burden in liver, kidney, and the eye tumor site. eGFP (0PM2 cells) is shown on the y-axis and PerCP signal control is shown on the x-axis.

[00017] FIGs. 8A - 8C are a set of flow cytometry plots showing human CD45 and CAR- T cells in mice after CAR T cell infusion and CAR-enhancer treatment. FIG. 8A is a set of flow cytometry plots that shows CAR T cells in blood, spleen, and lymph nodes. FIG. 8B is a set of flow cytometry plots that shows CAR T cells in bone marrow and lung. FIG. 8C is a set of flow cytometry plots that shows CAR T cells in the liver, kidney, and the eye tumor site. CD45 staining is shown on the y- axis and CAR-enhancer labeled with AF647 staining is shown on the x-axis.

[00018] FIGs. 9A -9E are a set of schematics, line plots, and box plots showing that CAR- enhancer treatment results in enhanced activity and persistence of CAR T cells in vivo. FIG. 9A is a line plot that shows circulating half-life of the BCMA CAR-enhancers. FIG. 9B schematically illustrates the experimental design. FIGs. 9C and 9D are a set of flow cytometry plots and box plots that show selective expansion and persistence of BCMA CAR T cells. FIG. 9E is a box plot that shows the percentage of CD8¹ CAR T cells after CAR-enhancer treatment.

[00019] FIGs. 10A - 10J are a set of schematics, survival, line, bar, and t-distributed stochastic neighbor embedding (tSNE) plots and photographs showing that CAR-enhancer treatment lowers the required dose of CAR T cells. FIG. 10A schematically illustrates the experimental design. FIG. 10B is a set of photographs that shows tumor burden in mice before and after CAR T cell infusion and CAR-enhancer treatment. FIG. 10C is a Kaplan-Meier plot that shows survival analysis. FIG. 10D is a line plot that shows flow cytometric analyses of CAR T cells in blood samples. FIG. 10E is a set of bar plots that show generation of memory CAR T cells. FIGs. 10F and 10G are a set of flow cytometric plots and bar plots showing that a substantial number of CAR T cells two months post-CAR T cell injection. FIG. 1 OH is a line plot showing that mice maintained consistent body weight throughout the experiment. FIG. 101 is a set of flow cytometric plots that show CAR T cells from CAR-enhancer treated mice have a stem-cell memory phenotype. FIG. 10J is a set of tSNE plots displaying FlowSOM defined clusters among persisting BCMA CAR T cells.

[00020] FIGs. 11A - HE are a set of schematics, photographs, and line, bar, and tSNE plots showing that CAR-enhancer treatment results in CAR T cell persistence in vivo. FIG. 11A schematically illustrates the experimental design. FIG. 1 IB is a set of photographs that show tumor burden in mice before and after CAR T cell infusion and CAR-enhancer treatment. FIG. 11C is a set of flow cytometric plots showing persistence of CAR T cells. FIG. 1 ID is a set of bar plots showing in vitro killing assays of persistent T cells. FIG. 1 IE is a set oft-SNE plots showing immune cell markers from CD8⁺ T cells.

[00021] FIGs. 12A - 12B are a schematic and a set of bar plots showing that CAR-E treatment expands CAR T cells in vivo in the absence of tumor cells. FIG. 12A schematically illustrates the experimental design. FIG. 12B is a set of bar plots that show counts of CAR T cells 30 days-post injection.

[00022] FIGs. 13A- 13B are a set of flow cytometry plots showing that neither the BCMA-muIL2 nor the VHH-muIL2 treatment exhibited binding to any specific population within human PBMCs. PBMCs were labeled with various markers to pre-gate B cells (CD20), T cells (CD3), or myeloid cells (CDl lb). Cells were stained using different concentrations of the treatments followed by an anti -FLAG- Al exa647 secondary staining. FIG. 13 A is a set of flow cytometry plots showing that BCMA-muIL2 does not bind to human PBMCs. FIG. 13B is a set of flow cytometry plots showing that VHH-muIL2 does not bind to human PBMCs.

[00023] FIGs. 14A - 14C are a set of photomicrographs and dot plots showing specific binding and gradual internalization of the BCMA-muIL2 in CAR T cells. FIG. 14A is a set of photomicrographs that show cells stained with CellTracker Blue CMAC, incubated with the indicated treatment, each treatment labeled with Alexa647 (BCMA-muIL2) or dsRed (VHH- muIL2) for 1 to 5 hours and imaged. Photomicrographs are representative of >100 cell images. FIG. 14B is a dot plot that shows quantitative analysis of the imaged cells. FIG. 14C is a dot plot that shows the correlation between Alexa647 mean intensity (BCMA-muIL2) and dsRed mean intensity (VHH-muIL2).

[00024] FIGs. 15A - 15C are a set of flow cytometry plots showing individual flow cytometric data corresponding to the pooled data presented in FIG. 9D. FIG. 15A is a set of flow cytometry results from mice treated solely with CAR T cells. FIG. 15B is a set of flow cytometry results from mice treated with CAR T cells and VHH-muIL2. FIG. 15C is a set of flow cytometry results from mice treated with CAR T cells and BCMA-muIL2.

[00025] FIGs. 16A - 16C are a set of flow cytometry plots showing individual flow cytometric data of the mice shown in FIGs. 10A - 10J. FIG. 16A is a set of flow cytometric results from mice treated solely with CAR T cells. FIG. 16B is a set of flow cytometry results from mice treated with CAR T cells and VHH-muIL2. FIG. 16C is a set of flow cytometry results from mice treated with CAR T cells and BCMA-muIL2. [00026] FIGs. 17A - 17C are a set of bar, line, and tSNE plots showing human T cell-derived cytokines in the serum of mice that received 0PM2 cancer cells followed by a low dose of CAR T cells. FIG. 17A is a bar plot that shows levels of IFNy, GM-CSF, and TNFa. Serum samples were diluted at a ratio of 1 :40. The same plates were used to incubate both the standard samples and the serum samples, and a standard curve was plotted for each cytokine. FIG. 17B is a set of line plots that shows IFNy levels between the BCMA-muZL2 group and the VHH-muIL2 group (error bars represent mean with standard deviation). FIG. 17C is a set of Flt-SNE mapping of CAR T cells derived from the PBS, BCMA-muIL2 and VHH-muIL2 treated mice showing the expression of ten immune cell markers.

[00027] FIG. 18 is a set of t-SNE mapping of CD4⁺ CAR⁺ T cells derived from the five BCMA- muIL2 CAR-E treated mice showing the expression of nine immune cell markers.

[00028] FIGs. 19A - 19G are a set of flow cytometry, tSNE, bar, violin, and pie plots and heatmaps showing single-cell RNA sequencing analyses elucidate BCMA-muIL2 effect on CAR T cells. FIG. 19A is a set of flow cytometry plots showing CAR¹ cells analyzed 89 days after CAR-T administration. FIG. 19B is a tSNE plot that shows data after Harmony algorithm, showing proportion of CD4, CD8, and proliferating (CD4 and CD8) cells. FIG. 19C is a tSNE plot that shows split between the groups treated with BCMA-muIL2 or VHH-muIL2 treatments. FIG. 19D is a set of heatmaps of significantly differentially expressed genes in CD4⁺ CAR T cells and CD8⁺ CAT T cells after the indicated treatment. FIG. 19E is a set of violin plots of the gene scores between CD8 and CD4 cells, the scores being constructed using the normalized expression of the different genes for each phenotype in FIG. 19D. FIG. 19F is a set of pie plots that shows the diversity of T-cell receptor (TCR) clonotypes. FIG. 19G is a bar plot that shows clonotype diversity within a sample’s total cell count.

[00029] FIGs. 20A - 20B are a set of schematics and flow cytometry plots showing that CAR- enhancer treatment results in enhanced organ trafficking of CAR T cells in vivo. FIG. 20A schematically illustrates the experimental design. FIG. 20B is a set of flow cytometry plots that show selective trafficking, expansion, and persistence of BCMA CAR T cells.

[00030] FIGs. 21 A - 21 C are a set of line and bar plots showing the effects of CAR-enhancers on non-transduced T cells and CAR T cells. FIGs. 21A and 21B are a line and bar plot, respectively, that together show dose-dependent activation of CAR T cells after CAR-enhancer treatment (FIG. 21A) and that the CAR-enhancer does not activate non-transduced T cells (FIG. 21B). FIG. 21 C is a line plot showing that the CD19-muIL2 CAR-enhancer does not block the killing efficacy of the CD 19 CAR T cells or non-transduced T cells (NT T cells).

[00031] FIGs. 22A - 22G are a set of schematics, photographs, line, and pie graphs showing that lower doses of CAR-enhancers enhance CAR T cell activity and promote functional memory. FIG. 22A schematically illustrates the experimental design. FIG. 22B is a set of photographs that shows bioluminescence imaging (BLI) of monitored tumor burdens. FIG. 22C is a survival analyses showing that all CAR-enhancer treated mice survived for the duration of the experiment. FIG. 4D is a line graph that shows the quantification of BLI analyses from FIG. 22B. FIG. 22E is a line graph that shows flow cytometric analyses of CAR T-cell presence in blood. FIG. 22F is a line graph that shows IFN-y levels. FIG. 22G is a set of pie graphs that show the CAR T cells in the bone marrow and spleen of mice treated with CAR-enhancer.

[00032] FIGs. 23 A - 23 G are a set of schematics, bar, and line graphs showing treatment of CAR- enhancer expands CAR T cells in in vivo in the absence of tumor cells in a dose-dependent manner. FIG. 23A schematically illustrates the experimental design. FIGs. 23B - 23C are a set of bar and line graphs showing flow cytometric analysis of spleen and bone marrow tissues harvested 30 days post-injection of CAR T cells. FIGs. 23D - 23E are a set of bar graphs that show analyses of persisting CAR T cells and different subsets of memory CAR T cells in the spleen and bone marrow of the CAR-E-treated mice. FIG. 23F is a bar graph showing that both the antigen and the low- affinity IL-2 components of CAR-enhancer are essential for its impact.

[00033] FIGs. 24A - 24M are a set of schematics, line, bar, and dot plots showing substantial activation and transcriptomic changes in CAR T cells after CAR-E treatment. FIG. 24A is a line plot that shows CAR-E induction of pSTAT5 activity in CAR T cells with either the full CAR construct or the CAR-ICD-A construct. FIGs. 24B - 24D are a set of line plots that show CAR-E CD69 (FIG. 24B), IFN-y (FIG. 24C), and TNF-a (FIG. 24D) staining in BCMA CAR T cells and BCMA CAR-ICD-A T cells. FIGs. 24E - 24G are a set of line plots that show CD69 (FIG. 24E), IFN-y (FIG. 24F), and TNF-a (FIG. 24G) staining after CAR-E treatment with dasatinib or ruxolitinib on CAR T cells. FIG. 24H schematically illustrates the experimental design for in vivo assessment of the efficacy of CAR-E on BCMA CAR T cells and BCMA CAR-ICD-A T cells. FIG. 241 is a set of bar plots that show expansion and persistence of BCMA CAR T cells and BCMA CAR-ICD-A T cells in mouse organs 1 month after CAR T cell injection; **** P<0.0001. FIG. 24J is a volcano plot that shows the highest upregulated genes in CD8⁺ CAR T cells 4 hours after CAR- E. treatment. FIG. 24K is a volcano Plot that shows the highest upregulated genes in CD8⁺ CAR- ICD-A T cells 4 hours after CAR-E treatment. FIG. 24L is a heatmap that shows gene expression changes in CD8⁺ and CD4⁺ T cells after 4 hours of CAR-E treatment. FIG. 24M is a heatmap that shows gene expression changes in CD8⁺ and CD4⁺ T cells after 4 and 24 hours of CAR-E treatment. [00034] FIGs. 25 a set of flow cytometry plots showing anti-human-CD45 and BCMA-CAR staining in blood samples of mice that received human CAR T cells with and without CAR-E treatment.

[00035] FIGs. 26A - 26B are a set of flow cytometry plots showing anti-human-CD45 and BCMA-CAR staining in individual mice that received human CAR T cells with and without CAR- E treatment. FIG. 26A is a set of flow cytometry plots of mouse organs collected at different time points after CAR T cell administration. FIG. 26B is a set of flow cytometry plots of mouse organs collected at different time points after CAR T cell and CAR-E administration.

[00036] FIGs. 27A - 27D are a set of heatmaps and tSNE plots showing CAR-E promotion of phyotypic diversity of bone marrow and splenocyte-derived CAR T cells. FIG. 27A is a heatmap that shows eight FLOW SOM-derived metaclusters in bone marrow samples. FIG. 27B is a heatmap that shows eight FLOW SOM-derived metaclusters in spleen samples. FIG. 27C is a tSNE plot that shows CAR T populations in bone marrow samples. FIG. 27D is a tSNE plot that shows CAR T populations in spleen samples.

[00037] FIGs. 28A - 28D are a set of flow cytometry plots showing anti-human-CD45 and BCMA-CAR staining in individual mice that received human CAR T cells and CAR-E treatment. FIG. 28A is a set of flow cytometry plots of mice that received CAR T cells and PBS control. FIG. 28B is a set of flow cytometry plots of mice that received CAR T cells and 2 mg/kg BCMA-muIL2. FIG. 28C is a set of flow cytometry plots of mice that received CAR T cells and 4 mg/kg BCMA- muIL2. FIG. 28D is a set of flow cytometry plots of mice that received CAR T cells and 8 mg/kg BCMA-muIL2.

[00038] FIGs. 29A - 29E are a set of flow cytometry plots showing anti-human-CD45 and BCMA-CAR staining in individual mice that received human CAR T cells and CAR-E treatment. FIG. 29A is a set of flow cytometry plots of mice that received CAR T cells and BCMA-CH3 control. FIG. 29B is a set of flow cytometry plots of mice that received CAR T cells and low dose IL-2. FIG. 29C is a set of flow cytometry plots of mice that received CAR T cells and VHH-muIL2. FIG. 29D is a set of flow cytometry plots of mice that received CAR T cells and BCMA-muIL2.

FIG. 29E is a set of flow cytometry plots of mice that received CAR T cells only.

[00039] FIG. 30 is a set of bar plots showing transcriptome changes in CAR T cells after CAR-E treatment.

DETAILED DESCRIPTION OF THE DISCLOSURE

[00040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated to facilitate the understanding of the present disclosure. [00041] As used in the description and the appended claims, the singular forms “a”, “an”, and “the” mean “one or more” and therefore include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an inhibitor” includes mixtures of two or more such inhibitors, and the like.

[00042] Unless stated otherwise, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term “about.”

[00043] The term “approximately” as used herein refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[00044] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of’ excludes any element or method step not specified in the claim (or the specific element or method step with which the phrase “consisting of’ is associated). The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified elements and method or steps and “unrecited elements and method steps that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure.

CAR-enhancer

[00045] The present disclosure provides a method of enhancing activity of CAR immune cells. The method entails, administering to a subject having had CAR immune cell therapy that targets an antigen present on a cancer cell, a first course of an effective amount of CAR-enhancer therapy, wherein the CAR-enhancer comprises a first moiety that binds an epitope on an extracellular domain (ED) of the CAR connected to a second moiety comprising a first immune cell effector domain, wherein administration of the first course of CAR-enhancer therapy is initiated at any time up to about 6 months after the subject received the CAR immune cell therapy.

[00046] The CAR-enhancer, also referred to herein as a CAR-engager or CAR-E, contains a first proteinaceous moiety and a second proteinaceous moiety. The first proteinaceous moiety binds an epitope on an extracellular domain (EB) of the CAR. In some embodiments the first proteinaceous moiety binds an epitope on the extracellular binding domain (EBD) of the CAR that binds a cancer antigen on the surface of a cancer cell. In some embodiments the first proteinaceous moiety binds an epitope on the ED of the CAR that does not bind the cancer antigen, e.g., a linker, such as a G4S (GGGGS (SEQ ID NO: 71)), or the framework of the antibody-fragment that is used as the CAR binding moiety, or any other part of the CAR. In some embodiments, the first moiety comprises an ectodomain of an antigen present on a cancer cell (also referred to herein as a cancer antigen). In some embodiments, the first moiety is an antibody that binds an epitope on the ED of the CAR or an ED-binding derivative thereof, such as a fragment of an antibody. The second proteinaceous entity comprises an immune cell effector domain connected to the first moiety. The ectodomain of an antigen is at least a portion of the antigen that is exposed on the cancer cell surface. The first moiety (e.g., an ectodomain) binds an extracellular domain of the CAR presented on the immune cell. The immune cell effector domain binds a cognate receptor on the same immune cell.

[00047] In some embodiments, CAR-enhancer is a contiguous protein, where the first proteinaceous moiety and the second proteinaceous moiety are connected by a peptide bond. In some embodiments, the first proteinaceous moiety and the second proteinaceous moiety are covalently connected by click chemistry.

[00048] In some embodiments, the CAR-enhancer is formulated and administered as a monomeric protein or proteinaceous entity. In other embodiments, the CAR-enhancer is formulated and administered in the form of a dimer, either as a homodimer or a heterodimer protein or proteinaceous entity.

Ectodomain

[00049] In some embodiments, the first moiety of the CAR-engager is an ectodomain that binds an EBD of a CAR presented on an immune cell. As is known in the art, the ectodomain of a cancer antigen is the portion of the antigen on the surface of a cancer cell that binds a T cell receptor or a CAR on an immune cell. The binding between CAR and cancer antigen may be direct or indirect. In direct binding embodiments, the ectodomain may be formed by contiguous or non-contiguous amino acid residues in the extracellular domain of a cancer antigen or may be an antibody or antibody fragment (including nanobody and nanobody fragments) that binds the CAR presented on an immune cell. In some embodiments, the CAR-enhancer may include the entire extracellular domain of a cancer antigen. Ectodomains may be derived from (e.g, identified in) a cancer antigen in accordance with standard techniques. See, e.g., and Gershoni el al., Biodrugs 2/(3 145-156 (2007) and Francino-Urdaniz and Whitehead, RSC Chem. Biol. 2 (6) : 1580-1589 (2021). The term “derived from” as used herein when referring to a protein and nucleic acid refers to a sequence that originates and is identified from the sequence of a parent (e.g., wild-type or endogenous) protein and nucleic acid, respectively. A sequence derived from a parent sequence may be identical, may be a portion of the parent sequence, or may have at least one variant from the parent sequence. Variants may include substitutions, insertions, or deletions. Thus, for example, an amino acid sequence derived from a parent sequence may be identical for a specific range of amino acids of the parent but does not include amino acids outside that specific region.

The amino acid sequences of representative cancer antigens from which an ectodomain may be derived are provided at the NCBI Accession numbers set forth in [00050] Table 1, and are incorporated herein by reference.

Table 1 : Gene Name, Symbols, and NCBI Accession Numbers of Representative Cancer Antigens

[00051] The ectodomains are not limited to known cancer antigens. Unique cancer antigens (neoantigens) may be determined by known methods. For example, cancer genomes can be compared with normal cell genomes to identify neoantigens. In some embodiments, caner transcriptomes are compared to normal cell transcriptomes. Computational methods may then be utilized to identify suitable binding sites for a CAR. Most often the CAR binds a portion of the extracellular domain of an antigen. In some embodiments, the CAR and the corresponding cancer antigen are known in the art.

[00052] In some embodiments, the ectodomain of the CAR-enhancer contains the entire extracellular domain of a cancer antigen. In some embodiments, the CAR-enhancer contains a portion of the extracellular domain of a cancer antigen which is targeted by a CAR.

[00053] In some embodiments, the ectodomain of the CAR-enhancer contains the extracellular domain of BCMA. The amino acid sequence of a representative CAR-enhancer that contains a BCMA extracellular domain is

NO: 1).

[00054] In some embodiments, the ectodomain of the CAR-enhancer contains two repetitions of the extracellular domain of BCMA. The amino acid sequence of a representative CAR-enhancer that contains two repetitions of the BCMA extracellular domain is set forth below (SEQ ID NO: 2):

[00055] In some embodiments, the ectodomain of the CAR-enhancer contains a variant of the extracellular domain of CD 19. The amino acid sequence of a representative CAR-enhancer that contains a variant of CD19 extracellular domain is set forth below (SEQ ID NO: 3):

[00056] In some embodiments, the ectodomain has at least 85% sequence identity to SEQ ID NO: 3, at least 90% sequence identity to SEQ ID NO: 3, at least 95% sequence identity to SEQ ID NO: 3, at least 98% sequence identity to SEQ ID NO: 3, at least 99% sequence identity to SEQ ID NO: 3.

[00057] The amino acid sequence of a representative CAR-enhancer that contains a second variant of CD19 extracellular domain is set forth below (SEQ ID NO: 4):

[00058] In some embodiments, the ectodomain of the CAR-enhancer contains the extracellular domain of CD 19. The amino acid sequence of a representative CAR-enhancer that contains a CD 19 extracellular domain set forth below (SEQ ID NO: 5): [00059] In some embodiments, the ectodomain of the CAR-enhancer contains a portion extracellular domain of CD 19. In some embodiments, the ectodomain of the CAR-enhancer is KDRPEIWEGEPP (SEQ ID NO: 103), which corresponds to positions 142-153 of SEQ ID NO: 5. [00060] In some embodiments, the ectodomain of the CAR-enhancer contains the extracellular domain of CD20. The amino acid sequence of a representative CAR-enhancer that contains a CD20 extracellular domain is KISHFLKMESLNEIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQS (SEQ ID NO: 6).

[00061] In some embodiments, the ectodomain of the CAR-enhancer contains the extracellular domain of CD22. The amino acid sequence of a representative CAR-enhancer that contains a CD22 extracellular domain is set forth below (SEQ ID NO: 7):

[00062] In some embodiments, the ectodomain of the CAR-enhancer contains a portion of the extracellular domain of CD22 (SEQ ID NO: 7). In some embodiments, the ectodomain of the CAR- enhancer contains the Ig domains 2-3 of CD22. The amino acid sequence of a representative CAR- enhancer that contains Ig domains 2-3 of CD22 is set forth below (SEQ ID NO: 104):

[00063] In some embodiments, the ectodomain of the CAR-enhancer contains the Ig domain 3 of CD22. The amino acid sequence of a representative CAR-enhancer that contains an Ig domain 3 of CD22 is set forth below (SEQ ID NO: 105):

[00064] In some embodiments, the ectodomain of the CAR-enhancer contains the Ig domains 5-7 of CD22. The amino acid sequence of a representative CAR-enhancer that contains Ig domains 5-7 of CD22 is set forth below (SEQ ID NO: 106):

[00065] In some embodiments, the ectodomain of the CAR-enhancer contains the Ig domains 5-7 of CD22. The amino acid sequence of a representative CAR-enhancer that contains Ig domains 6-7 of CD22 is set forth below (SEQ ID NO: 107):

[00066] In some embodiments, the ectodomain of the CAR-enhancer contains an extracellular domain of Claudin 18.2. The amino acid sequence of a representative CAR-enhancer that contains a Claudin 18.2 first extracellular domain is set forth below (SEQ ID NO: 8):

[00067] The amino acid sequence of a representative CAR-enhancer that contains a Claudin 18.2 second extracellular domain is set forth below (SEQ ID NO: 9):

1 vtnfwmstan mytgmggmvq tvqtrytfga a

[00068] In some embodiments, the ectodomain of the CAR-enhancer contains the extracellular domain of SLAMF7. The amino acid sequence of a representative CAR-enhancer that contains a SLAMF7 extracellular domain is set forth below (SEQ ID NO: 10):

[00069] In some embodiments, the ectodomain of the CAR-enhancer contains the extracellular domain of PD-1. The amino acid sequence of a representative CAR-enhancer that contains a PD-1 extracellular domain is set forth below (SEQ ID NO: 11):

[00070] In some embodiments, the ectodomain of the CAR-enhancer contains a variant of the extracellular domain of PD-1. In some embodiments, the ectodomain of the CAR-enhancer contains the N-loop of PD-1. The amino acid sequence of a representative CAR-enhancer that contains the N-loop of the PD-1 extracellular domain is LDSPDRPWNP (SEQ ID NO: 108), which corresponds to positions 2-11 of SEQ ID NO: 11. [00071] In some embodiments, the ectodomain of the CAR-enhancer contains the CD-loop of PD- 1. The amino acid sequence of a representative CAR-enhancer that contains the CD-loop of the PD- 1 extracellular domain is NQTDKLAAFPEDRSQPGQDCRFRVTQ (SEQ ID NO: 109), which corresponds to positions 51-76 of SEQ ID NO: 11.

[00072] In some embodiments, the ectodomain of the CAR-enhancer contains the extracellular domain of KIT. The amino acid sequence of a representative CAR-enhancer that contains a KIT extracellular domain is set forth below (SEQ ID NO: 12):

[00073] In some embodiments, the ectodomain of the CAR-enhancer contains the extracellular domain of TROP2. The amino acid sequence of a representative CAR-enhancer that contains a

TROP2 extracellular domain is set forth below (SEQ ID NO: 13):

[00074] In some embodiments, the ectodomain of the CAR-enhancer contains the extracellular domain of CD38. The amino acid sequence of a representative CAR-enhancer that contains a CD38 extracellular domain is set forth below (SEQ ID NO: 14):

[00075] In some embodiments, the ectodomain of the CAR-enhancer is derived from mesothelin

(MSLN). MSLN is a GPI-anchored protein, therefore the entire MSLN protein is extracellular. The amino acid sequence of a representative MSLN is set forth below (SEQ ID NO: 15): [00076] In some embodiments, the ectodomain c ^? the CAR-enhancer contains a portion of an extracellular domain of a cancer antigen. In some embodiments, the ectodomain of the CAR- enhancer contains a portion of the MSLN protein. In some embodiments, the ectodomain of the CAR-enhancer is IPNGYLVLDLSMQEALS (SEQ ID NO: 16). In some embodiments, the ectodomain of the CAR-enhancer is YNVNDLSMQEL (SEQ ID NO: 17), where N is any amino acid.

CAR-binding antibodies

[00077] The first moiety of the CAR-enhancer may be an antibody that binds an epitope on the ED of the CAR, or an ED-binding derivative thereof. Antibody derivatives may be an antibody fragment (e.g., a scFv) which includes nanobody fragments.

[00078] In some embodiments, the first moiety of the CAR-enhancer is an anti-anti-CD19 antibody binding moiety that binds an epitope on a CAR the EBD of which binds CD 19. A representative anti-anti-CD19 binding moiety is set forth below (SEQ ID NO: 111):

[00079] In some embodiments, the first moiety of the CAR-engager binds another an epitope on portion of the ED that does not engage the cancer antigen, such as a linker. In some embodiments, for example, the EBD of the CAR includes a linker containing a G4S motif, and the first moiety of the CAR-engager may be an anti-(G4S) binding moiety that binds an epitope on a CAR the linker of which has at least two repeats of GGGGS (SEQ ID NO: 71). A representative heavy chain variable region (VH) of an anti-(G4S) binding moiety is set forth below (SEQ ID NO: 112):

[00080] A representative light chain variable region (VL) of an anti-(G4S) binding moiety is set forth below (SEQ ID NO: 113): [00081] In some embodiments, the first moiety of the CAR-enhancer is an anti-K light chain antibody binding moiety that binds an epitope on a CAR the EBD of which is contains a K light chain. A representative anti-K light chain binding moiety is set forth below (SEQ ID NO: 110).

[00082] In some embodiments, the first moiety of the CAR-enhancer is an anti-anti-mouse antibody binding moiety that binds an epitope on a CAR the EBD of which is derived from antibodies originating from a mouse. Representative anti-anti-mouse binding moieties are known in the art, see, Kochenderfer et al., J. Immunother. 32(7) :689-702 (2009) and Cheng etal, Cytometry A. 703(7): 16-26 (2023).

[00083] Additional antibodies and derivatives thereof that bind CAR EDs that may be useful are known in the art, see, e.g., U.S. Patent 9,701,758 and U.S. Patent Application Publication 2005/0287148, both of which are incorporated by reference in their entirety herein.

Second Moiety - Immune cell effector domain

[00084] The second moiety is an immune cell effector domain binds a cognate receptor on the immune cell that expresses the CAR-encoding nucleic acid. This binding event modulates the activity of the CAR-immune cell. The terms “modulate(s),” and “modulation” as used herein embrace both activation and inhibition of the CAR immune cell. Accordingly, the immune cell effector domain may be a cytokine, an immune cell-activating moiety, or an immune cell-inhibiting moiety, and variants and fragments thereof that bind to their cognate targets. The term “cytokine”, as is known in the art, includes low molecular weight extracellular polypeptides/glycoproteins that promote, modulate, and regulate the immune response (i.e., increase or decrease activity, differentiation, or proliferation). Representative examples of cytokines include chemokines, interferons (IFNs), interleukins (Ils), lymphokines and tumor necrosis factors (TNFs). The term “immune cell-activating variant” of a cytokine as used herein refers to non-naturally occurring variant of a cytokine capable of binding to a cytokine receptor on an immune cell and initiating signal transduction through that receptor to achieve substantially the same effect as the naturally occurring cytokine.

[00085] In some embodiments, the CAR-enhancer contains a plurality (z.e., two or more) of immune cell effector domains, any two or more of which may be the same as or different from each other. In some embodiments, the CAR-enhancer contains two immune cell effector domains. In some embodiments, the CAR-enhancer contains three immune cell effector domains.

[00086] In some embodiments, the immune cell effector domain is an immune cell activating moiety, e.g, immune cell activating cytokines and immune cell-activating variants and fragments thereof. Immune cell activating moi eties activate, promote, or maintain the activity of immune cells. [00087] In some embodiments, the immune cell effector domain is derived from CD40, CD48, CD58, CD70, CD80, CD86, CD112, glucocorticoid-induced TNFR-related protein ligand (GITRL; TNFSF18), herpesvirus entry mediator (HVEM; TNFSF14), Semaphorin 3B (SEMAA; SEMA3B), Signaling lymphocytic activation molecule family member 1 (SLAM; SLAMF1; CD 150), T cell immunoglobulin and mucin domain containing 4 (TIM4), TNF superfamily member 4 (TNFSF4; OX40L), TNF superfamily member 8 (TNFSF8; CD30L), interleukin-2 (IL-2), IL-7, IL-9, IL-10, IL-12, IL-15, IL-18, IL-21, IL-27, CCL21, 4-1BBL (also known as TNF superfamily member 9; TNFSF9), or an immune cell-activating variant thereof.

[00088] The amino acid sequences of representative immune cell activating moieties (c.g, cytokines) are from which the immune cell effector domain may be derived are provided at the NCBI Accession numbers set forth in Table 2, and are incorporated herein by reference.

Table 2: Gene Name, Symbols, and NCBI Accession Numbers of Representative Immune Cell- Activating Proteins

[00089] In some embodiments, the immune cell effector domain is the wild-type IL-2, having the amino acid sequence set forth below (SEQ ID NO: 102; NCBI Accession No. NP_000577):

[00090] In some embodiments, the immune cell effector domain is a synthetic, i.e., non-naturally occurring IL-2, which is a variant of the wild-type IL-2 (SEQ ID NO: 102) in that it has an amino acid substitution at positions 16 and/or 42.

[00091] In some embodiments, the immune cell effector domain has an H16A substitution (i.e., an alanine (A) at position 16 in place of the histidine (H)) relative to SEQ ID NO: 102 and/or an F42A substitution (i.e., an alanine (A) at position 42 in place of the phenylalanine (F)) relative to SEQ ID NO: 102, both substituted alanine residues shown as boxed amino acids in SEQ ID NO: 19). In some embodiments, the immune cell effector domain is the weak affinity variant of IL-2 (muIL2), having the amino acid sequence set forth below (SEQ ID NO: 19), which contains the H16A and F42A substitutions, as follows:

[00092] By “weak affinity,” it is meant that the natural (wild-type) sequence of human IL-2 (SEQ ID NO: 102) has higher affinity for the IL -2 receptor (IL-2R) than SEQ ID NO: 19. [00093] More specifically, the muIL2 (SEQ ID NO: 19) immune cell effector domain has a dissociation constant (KD) of about 1200 nM for IL-2Ra (CD25), representing a 110-fold decrease as compared to wild type IL-2, and a KD of about 610 nM for IL-2RP, representing a 3-fold decrease as compared to wild type IL-2.

[00094] In some embodiments, the CAR-enhancer contains more than one immune cell effector domain. The immune cell effector domains in the CAR-enhancer can be the same or different.

[00095] In some embodiments, the CAR-enhancer contains two immune cell effector domains, c.g., first and the second immune cell effector domains that are the weak affinity IL-2 variants, having together, the amino acid sequence set forth below (SEQ ID NO: 20):

[000101] In some embodiments, the immune cell effector domain is an immune cell-activating variant of a cytokine. In some embodiments, the immune cell effector domain is neoleukin-2/15 (Neo-2/15), which binds to the IL-2R-0, having the amino acid sequence set forth below (SEQ ID NO: 26).

[000102] In some embodiments, the CAR-enhancer contains Neo-2/15 two immune cell effector domains, each having the amino acid sequence of SEQ ID NO: 26.

[000103] In some embodiments, the immune cell effector domain may be derived from 4-1BBL. 4- 1BBL is also known as TNF ligand superfamily member 9 (TNFSF9). The amino acid sequence of a representative 4-1BBL is provided at NCBI Accession No. NP_003802, incorporated herein by reference. In some embodiments, the immune cell effector domain may be derived from the extracellular domain of 4-1BBL. In some embodiments, the immune cell effector domain contains a portion of the extracellular domain of 4-1 BBL, having the amino acid sequence set forth below (SEQ ID NO: 27):

[000104] In some embodiments, the CAR-enhancer contains three immune cell effector domains, e.g., wherein all of the first, the second, and the third immune cell effector domains are the extracellular domain of 4-1BBL, each having the amino acid sequence of SEQ ID NO: 27.

[000105] In some embodiments, the immune cell effector domain may be a fragment, e.g., a singlechain variable antibody fragment (scFv), that binds and activates the CAR immune cell. In some embodiments, the immune cell effector domain is an scFv that binds an epitope on to 4- IBB, CD2, CD27, CD28, CD30 (TNFRSF8), CD40L, CD226, CTLA4, GITR, IL-2R, LIGHT, 0X40, PD-1, TIM2, SLAM, or TIM1.

[000106] In some embodiments, the immune cell effector domain is a scFv that binds CTLA4. In some embodiments, the immune cell effector domain is derived from a commercially available anti- CTLA4 antibody, antibody fragment, or derivative thereof, e.g., bavunalimab (formerly pavunalimab/XmAb 22841), botensilimab, cadonilimab, ipilimumab (Yervoy®), quavonlimab, tremelimumab (Imjudo®), volrustomig, vudalimab, or zalifrelimab. The amino acid sequences of representative heavy and light chains of antibodies that bind CTLA4 are set forth in Table 3.

Table 3: Amino acid Sequences of Representative anti-CTLA Antibody Heavy and Light Chains

[000107] In some embodiments, the immune cell effector domain contains the VL having the amino acid sequence set forth below (SEQ ID NO 36):

1 eivltqspgt Islspgerat Iscraqsvsr ylgwyqqkpg qaprlliyga stratgipdr 61 fsgsgsgtdf tltitrlepe dfavyycqqy gsspwtfgqg tkveik

[000108] In some embodiments, the immune cell effector domain contains the VH having the amino acid sequence set forth below (SEQ ID NO 37):

1 evqlvesggg Ivkpggslrl scaasgftfs sysmnwvrqa pgkglewvss isssssyiyy 61 aesvkgrfti srdnaknsly Iqmnslraed tavyycarvg Ifgpfdiwgq gtlvtvss [000109] In some embodiments, the immune cell effector domain binds 0X40. In some embodiments, the immune cell effector domain is derived from a commercially available anti-OX40 antibody, antibody fragment (e.g., scFv), or derivative thereof, e.g., tavolimab, or vonlerolizumab (Pogalizumab; MOXR 0916). The amino acid sequences of representative heavy and light chains of which are set forth in Table 4.

Table 4: Amino acid Sequences of Representative anti-OX40 Antibody Heavy and Light Chains

[000110] In some embodiments, the immune cell effector domain contains the VL having the amino acid sequence set forth below (SEQ ID NO 42):

1 diqmtqspss Isasvgdrvt itcrasqdis nylnwyqqkp gkapklliyy tsrlrsgvps 61 rfsgsgsgtd ftltisslqp edfatyycqq ghtlpptfgq gtkveik

[000111] In some embodiments, the immune cell effector domain contains the VH having the amino acid sequence set forth below (SEQ ID NO 43):

1 evqlvqsgae vkkpgasvkv sckasgytft dsymswvrqa pgqglewigd mypdngdssy 61 nqkfrervti trdtststay lelsslrsed tavyycvlap rwyfsvwgqg tlvtvss

[000112] In some embodiments, the immune cell effector domain binds PD-1. In some embodiments, the immune cell effector domain is derived from a commercially available anti-PD- 1 antibody, antibody fragment (e.g, scFv), or derivative thereof, e.g, atezolizumab, avelumab, bintrafusp alfa, cosibelimab, danburstotug, durvalumab (Imfinzi®), inbakicept, lodapolimab, pimivalimab, or socazolimab. The amino acid sequences of representative heavy and light chains of which are set forth in Table 5.

Table 5: Amino Acid Sequences of Representative anti-PD-1 Antibody Heavy and Light Chains

[000113] In some embodiments, the immune cell effector domain contains the VL having the amino acid sequence set forth below (SEQ ID NO 52):

1 eivmtqspat Isvspgerat Iscrasqsvs snlawyqqkp qqaprlliyq astratqipa 61 rfsgsgsgte ftltisslqs edfavyycqq ynnwprtfgq gtkveik

[000114] In some embodiments, the immune cell effector domain contains the VH having the amino acid sequence set forth below (SEQ ID NO 53):

1 qvqlvesggg wqpgrslrl scaasgftfs sygmhwvrqa pgkglewvav iwydgsnkyy 61 adsvmgrfti srdnskntly Iqmnslraed tavyycasng dhwgqgtlvt vss

[000115] In some embodiments, the immune cell effector domain is an immune cell-inhibiting moiety, representative types of which include immune cell inhibiting cytokines and immune cell- inhibiting variants and fragments thereof. Immune cell inhibiting moieties repress or block immune cell activity and function. In some embodiments, the immune cell-inhibiting moiety may be derived from CD80, CD86, CD112, CD155, CD276 (B7-H3), Ceacam-1, FGL1, galectin-3, HLA-E, HVEM, PD-L1, PD-L2, VISTA, or VTCN1 (B7-H4). The amino acid sequences of representative immune cell-inhibiting proteins from which the immune cell effector domain may be derived are provided at the NCBI Accession numbers set forth in Table 6, and are incorporated herein by reference.

Table 6: Gene Name, Symbols, and NCBI Accession Numbers of Immune Cell-Inhibiting Proteins

[000116] In some embodiments, the immune cell effector domain is the extracellular domain of CD80. The amino acid sequence of a representative CD80 extracellular domain is set forth below

(SEQ ID NO: 54): [000117] In some embodiments, the immune cell effector domain is the extracellular domain of CD86. The amino acid sequence of a representative CD86 extracellular domain is set forth below

(SEQ ID NO: 55):

[000118] In some embodiments, the immune cell effector domain is the extracellular domain of

CD155 (nectin-5; PVR). The amino acid sequence of a representative CD155 extracellular domain is set forth below (SEQ ID NO: 56):

[000119] In some embodiments, the immune cell effector domain is the extracellular domain of

CD276 (B7-H3). The amino acid sequence of a representative CD276 extracellular domain is set forth below (SEQ ID NO: 57):

[000120] In some embodiments, the immune cell effector domain is the extracellular domain of

Ceacam-1. The amino acid sequence of a representative Ceacma-1 extracellular domain is set forth below (SEQ ID NO: 58):

[000121] In some embodiments, the immune cell effector domain is the extracellular domain of FGL1. The amino acid sequence of a representative FGL1 extracellular domain is set forth below

(SEQ ID NO: 59): [000122] In some embodiments, the immune cell effector domain is the extracellular domain of gal ectin-3. The amino acid sequence of a representative galecin-3 extracellular domain is set forth below (SEQ ID NO: 60): [000127] In some embodiments, the immune cell effector domain is the extracellular domain of PD-L2. The amino acid sequence set of a representative PD-L2 extracellular domain is forth below

(SEQ ID NO: 65):

[000128] In some embodiments, the immune cell effector domain is the extracellular domain of

VTCN1 (B7-H4). The amino acid sequence of a representative VTCN1 is set forth below (SEQ ID

NO: 66):

Dimerization domain

[000129] In some embodiments, the CAR-enhancer further includes a dimerization domain. In these cases, the CAR-enhancer forms and is administered in the form of a homodimer or a homomultimer. The homodimer thus contains two CAR-enhancer entities. The order of the ectodomain, the immune effector domain and the dimerization domain is not critical. In some embodiments, the dimerization domain is disposed between an ectodomain and an immune cell effector domain.

[000130] In some embodiments, the CAR-enhancer is in the form of a heterodimer, which contains a first entity containing an ectodomain of an antigen present on a cancer cell connected to a first dimerization domain and a second entity containing an immune cell effector domain connected to a second dimerization domain. In these embodiments, the first and second dimerization domains dimerize the first and second entities to form a heterodimer.

[000131] In some embodiments, the first and second dimerization domains contain a knob-in-hole configuration. One of the dimerization domains contains a protuberance (knob) and the other dimerization domain contains a cavity (hole) that is sterically compensatory to the protuberance, where the tertiary structure of the protuberance is positionable within the tertiary structure of the cavity. Dimerization domains with knob-in-hole configurations may have directed amino acid mutations where the protuberance is an amino acid that has a larger side chain volume than present on a dimerization domain derived from a natural source (e.g., IgA, IgD, IgG, IgM, or IgE) and the cavity is an amino acid that has a smaller side chain volume than present on a dimerization domain derived from a natural source.

[000132] In some embodiments, the protuberance is an amino acid change from a threonine (T) to a lysine (K) and the corresponding cavity is an amino acid change from a leucine (L) to an aspartic acid (D) or a lysine (K). In some embodiments, the first dimerization domain contains two amino acid substitutions, for example, a threonine (T) to a lysine (K) and a leucine (L) to a lysine (K), while the second dimerization domain contains a leucine (L) to an aspartic acid (D) or a glutamic acid (E) and a tyrosine (Y) to a glutamic acid (E) or aspartic acid (D).

[000133] In some embodiments, the knob-in-hole dimerization domains are based on opposed charges. In some embodiments, the first dimerization domain contains a positively charged amino acid and the second dimerization domain contains a negatively charged amino acid sterically opposable to the positively charged amino acid on the first dimerization domain.

[000134] Additional protuberance and cavity arrangements are known in the art. See, e.g., U.S. Patents 5, 821, 333, %7, 183, 076, 8,642,745, 9,248,182, 9,309,311, 9,527,927, 9,562, 109, 9,890,204, 10,138,303, and 11,168,344 and U.S. Patent Application Publications 2005/0079170, 2006/0025576, 2013/0089554, and 2014/0024111.

[000135] In some embodiments, the dimerization domains may be derived from IgA, IgD, IgG, IgM, or IgE. The first and the second dimerization domains may contain the same or different amino acid sequences, provided that they bind each other. In some embodiments, the first and the second dimerization domains are the IgGl constant heavy (CH) 3 domain. The amino acid sequence of a representative IgGl CH3 domain is set forth below (SEQ ID NO: 67):

[000136] In some embodiments, the first and the second dimerization domains are the IgGl constant heavy CH2 domain. The amino acid sequence of a representative IgGl CH2 domain is set forth below (SEQ ID NO: 68):

[000137] In some embodiments, the first and the second dimerization domains are the IgGl CH2 and CH3 domains. The CH2 and CH3 domains may be interconnected by a linker. In some embodiments, the first, the second, or both the first and the second dimerization domains contain a fragment crystallizable region (Fc). Tn some embodiments the Fc contains L234A, L235A, and P329G substitutions relative to wild-type Fc that abolishes binding of the Fc to (1) the Fc-y receptor and (2) the complement component Iq (Clq), referred herein as a “silent Fc”. The silent Fc maintains binding to the neonatal Fc receptor (FcRn) (and therefore extending circulatory half-life of CAR-E which contains the silent Fc to several days). Silent Fc also provides a stabilizing effect to the CAR-E (comparable to the stabilizing effect of wild-type Fc). The three L234A, L235A, and P329G substitutions are also commonly referred to as PG-LALA.

Linkers

[000138] In some embodiments, the CAR-enhancer contains one or more linkers. A linker may provide flexibility in terms of allowing the ectodomain and the immune cell effector domains to bind to their respective cognate receptors on the CAR-expressing immune cell or steric spacing (z.e., a spacer) between the ectodomain and the immune cell effector domain.

[000139] A linker may be disposed between any two CAR-enhancer components (also referred to herein as domains, entities or moi eties or portions) (e.g., the ectodomain and the immune cell effector domain).

[000140] A linker may be disposed between the dimerization domain and the adjacent domain. In some embodiments, a linker may be disposed between the dimerization domain and the immune cell effector domain. In some embodiments, the CAR-enhancer contains two linkers, where a first linker is disposed between the ectodomain and the dimerization domain, and a second linker is disposed between the dimerization domain and the immune cell effector domain.

[000141] In some embodiments, the linker comprises an amino acid having the sequence GGGX, GGGGX (SEQ ID NO: 69), or GSSGSX (SEQ ID NO: 70), where X is any nucleotide, typically either cysteine (C) or serine (S), or repeating sequence thereof. In some embodiments, the linker has the amino acid sequence GGGGS (SEQ ID NO: 71), GSPRG (SEQ ID NO: 72), GGGGSGGGGS (SEQ ID NO: 73), GGGGSGGGGSGGGGS (SEQ ID NO: 74), GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 75), GSPRGGGGSGGGGSGGGGS (SEQ ID NO: 76), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 77), KESGSVSSEQLAQFRSLD (SEQ ID NO: 78), EGKSSGSGSESKST (SEQ ID NO: 79), or GSAGSAAGSGEF (SEQ ID NO: 80).

[000142] In some embodiments, the linker may be derived from IgA, IgD, IgE, IgG, or IgM. In some embodiments, the linker may be derived from the hinge region of CD3^, CD4, CD8a, CD28, IgGl, IgG2, or IgG4. Amino acid sequences of representative linkers are listed in Table 7. Table 7: Amino acid Sequences of Representative Linkers

[000143] In some embodiments, the CAR-enhancer is in the form of a fusion protein, where the components are linked by peptide bonds. In other embodiments, the CAR-enhancer contains proteinaceous entities interconnected by click chemistry, a chemical connection formed by a method of controlled protein ligation. The connection may be an azide-alkyne connection, an oxime or hydrazine connection, a tetrazine-transcyclooctene connection, an azide-nitrone connection, a thiolalkene connection, an alkene-tetrazole connection, an alkene-tetrazine connection, an alkene-azide connection, a conjugated diene-alkene connection, or an isonitrile-tetrazine connection.

[000144] Additional controlled protein ligation chemistries, systems, and methods are known in the art. See, e.g., U.S. Patents 7,375,234, 7,763,736, 8,101,238, 8,372,986, 8,394,914, 8,877,170, 8,927,682, 8,927,736, 9,302,997, 9,896,547, 11,028,185, 11,091,588, and 11,352,460, and U.S. Patent Application Publication 2009/0069561 .

Nucleic Acids

[000145] In some embodiments, the CAR-enhancer may be encoded in a nucleic acid and used to express the CAR-enhancer. The term “nucleic acid” as used herein refers to a polymer of nucleotides, each of which are organic molecules consisting of a nucleoside (a nucleobase and a five-carbon sugar) and a phosphate. The term nucleotide, unless specifically stated or obvious from context, includes nucleosides that have a ribose sugar (/>., a ribonucleotide that forms ribonucleic acid, RNA) or a 2’ -deoxyribose sugar (z.c., a deoxyribonucleotide that forms deoxyribonucleic acid, DNA). Nucleotides serve as the monomeric units of nucleic acid polymers or polynucleotides. The four nucleobases in DNA are guanine (G), adenine (A), cytosine (C) and thymine (T). The four nucleobases in RNA are guanine (G), adenine (A), cytosine (C) and uracil (U). Nucleic acids are linear chains of nucleotides (e.g., at least 3 nucleotides) chemically bonded by a series of ester linkages between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar (/.<?., ribose or 2’-deoxyribose) in the adjacent nucleotide.

[000146] In some embodiments, the CAR-enhancer is encoded by two nucleic acids, e.g., the first moiety is encoded by a first nucleic acid and the second moiety is encoded by a second nucleic acid. [000147] In some embodiments, nucleic acid encoding the CAR-enhancer includes a signal peptide-encoding nucleic acid disposed 5’ to the nucleic acid encoding the first moiety. The term “signal peptide” as used herein refers to a short (e.g., 5-30 or 10-100 amino acids long) stretch of amino acids that directs the transport of the protein during translation. CAR-enhancers containing a signal peptide will be secreted from the cell. Typically, the signal peptide is cleaved from the CAR- enhancer before secretion. The signal peptide may be connected to the nucleic acid encoding the first moiety or the nucleic acid encoding the second moiety.

[000148] In some embodiments, the signal peptide may be derived from Ig-y-3 heavy chain (IGHG3), albumin, CD8a, CD33, erythropoietin (EPO), IL-2, human or mouse Ig-kappa chain V- III (IgK VIII), tissue plasminogen activator (tPA), or secreted alkaline phosphatase (SEAP). Signal peptides may also be synthetic (i.e., non-naturally occurring). Amino acid sequences of representative signal peptides are listed in Table 8.

Table 8: Amino acid Sequences of Representative Signal Peptides

Vectors

[000149] The CAR-enhancer-encoding nucleic acid may be introduced to a cell by a suitable vector. In embodiments, wherein the first moiety and the second moiety are linked chemically, e.g., via click chemistry, the CAR-encoding nucleic acids may be introduced into one or more cells by separate vectors. A vector is configured so as to contain the elements necessary to effect transport into the immune cell and effect expression of the nucleic acid(s) after transformation. Such elements include an origin of replication, a poly-A tail sequence, a selectable marker, and one or more suitable sites for the insertion of the nucleic acid sequences, such as a multiple cloning site (MCS), one or more suitable promoters, each promoter operatively linked to the insertion sites of the nucleic acid sequences and the selectable marker, and additional optional regulatory elements.

[000150] The term “promoter” as used herein refers to a nucleic acid sequence that regulates, directly or indirectly, the transcription of a corresponding nucleic acid coding sequence to which it is operably linked, which in the context of the present disclosure, is a CAR-enhancer protein. A promoter may function alone to regulate transcription, or it may act in concert with one or more other regulatory sequences (e.g., enhancers or silencers, or regulatory elements that may be present in the nucleic acid sequences or the vector). Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (toward the 5’ region of the sense strand). Promoters typically range from about 100-1000 base pairs in length.

[000151] The term “operatively linked” as used herein is to be understood that a nucleic acid sequence is spatially situated or disposed in the vector relative to another nucleic acid sequence, e.g., a promoter is operatively linked to drive the expression of a nucleic acid coding sequence (e.g., the CAR-enhancer-encoding nucleic acid sequence).

[000152] In some embodiments, a single vector contains a single promoter operatively linked to the CAR-enhancer-encoding nucleic acid. In some embodiments, a single vector contains a single promoter operatively linked to the first moiety-encoding nucleic acid and the second moiety- encoding nucleic acid. In some of these embodiments, the nucleic acids are separated by a nucleic acid encoding a self-cleaving peptide or an internal ribosome entry site (IRES). In some embodiments, the single vector contains a first promoter operatively liked to the first moiety- encoding nucleic acid and a second promoter operatively liked to the second moiety-encoding nucleic acid.

[000153] In some embodiments, two vectors are provided. In some embodiments, a first vector contains a promoter operatively linked to the first moiety-encoding nucleic acid and a second vector contains a promoter operatively linked to the second moiety-encoding nucleic acid.

[000154] In some embodiments, the vector contains a strong mammalian promoter, for example a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, synthetic promoters (e.g., RPBSA (synthetic, from Sleeping Beauty), or CAG (synthetic, CMV early enhancer element, chicken P-Actin, and splice acceptor of rabbit P-Globin)) or promoters derived from the P-actin, phosphoglycerate kinase (PGK), or factor EFla genes. In some embodiments, the promoter may contain a core region located close to the nucleic acid coding sequence. In some embodiments, the promoter is modified to remove methylation sensitive motifs (e.g., a cytosine nucleotide is followed by a guanine nucleotide, or “CpG”), or by the addition of a regulatory sequence that binds transcriptional factors that repress DNA methylation. In some embodiments, the vector includes A/T-rich, nuclear matrix interacting sequences, known as scaffold matrix attachment regions (S/MAR), which enhance transformation efficiency and improve the stability of transgene expression.

[000155] In some embodiments, the vector is a viral vector, for example, a retroviral vector, a lentiviral vector, an adenoviral vector, a herpesvirus vector, an adenovirus, or an adeno-associated virus (AAV) vector. The construction of lentiviral vectors has been described, for example, in U.S. Patents 5,665,577, 5,981,276, 6,013,516, 7,090,837, 8,119, 119 and 10,954,530.

[000156] In other embodiments, the vector is a non-viral vector, representative examples of which include plasmids, mRNA, linear single stranded (ss) DNA or linear double stranded (ds) DNA, minicircles, and transposon-based vectors, such as Sleeping Beauty (SB)-based vectors and piggyBac(PB)-based vectors. In yet other embodiments, the vector may include both viral and non- viral elements.

[000157] In some embodiments the vector is a plasmid. In addition to a promoter operatively linked to the nucleic acids, the plasmid may also contain other elements e.g., that facilitate transport and expression of the nucleic acid in an immune cell. The plasmid may be linearized with restriction enzymes, in vitro transcribed to produce mRNA, and then modified with a 5’ cap and a 3’ poly-A tail. Tn some embodiments, the vector multiple plasmids, a first plasmid encoding a first proteinaceous entity (e.g., the ectodomain of the CAR-enhancer) and a second plasmid encoding a second proteinaceous entity (e.g., the immune effector domain of the CAR-enhancer).

Cells

[000158] The CAR-enhancer may be expressed in a genetically modified (or transformed) cell containing a vector that contains a nucleic acid encoding the CAR-enhancer or components of the CAR-enhancer for the purpose of making and purifying the CAR-enhancer protein.

[000159] Cells useful for the cloning and other manipulations of these vectors are conventional. Cells from various strains of E. coli may be used for replication of the vectors and other steps in the construction of the CAR-enhancers of this disclosure.

[000160] Suitable host cells or cell lines for the expression of the nucleic acid-encoding CAR- enhancers include eukaryotic cells. In some embodiments, the cells are a mammalian cell line. In some embodiments, the cells are mammalian cells such as CHO (e.g., DG44, CHO-S), fibroblast cells (e.g., 3T3, COS), embryonic cells (e.g., PER.C6, HEK (e.g., HEK.293)), somatic cell hybrids (e.g., Sp2/0), and cancer cells, for example, myeloma cells (e.g., NS0 (NS zero)). In some embodiments, the nucleic acids encoding the CAR-enhancer is expressed in a CHO or a myeloma cell. Human cells may be used, thus enabling the expressed CAR-enhancer to be modified with human glycosylation patterns. The selection of suitable mammalian cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Green et al. , eds., Molecular Cloning: A Laboratory Manual, 5^th ed., Cold Spring Harbor Laboratory Press, New York, 2012.

[000161] In some embodiments, the cells are prokaryotic. Prokaryotic (i.e., bacterial) cells may prove useful as host cells suitable for the expression of the nucleic acids encoding CAR-enhancers (see, e.g., Pluckthun, Immunol. Rev. 130'.151-188 (1992)). However, due to the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded form or in a non-glycosylated form, any CAR-enhancers produced in a bacterial cell would be screened for retention of function (e.g., CAR binding ability). If the CAR-enhancer expressed by the bacterial cell was produced in a properly folded form, that bacterial cell would be a desirable host, or in alternative embodiments the CAR-enhancer may express in the bacterial host and then be subsequently re-folded. For example, various strains of E. Coli used for expression are well-known as host cells in the field of biotechnology. Various strains of B. Subtilis, Streptomyces, other bacilli and the like may also be employed.

[000162] After expression in a cell, the CAR-enhancers are isolated from the cell (e.g., cell lysates) or from the medium in which the cell is cultured. Protein isolation techniques are known in the art. Representative isolation techniques include chromatography, affinity chromatography, nickel- % nitrilotri acetic acid (Ni-NTA) affinity chromatography, high performance liquid chromatography (HPLC), hydroxylapatite chromatography, protein A-Sepharose, gel electrophoresis, and dialysis. In some embodiments, the affinity chromatography resin is a Protein A affinity chromatography resin or a Protein G affinity chromatography resin. Additional protein isolation systems and methods are known in the art. See, e.g., U.S. Patents 516,9936, 6,267,958, 8,357,778, 9,630,165, 9,708,399, 10,023,608, 10,207,229, 11,369,703, and 11,390,668, U.S. Patent Application Publications 2008/0090995, 2012/0244075, 2017/0158760, 2019/0276492, and 2021/0206815, and Traunecker et al., Embo J. 10(12)3655-9 (1991).

[000163] In some embodiments, the CAR-enhancer is encoded by two or more nucleic acids, e.g., the ectodomain-containing moiety is encoded by one nucleic acid and the immune cell effector domain is encoded by a second nucleic acid. In these embodiments, after expression in a suitable cell and purification, the purified ectodomain and the purified immune cell effector may be connected by a suitable chemical connection reaction, described above.

Pharmaceutical compositions

[000164] For purposes of practicing the disclosed methods, the CAR-enhancers may be formulated in a pharmaceutically acceptable carrier. The CAR-enhancer in the pharmaceutical composition may be in the form of a monomer (in embodiments lacking a dimerization domain), homodimer, or heterodimer, as described herein.

[000165] Compositions may be provided as sterile solid or liquid preparations. Solid preparations may be reconstituted and diluted into a liquid preparation before use, e.g., with carriers containing isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous solutions, which may be buffered to a selected pH. Liquid carriers include aqueous or non-aqueous carriers alike. Representative examples of liquid carriers include sterile water for injection, saline, Lactated Ringer Injection solution, phosphate buffered saline, a soluble protein, soluble sugars (e.g., dextrose), dimethyl sulfoxide (DMSO), polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), ethanol, and suitable mixtures thereof. In some embodiments, the liquid carrier includes a protein dissolved or dispersed therein, representative examples include serum albumin (e.g., human serum albumin, recombinant human albumin), gelatin, and casein. The compositions are typically isotonic, i.e., they have the same osmotic pressure as blood. Citric acid, sodium chloride, sugars, polyalcohols, and isotonic electrolyte solutions (e.g., Plasma-Lyte®) may be used to achieve the desired isotonicity. Depending on the carrier, other excipients may be added, e.g., wetting, dispersing, or emulsifying agents, gelling and viscosity enhancing agents, preservatives and the like as known in the art. In some embodiments, the compositions include citric acid, ethylenediaminetetraacetic acid (EDTA), and polysorbate 20 with a pH range between about 6.8 to about 7.2.

Cancer Subjects

[000166] Cancers treatable in accordance with the disclosed methods broadly include hematopoietic cancers and cancers characterized by the presence of a solid tumor.

[000167] The term “subject” (or “patient”) as used herein includes all members of the animal kingdom prone (or disposed) to or suffering from the indicated cancer. In some embodiments, the subject is a human. Therefore, a subject “having a cancer” or “in need of’ treatment according to the present disclosure broadly embraces subjects who have been positively diagnosed, including subjects having active disease who may have been previously treated with one or more rounds of therapy, and subjects who are not currently being treated (e.g., in remission) but who might still be at risk of relapse, and subjects who have not been positively diagnosed but who are predisposed to a cancer (e.g., on account of the basis of prior medical history and/or family medical history, or who otherwise present with a one or more risk factors such that a medical professional might reasonably suspect that the subject was predisposed to cancer).

[000168] The terms “treat”, “treating”, and “treatment” as used herein refer to any type of intervention, process performed on, or the administration of an active agent to the subject in need thereof with the therapeutic objective (“therapeutic effect”) of reversing, alleviating, ameliorating, inhibiting, diminishing, slowing down, arresting, stabilizing, or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a cancer.

[000169] In some embodiments, the cancer is a hematopoietic cancer. Representative hematological cancers include plasma cell neoplasm (e.g., myeloma, multiple myeloma, relapsed or refractory multiple myeloma, plasma cell myeloma, extramedullary multiple myeloma, monoclonal gammopathy of unknown significance (MUGS), asymptomatic smoldering multiple myeloma, or solitary plasmacytoma), lymphoma (e.g., Hodgkin’s lymphoma, non-Hodgkin’ s lymphoma, Burkitt’s lymphoma, plasmablastic lymphoma, plasmacytoid lymphoma, or diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL)), leukemia (e.g., relapsed or refractory acute B lymphocytic leukemia (ALL), or relapsed or refractory acute lymphoblastic leukemia), and carcinomas (e.g., Waldenstrom macroglobulinemia and glioblastoma (astrocytoma)). In these embodiments, the therapeutic effect might include on or more art- recognized indicia of therapeutic efficacy, representative examples of which include prevention or prolongation of metastases, improvement in survival time, total/complete or partial remission of a cancer, e.g., no detectable cancer cells and less tumor cells or smaller tumors, respectively, or a reduction in tumor cell number. In some embodiments, the hematopoietic cancer is multiple myeloma, lymphoma, or leukemia. Representative examples of CAR immune cell therapy appropriate for or otherwise known to treat hematopoietic cancers include ALL, DLBCL, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, non-Hodgkin lymphoma, FL, MCL, and multiple myeloma.

[000170] In some embodiments, the cancer is characterized by the presence of a solid tumor. In some embodiments, the cancer is a bladder cancer (e.g. transitional cell carcinoma, also called urothelial carcinoma), kidney cancer (e.g., renal cell carcinoma (RCC), kidney renal clear cell carcinoma (KIRC), transitional cell cancer, or Wilms tumor), skin cancer (e.g., melanoma, skin cutaneous melanoma (SKCM), basal cell carcinoma, and squamous cell carcinoma of the skin), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, including lung adenocarcinoma (LU AD) and lung squamous cell carcinoma (LUSC)), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN) also called head and neck squamous cell carcinoma (HNSC), laryngeal and hypopharyngeal cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, oral and oropharyngeal cancer, and salivary gland cancer), colon or rectal cancer (e.g., colorectal carcinoma (CRC), colon adenocarcinoma (COAD), rectum adenocarcinoma (READ)), ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma, epithelial ovarian carcinomas, fallopian tube cancer, and primary peritoneal cancer), endometrial cancer, cervical cancer (e.g., cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC)), prostate cancer (e.g., prostate adenocarcinoma (PRAD)), and stomach cancer (e.g., stomach adenocarcinoma (STAD)). Representative examples of CAR immune cell therapy appropriate for or otherwise known to treat solid tumors include malignant mesothelioma, ovarian cancer, breast cancer (e.g. , triple-negative breast cancer (TNBC)), pancreatic cancer, lung cancer, liver cancer, glioblastoma, gastric cancer, endometrial cancer, cervical cancer, biliary cancer, uterine serous carcinoma, cholangiocarcinoma, neuroblastoma, sarcoma, lung cancer, and melanoma.

[000171] In some embodiments, the cancer is characterized as being in a state of minimal residual disease (MRD). MRD is a state at which a cancer patient has a small number of cancer cells that remain in the body after treatment. The number of remaining cells may be so small that they do not cause any physical signs or symptoms of the cancer, and often may not be detectable through traditional methods, such as viewing cells under a microscope and/or by tracking abnormal serum proteins in the blood.

[000172] The amount of cancer antigens present in a subject in a state of MRD are limited. And this limited presence of the cancer antigen may not adequately support the proliferation and efficacy of CAR immune cells. The additional presence of a CAR-enhancer presents the CAR immune cells with not only additional cancer antigen, but also a supportive immune cell effector domain that may modulate the activity of the CAR immune cell to promote proliferation, efficacy, and/or persistence. [000173] In some embodiments, the subject receiving an administration of CAR-enhancer is in a state of MRD. In some embodiments, the method of treating cancer involves treatment of a state of minimal residual disease (MRD) in the subject. In some embodiments, the method of treating cancer involves elimination of MRD in the subject.

[000174] To test for MRD, samples from either a blood draw or a bone marrow aspiration may be used. The most widely used tests to measure MRD are flow cytometry, polymerase chain reaction (PCR) and next-generation sequencing. Methods that may be suitable for use in measuring MRD are described in, e.g, U.S. Patents 8,124,353, 9,528,160, 10,280,462, 11,618,787, and 11,633,426, and U.S. Patent Application Publications 2011/0294148, and 2022/0380852.

Prior CAR immune cell therapy

[000175] The methods of the present disclosure entail administration of the CAR-enhancer to a subject having had CAR immune cell therapy. As known in the art, CAR immune cells contain a synthetic CAR molecule that binds a cancer antigen. Typically, CARs contain an extracellular domain to which the CAR-engager binds, a transmembrane domain, and an intracellular domain comprising a stimulatory domain. [000176] The extracellular domain of the CAR that binds the ectodomain of a cancer antigen may contain an antibody fragment. In some embodiments, the CAR binds BCMA. CAR extracellular domains that bind to BCMA are known in the art. See, e.g., FDA-approved CAR-expressing immune cells ciltacabtagene autoleucel (Carvykti®, also referred to herein as “cilta-cel”), and idecabtagene vicleucel (Abecma®, also referred to herein as “ide-cel”), U.S. Patents 10,072,088, 10,683,369, 11,084,880, and 10,174,095, and U.S. Patent Application Publications 2016/0131655, 2017/0226216, 2018/0133296, 2019/0151365, 2019/0359727, 2019/0381171, 2020/0339699, 2020/0360431, 2020/0055948, and 2022/0064316. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-BCMA antibody, BCMA-binding fragment, or derivative thereof, e.g., belantamab (Blenrep®), linvoseltamab (REGN5458), pacanalotamab (AMG 420), pavurutamab (AMG 701), and teclistamab (Tecvayli®). In some embodiments, the extracellular domain of the CAR will bind the BCMA ectodomain of the CAR-enhancer that has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[000177] In some embodiments, the CAR binds CD 19. CAR extracellular domains that bind to CD19 are known in the art. See, e.g., FDA-approved CAR-expressing immune cells lisocabtagene maraleucel (Breyanzi®), tisagenlecleucel (Kymriah®), brexucabtagene autoleucel (Tecartus®), and axicabtagene ciloleucel (Yescarta®), U.S. Patents 9,629,877, 10,273,300, and 10,533,055, and U.S. Patent Application Publications 2020/0392248, and 2021/0238253. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-CD19 antibody, anti-CD19- binding fragment, or derivative thereof, e.g., loncastuximab (Zynlonta®), tafasitamab (Monjuvi®), denintuzumab (SGN-CD19A), and inebilizumab (Uplizna®). In some embodiments, the extracellular domain of the CAR will bind the CD 19 ectodomain of the CAR-enhancer having the amino acid sequence of any one of SEQ ID NOs: 3-5, or 103.

[000178] In some embodiments, the CAR binds CD20. CAR extracellular domains that bind to CD20 are known in the art. See, e.g., U.S. Patents 10,189,903, 10,442,867, 10,934,363, 11,066,457, 11,160,833, and 11,439,665, and U.S. Patent Application Publication 2018/0187149. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-CD20 antibody, anti-CD20-binding fragment, or derivatives thereof, e.g., ofatumumab (Arzerra®, Kesimpta®), veltuzumab (IMMU-106), tositumomab (Bexxar®), and rituximab (Rituxan®, Riabni®, Truximab®). In some embodiments, the extracellular domain of the CAR will bind the CD20 ectodomain of the CAR-enhancer having the amino acid sequence SEQ ID NO: 6. [000179] Tn some embodiments, the CAR binds CD22. In these embodiments, the extracellular domain of the CAR will bind a CD22 ectodomain of the C AR-enhancer. CAR extracellular domains that bind CD22 are known in the art. See, e.g., U.S. Patents 9,139,649, 9,181,343, and 10,494,435, U.S. Patent Application Publications 2015/0175711, 2018/0086843, 2021/0047402, 2021/0095022, 2022/0220198, and 2022/0273710, and Fry et al., Nat. Med. 2#(7/20-28 (2018). In some embodiments, the CAR extracellular domain is derived from a commercially available anti-CD22 antibody, anti-CD22-binding fragment, or derivatives thereof, e.g., bectumomab, epratuzumab, inotuzumab, moxetumomab, and epratuzumab. In some embodiments, the CAR-enhancer contains a CD22 ectodomain that has the amino acid sequence SEQ ID NO: 7. In some embodiments, the CAR-enhancer contains a CD22 ectodomain that has any one of the amino acid sequences SEQ ID NOs: 104-107.

[000180] In some embodiments, the CAR binds SLAMF7. CAR extracellular domains that bind SLAMF7 are known in the art. See, e.g., U.S. Patent 10,799,536, and U.S. Patent Application Publications 2020/0024342, 2020/0283534, 2021/0230548, and 2021/0253729. In some embodiments, the CAR extracellular domain is derived from a commercially available anti- SLAMF7 antibody, anti-SLAMF7-binding fragment, or derivative thereof, e.g., elotuzumab (Empliciti®). In some embodiments, the extracellular domain of the CAR will bind the SLAMF7 ectodomain of the CAR-enhancer having the amino acid sequence SEQ ID NO: 10.

[000181] In some embodiments, the CAR binds PD-1. CAR extracellular domains that bind to PD- 1 are known in the art. See, e.g., U.S. Patents 10,124,023 and 11,136,392, and U.S. Patent Application Publications 2021/0061877, 2020/0281974, and 2022/0064595. In some embodiments, the CAR extracellular domain is derived from a commercially available anti -PD-1 antibody, anti- PD-1 -binding fragment, or derivative thereof, e.g., balstilimab, budigalimab, cadonilimab, cemiplimab (Libtayo®), cetrelimab, dostarlimab (Jemperli®), izuralimab, nivolumab (Opdivo®), pacmilimab, pembrolizumab (Keytruda®), penpulimab, peresolimab, pidilizumab, retifanlimab, rosnilimab, sintilimab, spartalizumab, tislelizumab, toripalimab, volrustomig, vudalimab, zeluvalimab, and zimberelimab. Therefore, in some embodiments, the extracellular domain of the CAR will bind the PD-1 ectodomain of the CAR-enhancer having the amino acid sequence SEQ ID NO: 11.

[000182] In some embodiments, the CAR binds receptor tyrosine kinase KIT proto-oncogene, (KIT). CAR extracellular domains that bind to KIT are known in the art. See, e.g., U.S. Patent Application Publications 2017/0335281, 2020/0048359, 2020/0071397, and 2021/0299177. In some embodiments, the CAR extracellular domain is derived from a commercially available anti- KIT antibody, anti-KIT-binding fragment, or derivative thereof, e.g., barzolvolimab. In some embodiments, the extracellular domain of the CAR will bind the KIT ectodomain of the CAR- enhancer having the amino acid sequence SEQ ID NO: 12.

[000183] In some embodiments, the CAR binds CD38. CAR extracellular domains that bind to CD38 are known in the art. See, e.g., U.S. Patents 10,709,775, 10,799,536, 10,836,998, and 11,365,394 and U.S. Patent Application Publications 2017/0296623, 2019/0135894, 2019/0135937, 2020/0308541, 2021/0046118, and 2022/0202859 In some embodiments, the CAR extracellular domain is derived from a commercially available anti-CD38 antibody, anti- CD38-binding fragment, or derivative thereof, e.g., daratumumab (Darzalex®), isatuximab (Sarclisa®), and mezagitamab. In some embodiments, the extracellular domain of the CAR will bind the CD38 ectodomain of the CAR-enhancer having the amino acid sequence SEQ ID NO: 14.

[000184] The intracellular domain of the CAR contains a signaling domain that enables intracellular signaling and immune cell function. The signaling domain may include a primary signaling domain and/or a co-stimulatory signaling domain. In some embodiments, the intracellular domain is capable of delivering a signal approximating that of natural ligation of an ITAM- containing molecule or receptor complex such as a TCR receptor complex.

[000185] In some embodiments, the signaling domain includes a plurality, e.g., 2 or 3, costimulatory signaling domains, e.g., selected from 4-1BB, CD3(^, CD28, CD27, ICOS, and 0X40. In some embodiments, the signaling domain may include a CD3(^ domain as a primary signaling domain, and any of the following pairs of co-stimulatory signaling domains from the extracellular to the intracellular direction: 4-1BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28-4-1BB; 0X40- CD28; CD28-OX40; 4-1BB-CD3Q CD3^-4-lBB; CD28-CD3i;; CD3<;-CD28; CD28-4-1BB and 4- 1BB-CD28. In some embodiments the primary signaling domain is derived from CD3(^, CD27, CD28, CD40, KIR2DS2, MyD88, or 0X40. In some embodiments, the co-stimulatory signaling domain is derived from one or more of CD3y, CD38, CD3e, CD3(^, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD68, CD72, CD80, CD86, CD137 (4-1BB; TNFRSF9), CD154, CLEC-1, 4-1BB, DAP10 (hematopoietic cell signal transducer ((HCST)), DAP12 (TYROBP), Dectin-1, FcaRI, FcyRI, FcyRII, FcyRIII, IL-2RB, ICOS, KIR2DS2, MyD88, 0X40, and ZAP70. [000186] A representative CAR with a CD3^ stimulatory signaling domain is the FDA-approved CAR-expressing immune cells tisagenlecleucel (Kymriah®). Representative CARs with CD3(^ and 4- IBB co-stimulatory signaling domains are the FDA-approved CAR-expressing immune cells idecabtagene vicleucel (Abecma®), lisocabtagene maraleucel (Breyanzi®), and ciltacabtagene autoleucel (Carvykti®). Representative CARs with CD28 and CD3^ co-stimulatory signaling domains are the FDA-approved CAR-expressing immune cells brexucabtagene autoleucel (Tecartus®) and axicabtagene ciloleucel (Yescarta®).

[000187] In some embodiments, the CAR immune cell is a T cell. In some embodiments, the CAR immune cell is a NK cell. Additional CAR immune cells are known in the art, e.g., U.S. Patents 5,906,936, 7,446,190, 7,741,465, 8,389,282, 8,399,645, 9,422,351, 9,790,267, 9,885,298, 10,124,023, 10,815,301, and 11,433,100, and U.S. Patent Application Publications 2019/0375815, 2020/0281973, 2021/0300986, 2022/0056101, and 2022/0193138.

[000188] The number of CAR immune cells administered to a subject will vary between wide limits, depending upon the location, type, and severity of the cancer, the age, body weight, and condition of the individual to be treated, etc. A physician will ultimately determine appropriate number of cells and doses to be used. Typically, the CAR immune cells will be given in a single, one-time dose.

[000189] Typical doses of CAR immune cells are known in the art. In some embodiments, the effective number of the CAR immune cells is between approximately 1 x 10⁴ to approximately U 10¹⁰ cells per subject. In some embodiments, the effective number of the CAR immune cells is about the number of cells given in FDA-approved CAR T cell therapies, between approximately l * 10⁶ to approximately l * 10¹⁰ cells per kg of subject body weight. Since the CAR-enhancer promotes functionality and persistence of CAR immune cells, CAR therapy that contemplates coordinate administration of the CAR-enhancer may entail use of fewer CAR immune cells compared to FDA-approved CAR T cell therapies. In these embodiments, the effective number of the CAR immune cells is between approximately l* 10⁴ to approximately l * 10⁷ cells per kg of subject body weight.

[000190] The CAR immune cells may be administered to a subject for the treatment of a cancer by any medically acceptable route. The CAR immune cells are typically delivered intravenously, although they may also be introduced into other convenient sites (e.g., to an affected organ or tissue) or modes, as determined by an attending physician. [000191] The CAR immune cells may be autologous or allogeneic. For example, immune cells or progenitors thereof can be isolated from a tissue of body fluid from one subject prior to administration to the same subject (autologous) or a different, compatible subject (allogeneic). Most typically, the CAR immune cells are administered once.

Administration of the CAR-Enhancer

[000192] The first course of CAR-enhancer therapy may be initiated up to about 6 months after administration of the CAR immune cell therapy. The term “effective amount” as used herein refers to a sufficient amount of CAR-enhancer to provide the desired effect, e.g., the amount of a CAR- enhancer to bind to a CAR-expressing immune cell.

[000193] In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 4 years after the CAR immune cell therapy. In some embodiments, the first course of CAR- engager therapy is initiated at any time up to about 3 years after the CAR immune cell therapy. In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 2 years after the CAR immune cell therapy. In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 1 year after the CAR immune cell therapy. In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 9 months after the CAR immune cell therapy. In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 6 months after the CAR immune cell therapy.

[000194] In some embodiments, the first course of CAR-enhancer therapy is initiated at any time up to about 5 months after the CAR immune cell therapy.

[000195] In some embodiments, the first course of CAR-enhancer therapy is initiated at any time up to about 4 months after the CAR immune cell therapy.

[000196] In some embodiments, the first course of CAR-enhancer therapy is initiated at any time up to about 3 months after the CAR immune cell therapy.

[000197] In some embodiments, the first course of CAR-enhancer therapy is initiated at any time up to about 2 months after the CAR immune cell therapy.

[000198] In some embodiments, the first course of CAR-enhancer therapy is initiated at any time up to about 1 month after the CAR immune cell therapy.

[000199] In some embodiments, the first course of CAR-enhancer therapy is initiated at any time up to about 4 weeks after the CAR immune cell therapy. [000200] Tn some embodiments, the first course of CAR-enhancer therapy is initiated at any time up to about 3 weeks after the CAR immune cell therapy.

[000201] In some embodiments, the first course of CAR-enhancer therapy is initiated about 2 weeks after the CAR immune cell therapy.

[000202] In some embodiments, the first course of CAR-enhancer therapy is initiated 2 weeks after the CAR immune cell therapy.

[000203] In some embodiments, the course of CAR-enhancer therapy is conducted over a period of time of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks. In some embodiments, the first course of CAR-enhancer therapy entails administering a total of about 1 to about 6 doses (e.g., 2 doses, 3 doses, 4 doses, 5 doses, or 6 doses) of the CAR-enhancer. In some embodiments, the first course of the CAR-enhancer therapy entails administration of about 1 dose per week, about 2 doses per week, about 3 doses per week, or about 4 doses per week.

[000204] In some embodiments, the first course of CAR-enhancer therapy is conducted over a period of time of about 1 to about 3 weeks with administration of about 1 to about 3 doses of CAR- enhancer per week.

[000205] The dosage amounts of the CAR-enhancer may range from about 1 to about 8 mg/kg of patient body weight. In some embodiments, the dosage (effective amount) of the CAR-enhancer is about 1 mg/kg, 2 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, or about 8 mg/kg.

[000206] In some embodiments, the present methods further include administration of a second, subsequent course of CAR-enhancer therapy to the subject. In these embodiments, the subject may have relapsed, or is at risk of relapse. The CAR-enhancer administered in the second course of CAR- enhancer therapy may be the same as or different from the CAR-enhancer administered in the first course of CAR-enhancer therapy. The CAR-engager administered in the second course of CAR- engager therapy may be administered within the same period of time after the CAR immune cell therapy as described above or in the same amounts of time described above, but after the first course of CAR-engager therapy.

[000207] In some embodiments, the CAR-enhancer is administered as an intravenous infusion over a period of time. Representative infusion times are 30 minutes, 60 minutes, and 90 minutes. In some embodiments, the infusion time is between 30 and 60 minutes. In some embodiments, the first administration is infused into a patient for 90 minutes and subsequent administrations are infused into a patient for 30 minutes.

Combination Therapy

[000208] In some embodiments, the present methods may include co-administration of another anti-cancer therapy. The term “co-administered” includes substantially contemporaneous administration, by the same or separate dosage forms, or sequentially, e.g., as part of the same treatment regimen or by way of successive treatment regimens. Thus, if given sequentially, at the onset of administration of the second therapy, the first of the two therapies is, in some cases, still detectable at effective concentrations at the site of treatment. The sequence and time interval may be determined such that they can act together (e.g., synergistically to provide an increased benefit than if they were administered otherwise). For example, the therapeutics may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they may be administered sufficiently close in time so as to provide the desired therapeutic effect, which may be in a synergistic fashion. Thus, the terms are not limited to the administration of the active agents exactly at the same time.

[000209] In some embodiments, the subject may also have had an additional anti-cancer therapy. The additional therapy may be (1) prior to CAR immune cell therapy, (2) after the CAR immune cell therapy but before the first course of CAR-enhancer therapy, (3) after the first course of CAR- enhancer therapy but before the second course of CAR-enhancer therapy, or (4) after the second course of CAR-enhancer therapy. In some embodiments, the additional anti-cancer therapy is chemotherapy, radiotherapy, immunotherapy, targeted therapy, pro-apoptotic therapy, or cell cycle regulation therapy, therapy with thalidomide, lenalidomide, bortezomib, and/or melphalan.

[000210] Expansion and differentiation agents may also be provided prior to, during, or after administration of the CAR immune cells to increase differentiation, expansion, and/or persistence of the CAR immune cells (e.g., T cells and NK cells). These and other aspects of the present application will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain embodiments of the application but are not intended to limit its scope, as defined by the claims.

EXAMPLES

Example 1 : Materials and Methods [000211] The cloning and expression of proteins were done following standard approaches. Other procedures, including flow cytometric analyses, BLI imaging, CAR T cells production, cell culture and animal handling were performed following standard protocols as briefly explained below.

[000212] Generation of CAR-enhancers. All genes were codon-optimized for mammalian expression in HEK293 cells, synthesized, and inserted into a vector expression system with a signal sequence for protein secretion into the supernatant. To facilitate production of the products, stable HEK293 cell lines were generated. Accordingly, HEK293 cells were transfected with pPAX2, pVSVG (packaging vectors), and the lentivirus plasmid containing the sequence of interest. The lentivirus was harvested at 48, 72, and 96 hours (h) post transfection, sedimented at 20,000 x g for 2h, and resuspended in optiMEM media. A new batch of HEK293 cells were then subjected to three rounds of transduction with the virus. Cells were allowed to recover in DMEM complete media and were subjected to puromycin selection to retain only cells that integrated the lentivirus plasmid. Cells were then expanded in four 15 cm culture dishes until they reached confluency, washed carefully with PBS, and incubated in serum-free DMEM for 24 to 48h. The supernatant was harvested, and protein expression was confirmed via SDS-PAGE and immunoblotting. Proteins were purified by adsorption onto a nickel nitriloacetic acid (Ni-NTA) metal affinity column. Non- specifically bound proteins were removed by washing with 40 mM imidazole. The imidazole concentration was increased to 250 mM, allowing recovery of the protein of interest. The protein was further purified via size-exclusion chromatography and were stored in 50 mM HEPES buffer, pH 7.5 at -80 °C until use.

[000213] Some CAR-enhancers were isolated by passage through an affinity chromatography resin, typically in the presence of a neutral phosphate buffer. The affinity chromatography resin was then subjected to an acidic buffer with a pH of about 3 to about 4, thereby washing CAR-enhancer off of the affinity chromatography resin. A basic buffer may be used to neutralize the acidic buffer, then a tangential flow filtration of the neutralized buffer can be performed with a formulation buffer, to isolate a concentrated and purified solution containing the CAR-enhancer.

[000214] Production of CAR T cells. The CAR construct that binds human CD 19 contains an scFv derived from the anti-human CD 19 antibody clone FMC69, followed by human CD28 and CD3(^ intracellular signaling domains. The CAR construct that binds human BCMA contains an scFv derived from the anti-human BCMA antibody clone MSK54, followed by human 41BB and CD3ij intracellular signaling domains. The human signaling CAR constructs were transduced into HeLa cells that stably produce gamma-retrovirus pseudotyped with the envelope of the feline endogenous virus (RD 114), which has been shown to transduce human hematopoietic cells (HSC) with high efficiency (Ward et al., Mol. Ther. S(5):804-12 (2003)). High viral titer clones were isolated by limiting dilution. The high expression clone was seeded and grown in DMEM complete media containing 10% FBS until 80% confluency. Media was exchanged with RPMI complete media containing 10% FBS. After 24 hours, the virus-containing media was harvested, sterile-filtered using a 0.45 pm PES filter, and utilized for producing CAR T cells.

[000215] The production of CAR T cells was adapted from previous studies. See, for example, Li etal., Methods Mol. Biol. 1514 A 11-118 (2017). In brief, whole blood was obtained from apheresis leukoreduction collars of platelet healthy donors, due to a high number of viable white blood cells. The whole blood was subjected to centrifugation through a Ficoll gradient to isolate PBMCs. Whole PBMCs were utilized without selecting for CD8⁺ T cells. PBMCs were resuspended in RPMI media containing 10% Fetal Bovine Serum (FBS), 200 lU/mL IL-2, 60 ng/mL IL-7, 10 ng/mL IL-15, 2 pg/mL anti-human CD3 (OKT3 clone), and 0.5 pg/mL anti-human CD28 (CD28.1 clone) at a cell concentration of 4 * 10⁶ cells/mL in 3 mL of media per well in a 6-well plate. After 24 hours, cells are harvested, spun down, and resuspended in the same volume of fresh media with FBS, IL-2, IL- 15, and IL-7 in addition to media harvested from anti-human BCMA CAR gamma-retrovirus producing cells, resulting in PBMC inoculation with the gamma-retrovirus. The PBMCs were then plated at 4 x 10⁶ cells/mL in 3mL into 6-well plates coated with 20 pg retronectin (coated with ImL of 20 pg/mL retronectin in PBS for 24 hours at 4 °C). The PBMC underwent spinoculation in a centrifuge for 1 h at 2000 * g at 30 °C and cultured at 37 °C. The transduction step was repeated with fresh gamma-retrovirus containing media, cytokines, and spinoculation. Flow cytometry analysis was utilized to assess the transduction efficiency of the CAR transgene using the dsRed reporter gene and recombinant BCMA labeled with AlexaFlour-647.

[000216] In vivo experiments. For all experiments NOD/SCID/Gamma (NSG; 1AO\D.C%-Prkdc^sc‘^d Il2rg^mlwJ^l/SzP) mice were used due to their immunocompromised status and ability to effectively engraft human cancer cell lines. Cells from the human multiple myeloma 0PM2 cell line were used to establish a multiple myeloma mouse model in NSG mice. In vivo experiments were initiated by tail vein intravenous injection of 1 x 10⁶ 0PM2 cells expressing GFP and Firefly Luciferase followed by biweekly Bioluminescent Imaging (BLI). Upon effective engraftment after 3 weeks, mice were intravenously injected through the tail vein with CAR T cells. BLI was performed biweekly afterwards to assess tumor burden. Quantification was measured using photons/sec using Aura software.

[000217] Organ analysis. At the conclusion of an experiment, the surviving mice were sacrificed and spleen, bone marrow, blood, liver, kidney, and lungs were harvested and weighed. The liver, kidney, and lungs were minced, digested with collagenase (final concentration of 1 pg/mL collagenase), and incubated at 37 °C for 1 h. Spleens were crushed, and bone marrow was aspirated using a 30-gauge insulin needle. All processed cells were pushed through a 70 pm strainer to produce a single cell suspension of cells. Cells were resuspended in 1 mb of ammonium-chloride- potassium (ACK) lysis buffer to deplete the sample of red blood cells for 2 m at room temperature. The resulting single cell suspensions were washed with fluorescence-activated single cell sorting (FACS) buffer of PBS and 0.5% BSA, stained, and analyzed using flow cytometry.

[000218] Cell lines and culture. The 0PM2 cell line, which endogenously express BCMA, were engineered to express green fluorescent protein (GFP) and Firefly Luciferase. Peripheral Blood Mononuclear Cells (PBMC) were obtained by Ficoll gradient of apheresis leukoreduction collars of platelet healthy donors. HEK293T cells were cultured in complete DMEM (Gibco), 1% L-glutamine (Gibco), 1% non-essential amino acids (NEAA) (Gibco), 1% pyruvate (Gibco), and 1% penicillin and streptomycin (Cytiva), and 10% fetal bovine serum (FBS). OPM2, and PBMCs were cultured in complete RPML1640 (Gibco), 1% L-glutamine (Gibco), 1% non-essential amino acids (NEAA) (Gibco), 1% pyruvate (Gibco), and 1% penicillin and streptomycin (Cytiva), and 10% fetal bovine serum (FBS). All cells were grown in 5% CO2, 95% air-humidified incubators at 37 °C.

[000219] Mouse studies. All experiments adhered to the pertinent ethical and safety protocols. The studies were carried out under the oversight of the Dana-Farber Cancer Institute Institutional Animal Care and Use Committee (protocol no. 20-006). The xenograft models utilized herein are described in Smith etal., Mol. Ther. 26(6) : 1447 -1456 (2018). Briefly, 8- to 12-week-old NOD-sc/t/IL2Ry^nu11 (NSG) and NOD-vcvr/ H2-Kl^nu11 H2-Abl^nu11 H2-Dl^nu11 IL2Rg^nu11 (NSG-MHC I/II double knockout (DKO)) were either purchased from Jackson Laboratory or bred in-house. All mice were sex and age matched into groups. Xenograft models were established by intravenous injection of 1 ^x 10⁶ cells of OPM2 or Nalm6 expressing GFP and Luciferase in 200 mL of PBS. Mice received indicated treatments in 300 mL of PBS through intraperitoneal injections. Tumor burden was assessed using the IVIS® Lumina Series III (Perkin Elmer) after intraperitoneal injection of D-Luciferin (150 mg/kg, from a 15 mg/ml solution) at the indicated time. Each mouse was imaged in groups of up to five mice in the supine position at the same time points (5 min). BLI intensity was analyzed by Aura imaging analysis software (Spectral Instruments Imaging). Peripheral blood from mice was obtained by submandibular bleeding in an EDTA-coated tube and analyzed for CAR T cell detection and expansion. In brief, volume of the blood was determined in order to calculate absolute values. Samples were then centrifuged, and serum harvested. Cell pellets were resuspended in 500-1000 mb of ACK Lysis Buffer (150 mM NH4CI, lOmM KHCO3, 0.1 mM EDTA) for 1 minute. Cells were then washed twice in FACS buffer, PBS + 1% Bovine Serum Albumin (BSA). Samples were then stained with anti-CD45-PacificBlue (1:50, Biolegend), anti-CD4-PE/Dazzle594 (1:50, Biolegend), anti-CD8-FITC (1 :50, Biolegend), anti-CCR7-AlexaFluor700 (1 :50, Biolegend), anti- CD62L-PE (1:50, Biolegend), anti-CD45RO-PerCP/Cy5.5 (1 :50, Biolegend), anti-CD45RA- APC/Fire750 (1 :50, Biolegend), anti-PDl-BV605 (1 :50, Biolegend), anti-HLA-DR-PE/Cy7 (1:50, Biolegend), anti-CD69-BV421 (1 :50, Biolegend), and recombinant BCMA-AlexaFlour647 (made in-house). Samples were processed on a Sony SP6800 Spectral Analyzer. Flow rate and acquisition time was noted to calculate absolute values. All experiments were performed in a blinded and randomized fashion. Animals were euthanized at the end of the experiment or when they met prespecified endpoints according to the IACUC protocols. Upon endpoint, major immune organs (spleen and bone marrow) as well as essential organs where possible metastatic lesions can form (liver, lung, and kidney) were harvested, weighed, and analyzed. In brief, spleen was crushed using a plunger and passed through a 40 pm strainer to acquire a single cell suspension. Bone marrow was aspirated using a 30-gauge insulin needle. Liver, lung, and kidney were diced using surgical scissors in 3 mL of Digestion buffer (ImL RPMI + 2mL PBS). Collagenase, Type 1 (Worthington) was added at a final concentration of 100 mg/mL and incubated at 37 °C for 1 hour. The resulting samples were passed through a 40 pm strainer to acquire a single-cell suspension. Samples were then stained with same antibodies used to stain blood samples and analyzed using a Sony SP6800 Spectral Analyzer. Flow rate and acquisition time were noted to calculate absolute values.

[000220] Microscopy. Cells were first stained with CellTracker Blue CMAC and seeded on poly- d-lysine-coated coverslips. Subsequently, the samples were incubated with the indicated treatment, labeled with Alexa647, at the specified time and temperature. After fixation using BD Cytofix buffer, the cells were imaged using the Leica THUNDER Imager. The intensity cut-off for the AlexaFluor647 channel was set at 2000 for all images, except for the VHH-muIL2 samples shown on the right (condition 4), for which, the image sensitivity was enhanced by 25-fold (intensity cut- off 80) to visualize the Alexa647 signal. Quantitative analysis of the fluorescence intensity was analyzed by aligning the base 2 logarithm of the ratio of integrated intensity of the membrane to the cytoplasm of the cells (see y-axis of FIG. 14B). For BCMA-muIL2 and BCMA-CH3 (conditions 1 and 2), only cells with a mean intensity to background ratio above 4 based on the dsRed channel were analyzed as they were identified as CAR⁺ T cells. For VHH-muIL2, cells with a mean intensity to background ratio greater than 2 based on the Alexa647 channel were selected to eliminate the background artifacts. Image quantification was performed using ImageJ software.

[000221] ELISA. ELISA analyses were performed to measure the levels of human T cell-derived cytokines in the serum of mice that received 0PM2 cancer cells followed by a low dose of CAR T cells, as shown in FIG. 17A. The cytokines analyzed with the ELISA MAX™ Standard Set (Biolegend) included ZFN-y, GM-CSF, and TNF-a following the manufacturer’s provided protein; however, only IFN-y was detectable in the collected samples. Serum samples were diluted at a ratio of 1:40. The same plates were used to incubate both the standard samples and the serum samples, and a standard curve was plotted for each cytokine.

[000222] pSTAT5 assay. Initially, CAR T were incubated in complete RPMI media without the presence of cytokines (rested) for 24 h. Cells were then stained with CellTrace™ Blue (Invitrogen) or CellTracker™ Red (Invitrogen) for 30 minutes at 37 °C for the indicated conditions. Cells were washed once with complete RPMI + 10% FBS media. CAR T cells stained with CellTracker™ Red were blocked using recombinant BCMA-CH3 (100 nM) for 20 minutes on ice while cells stained with CellTrace™ Blue were not blocked. Cells were then washed once with complete RPMI + 10% FBS media followed by seeding of approximately 2* 10⁵ cells (either the two separately stained cells co-cultured or separately cultured) per well of a 96 well plate in the presence of serial dilutions of treatments or cytokine controls at 37 °C. After 5 minutes of incubation, cells were immediately fixed with 1.5% formaldehyde in PBS for 10 minutes at room temperature. Cells were then permeabilized with ice cold 100% methanol for 20 minutes on ice 4 °C. Fixed and permeabilized cells were washed twice with FACS buffer, PBS + 1% Bovine Serum Albumin (BSA) and then incubated with anti- STAT5 pY694-PE/Cy7 (1 :200, Biolegend) for 30 minutes on ice. Cells were then washed twice with FACS buffer and analyzed using the BD FACSCanto™ II (Becton Dickinson). Flow cytometry data was analyzed using FlowJo™ (Becton Dickinson) and dose-response curves were fitted to a logistic sigmoidal model and half-maximal effective concentration (ECso) and 95% confidence intervals were calculated using Prism data analysis software (GraphPad). [000223] scRNA-seq Analysis. Gene counts for each sample were obtained using the CellRanger multi -function through lOx Cloud computing and pooled using the CellRanger aggr function to produce an ,h5 file that could be loaded into R as a Seurat object. Seurat pipeline was performed for QC filtering (number of total counts < 20’000, molecular identifiers [nUMI] < 6,000 and ribosomal RNA <10% of the reads). Data was then scaled and normalized using scTransform, and original samples were traced back using the demultiplexing function HTODemuxQ. Phenotype of the cells was then determined using projection of our sample to the Seurat pbmc multimodal dataset using the FindTransferAnchors() and FindQuery() workflow. The FindMarkers() function was used to find differentially expressed genes between chosen groups. The heatmaps were generated with the DoHeatmap() function with a downsampling of 500 cells. Gene Scores were obtained by creating a list of the genes of interest which was given as the feature for the function AddModuleScore().

[000224] Preparing samples for bulk RNA-sequencing. CAR T cells with and without the intracellular domain were incubated in complete RPMI media without the presence of cytokines for 24 hours. A 96 well plate was seeded with approximately 2x 10⁵ cells/well in the presence of 10 nM treatment or controls at 37 °C for 2 hours. Cells were washed with FACS buffer and left at 37 °C for 2 or 22 hours (4 hour and 24-hour timepoints). Cells were stained with anti-CD8-FITC (1:50, Biolegend), anti-CD4-PE/Dazzle594™ (1 :50, Biolegend) and Alexa647 labeled BCMA and sorted on the Sony Sorter MA900. 10,000 CD4 and 10,000 CD8 cells were sorted per condition. SMART - Seq mRNA library preparation kit (Takara Bio) was utilized to generate mRNA libraries, with each replicate tagged with a unique index. Libraries were pooled and sequenced through Novogene at a sequencing depth of 20 million reads per sample.

[000225] Bulk RNA Seq-Analysis. Gene counts for the samples were obtained by trimming the fastQ files and transcript quantification using the RNAlysis software. Gene names were obtained from the homo sapiens ensembl database with biomaRt, and differential expression between different conditions was determined using the DESeq2 pipeline. Volcano plots were drawn using the EnhancedVolcano library, with a cutoffs at logFC > |2| and p-value > 10'⁶. Heatmaps were generated using the pheatmap library. GSEA was performed using the pipeline of the fgsea package with the ranking metric being -logio(p-value)*sign(fold change) and basing the computations on the hallmark pathways of the MSigDB collection.

Example 2: BCMA-containing CAR-enhancer in vitro characterization. [000226] A Fusion protein consisting of the human BCMA ectodomain was fused with a two low- affinity mutated human IL-2 (muIL2) domains, and to improve pharmacokinetics and enhance stability, the CH3 domain (Feige etal., Trends Biochem. Sci. 35(4) : 189- 198 (2010)) of human IgGl (approximately 14 kDa in size) was incorporated between the antigen and the muIL2 (FIG. 2B) (Quayle etal., Clin. Cancer Res. 26(81:1953-1964 (2020)). Consequently, the BCMA-muIL2 CAR- enhancer preferentially delivers the low-affinity IL-2 to the surface of CAR T cells through antigen- to-CAR specific binding, minimizing effects on normal T cells, Tregs, or systemic toxicity.

[000227] It was recently demonstrated that IL-2 induces an alternative differentiation pathway of T cells, resulting in the generation of distinct “better effector” CD8⁺ T cells (Hashimoto et al., Nature 610(7930)'.173-181 (2022)). This process may rely, at least in part, on IL-2 binding to IL- 2Rot. Additionally, IL-2RPy-biased agonists may drive T cells towards a terminally differentiated state (Codarri et al., Nature 610(7930)'.161-172 (2022)). CAR-enhancers may be able to overcome the need for IL-2Ra in the alternative differentiation pathway by anchoring the low-affinity IL -2 on the surface of the CAR T cells via the antigen-to-CAR binding, thereby promoting the generation of memory CAR T cells. A potential synergistic effect between CAR signaling and IL-2 signaling may also exist.

[000228] To assess the binding affinity of BCMA-containing CAR-enhancers, flow cytometric analysis was performed by staining BCMA CAR T cells with varying concentrations of the BCMA CAR-enhancers. An ECso of about 0.21 nM was observed for the BCMA CAR-enhancer, which was comparable to that of a dimeric BCMA lacking the muIL2 (BCMA-CH3) (FIG. 2E), indicating binding was mainly due to the BCMA ectodomain rather than the muIL2. FIG. 2E shows the dosedependent staining of BCMA CAR T cells with the CAR-enhancer using flow cytometry (n=3 for each point), non-transduced T cells were used as controls, a secondary Alexa647-labeled anti-FLAG antibody was used for staining. Error bars represent mean with 95% confidence interval. Minimal binding of BCMA CAR-enhancer was observed to non-transduced T cells as well as minimal binding of VHH-muIL2, a control construct which replaces the BCMA ectodomain with an irrelevant nanobody (VHH) in the CAR-enhancer CH3-muIL2 construct. This observation further suggests that the binding of the CAR-enhancer to CAR T cells is primarily driven by the BCMA antigen, and that the muIL2 exhibits weak binding to both CAR T and non-transduced T cells. The BCMA-muIL2 CAR-enhancer did not exhibit binding to any immune cell populations in human peripheral blood mononuclear cells (PBMCs) (FIGs. 13A - 13B). [000229] Next, the functional effects of BCMA CAR-enhancer on BCMA CAR T cells were evaluated. After a 24-hour resting period without cytokines, BCMA CAR T cells were incubated with varying concentrations of the BCMA-muIL2 CAR-enhancer for 24 h, followed by assessment of the expression of the CD69 activation marker (Cibrian and Sanchez-Madrid, Eur. J. Immunol. 47(6):946-953 (2017)) using flow cytometric analysis. The results demonstrated a dose-dependent and selective increase in CD69 expression on CAR T cells (FIG. 2F), while no effect was observed on non-transduced T cells (FIG. 2G). Moreover, BCMA CAR-enhancer treatment resulted in a significantly higher increase in CD69 expression compared to VHH-muIL2, BCMA-CH3, or their combination suggesting the observed effect is only evident when the low-affinity IL-2 is fused to the antigen. An unpaired t-test indicated a statistically significant (P < 0.0001) increase in activation in the BCMA-muIL2 treatment group compared to VHH-muIL2, BCMA-CH3, or their combination control groups at a concentration of 0.1 nM or higher (error bars represent mean with 95% confidence interval) (FIG. 2F). Zero treatment and CD3/CD28 activation in FIG. 2G were used as negative and positive controls, respectively (error bars represent mean with 95% confidence interval).

[000230] BCMA CAR-E does not inhibit killing efficacy of BCMA CAR T cells. Since both the CAR-enhancers and cancer antigens bind the CAR, the potential inhibitory effect of BCMA CAR- enhancer on the killing activity of BCMA CAR T cells was investigated. To investigate, a killing assay was conducted using BCMA CAR T cells and patient-derived BCMA⁺ 0PM2 cancer cells in the presence of varying concentrations of the BCMA CAR-enhancer. Remarkably, the results demonstrated no inhibition of killing even at the highest tested concentration (100 nM of the CAR- enhancer) (FIG. 2H). 0PM2 cells were co-incubated with BCMA CAR T cells (shown in red) or non-transduced T cells (shown in gray) (E:T ratio 1 : 1; 30,000 cells of each) in the presence of varying concentrations of the BCMA-muIL2 CAR-E treatment. Live (PL) OPM2 cells were counted 48 hours later, with an N of 3 for each of the experiments. Error bars in FIG. 2H represent mean with standard deviation. Without being bound by theory, this finding might be attributed to the reversibility of CAR-enhancer binding to CAR, while the killing process, which involves the clustering effect and synapse formation between CAR and cancer antigen, is an irreversible event. Moreover, the binding avidity of CAR to membrane-bound BCMA might surpass the CAR binding avidity to the soluble antigen, contributing to this result. Notably, multiple myeloma patients exhibit high levels of soluble BCMA in their circulation due to shedding caused by y-secretase (Laurent et al., Nat. Commun. 6:7333 1 -12 (2015)). Despite this, BCMA CAR T cells produce remarkable initial responses in patients suggesting that the soluble BCMA antigen does not inhibit the activity of CAR T cells. The experimental findings disclosed herein are consistent with these earlier findings.

[000231] The BCMA CAR-enhancer selectively induces STAT5 activity in CAR T cells through the cis-delivery of the low-affinity IL-2 to the same targeted CAR T cells. IL-2 is known to exhibit strong activity on T cells, and the phosphorylation of STAT5 both serves as a reliable indicator of IL-2/IL-2R engagement and correlates with downstream effects such as phenotypic marker expression and cell proliferation (Jones et al., J. Immunol. 205(7/ 1721-1730 (2020)). To evaluate the impact of BCMA CAR-enhancer on STAT5 activity, BCMA CAR T cells were exposed to varying concentrations of the BCMA CAR-enhancer. Following a 5-minute incubation at 37 °C, cells were fixed and stained for pY694 STAT5. The results revealed that the BCMA-muIL2 CAR- enhancer induced phosphorylation of STAT5 in CAR T cells with an ECso of -0.014 nM (FIG. 21). In contrast, the VHH-muIL2 control required a higher concentration (EC so = 3.9 nM) to induce STAT5 phosphorylation, indicating that the BCMA-mediated delivery of the low-affinity IL-2 to CAR T cell surface significantly enhances the sensitivity of the muIL-2 by over 200-fold. Wild-type IL-2 exhibited a lower ECso (approximately 0.001 nM) suggesting a difference in signaling kinetics. The two-step process involved in STAT5 activity mediated by the BCMA-muIL2 CAR-enhancer involves: (i) binding of the antigen-to-CAR on T cell surfaces and (ii) subsequent interaction of the low-affinity IL-2 with nearby IL-2R, which, without being bound by theory, may be the reason for the measured difference. In contrast, wild-type IL-2 requires only binding to IL-2R, enabling it to more rapidly induce STAT5 activity. The VHH-muIL2 can activate STAT5 solely through the low- affinity IL-2, which may explain its requirement for higher concentrations to induce STAT5 activity in T cells.

[000232] BCMA CAR T cells pre-blocked with BCMA-CH3 showed a significant decrease in pSTAT5 levels to the same degree as control VHH-muIL2, validating that the potency of the CAR- enhancer is mediated by antigen-to-CAR binding (FIG. 21). To determine whether the CAR- enhancer binding to target cell can result in STAT5 signaling in an adjacent cell (/ra//.s-activation), non-blocked and pre-blocked BCMA CAR T cells were co-cultured in the presence of varying concentrations of CAR-enhancer. Pre-blocked CAR T cells had lower pSTAT5 levels compared to their co-cultured non-blocked CAR T cells, indicating that CAR-enhancer affects the targeted CAR T cells (cis-activation) but not adjacent cells. BCMA CAR T cells were treated with the indicated treatments for 5 min at 37 °C followed by STAT5 phosphorylation assessment. For the pre-blocking experiments, the BCMA CAR T cells were treated with BCMA-CH3 (100 nM) for 20 min at 4 °C prior to the 5 min exposure to the CAR-enhancer treatment at 37 °C (n=3 for each condition). Error bars in FIG. 21 represent mean with standard deviation. Taken together, the analysis of STAT5 activity supports the notion that the BCMA CAR-enhancer exerts its influence on targeted CAR T cells through the cis delivery of the low-affinity IL-2, with the effect mediated via antigen-to-CAR binding.

Example 3: The CAR-enhancer immune cell effector domain stimulates T cells independent of a CAR.

[000233] To show that CAR-enhancer immune cell effector domains stimulate immune cells, the following experiment was performed. In this experiment, the experimental setup of which is illustrated in FIG. 3A, peripheral blood mononuclear cells (PBMCs) were stimulated with anti-CD3, anti-CD28, IL-2, IL-7, and IL- 15 to produce activated T cells, which were not transduced with any exogenous transgenes. The activated T cells were treated with teceleukin (recombinant human IL -2 without glycosyl units), a CAR-enhancer that contains a N-terminal ectodomain that binds BCMA (~7 kDa), a CH3 domain (~14 kDa), and two repeats of the weak affinity variant of IL-2 immune cell effector domain, with the overall structure of BCMA-CH3-muIL2-muIL2 and referred herein as BCMA-muIL2, or a CAR-enhancer that contains an ectodomain that binds BCMA, a CH3 domain, and the Neo-2/15 immune cell effector domain, with the overall structure of BCMA-CH3- Neo-2/15, referred herein as BCMA-Neo-2/15, for 4 days. After treatment, T cells were counted and stained with carboxyfluorescein succinimidyl ester (CFSE) and analyzed for mean fluorescence intensity (MFI) of CFSE to determine T cell division.

[000234] T cells treated with the CAR-enhancers BCMA-muIL2 or BCMA-Neo-2/15 effected T cell counts and division (CFSE staining), similar to teceleukin, which is known to activate T cells; however, the CAR-enhancer treatment resulted in less sensitivity as compared to the teceleukin treatment. Systemic administration of IL-2 is associated with severe side effects (Rosenberg, J. Immunol. 792(72/5451-5458 (2014); Dutcher etaL, J. Immunother. Cancer. 2(7/26 1-23 (2014); Pachella et al., J. Adv. Pract. Oncol. 6(3/212-221 (2015)), including vascular leak syndrome and preferential expansion of CD4⁺CD25⁺ regulatory T (Treg) cells, that are known to result in immune suppression. The presently disclosed results indicate that the CAR-enhancers do not activate normal T cells when used at low concentrations, and that the stimulatory immune cell effector domains retain their normal function when attached to an ectodomain of a CAR-enhancer to activate T cells.

Example 4: CAR-enhancers activate CAR T cells specifically through the ectodomain

[000235] To show that CAR-enhancer immune cell effector domains stimulate immune cells, the following experiment was performed. As illustrated in FIG. 4A, PBMCs were stimulated with anti- CD3, anti-CD28, IL-2, IL-7, and IL-15 to produce activated T cells, which were then transduced with a vector containing a CAR. The activated CAR-expressing T cells (CAR T cells) were rested for 24 hours and then treated with CAR-enhancers containing an immune cell effector domain or CAR-enhancers lacking an immune cell effector domain as an ectodomain control.

[000236] CAR-enhancers containing BCMA ectodomain, a CH3 domain, and containing either 4- 1BBL (BCMA-41BBL), weak affinity IL-2 (BCMA-muIL2), or Neo-2/15 (BCMA-Neo-2/15) immune cell effector domains were tested for T cell activation. To test the ectodomain specificity of the CAR-enhancers for CAR T cells, as a control, irrelevant nanobody that binds the intracellular protein UBC6E (VHH6E) fused to a CH3 domain, and either 4-1BBL (VHH6E-41 BBL) or weak affinity IL-2 (VHH6E-muIL2) were tested for T cell activation. An additional ectodomain specificity control containing a nanobody that binds FN1 (clone NJB2, abbreviated NJB2-VHH) fused to a CH3 domain, and the Neo-2/15 stimulatory (NJB2-VHH-Neo-2/15) was tested for T cell activation. The ectodomain specificity controls have similar overall structure as the CAR-enhancers used in this experiment (protein domain-CH3-muIL2-muIL2 or protein domain-CH3-Neo-2/15). Ectodomain specificity controls and CAR-enhancers were incubated with CAR T cells for 10 hours, and the cells were stained for CD69 as an activation marker and measured by flow cytometry.

[000237] All of the BCMA ectodomain CAR-enhancers induced expression of CD69 in CAR T cells (FIG. 4B). The BCMA-CH3-Neo-2/15 had the lowest threshold of induced expression ofCD69 in CAR T cells (0.01 nM CAR-enhancer). The BCMA-CH3 protein (lacking an immune cell effector domain) had minimal effect on CAR T cell expression of CD69 at the highest concentration tested, 10 nM of BCMA-CH3 protein. None of the ectodomain specificity control proteins induced CAR T cell expression of CD69. These results indicate that CAR-enhancers containing a cancer antigen ectodomain specifically activates CAR T cells that expresses a CAR that recognizes CAR- enhancer’ s cancer antigen ectodomain.

Example 5: CAR-enhancers stimulate CAR T cell killing target cells [000238] To show that CAR-enhancers do not inhibit CAR T 1 cell killing, the following experiment was performed. CAR T cells were produced as described above and co-incubated with CAR-enhancers and BCMA⁺ multiple myeloma cancer cells. CAR T cells were incubated with OPM2 BCMA⁺ cells at an E:T ratio of 1 : 1 for 1 day and analyzed for target cell survival as compared to target cells without T cell coincubation (FIG. 5A).

[000239] BCMA ectodomain CAR-enhancers with either a muIL2 (BCMA-CH3-muIL2) or a 4- 1BBL (BCMA-CH3-41BBL) immune cell effector domain did not inhibit killing of 0PM2 cells (FIG. 5B). These results indicate that CAR-enhancers containing a cancer antigen ectodomain and a stimulatory immune cell effector domain do not inhibit CAR T cell killing of target cells that also express the same cancer antigen as the CAR-enhancer.

Example 6: CAR-enhancers reduce tumor burden, extend survival, and extend CAR T cell persistence in vivo

[000240] To show that CAR-enhancers reduce tumor burden, extend CAR T cell in vivo persistence, and extend survival, the following experiment was performed. 1 * 10⁶ OPM2 BCMA? multiple myeloma cancer cells were intravenously (i.v.) injected into NOD-scid fL2Ry^nu11 (NSG) mice 10 days before infusing a suboptimal dose of 5 * IO³ anti -BCMA CAR T cells by i.v. injection. After CAR T cell infusion, mice were treated twice weekly for two weeks, followed by once weekly with 200 pg/mouse a CAR-enhancer containing a BCMA ectodomain, a CH3 domain, and two weak affinity IL-2 immune cell effector domains (BCMA-CH3-muIL2-muIL2) by intraperitoneal (i.p.) injection (FIG. 6A).

[000241] Mice were subjected to bioluminescent imaging (BLI) for luciferase (indicating tumor burden of luciferase+ OPM2 cells) on days indicated in FIGs. 6B - 6D. Control mice that received OPM2 cells and no-CAR T cell infusion had progressively more tumor burden during the experiment and reached a humane end point on day 39 and 46. Mice that received OPM2 cells and the suboptimal dose of CAR T cells had controlled tumor growth until day 32, when they also saw progressively more tumor burden during the experiment and reached a humane end point on day 46. Mice that received OPM2 cells, CAR T cells, and CAR-enhancer therapy had reduced tumor burden (FIGs. 6B - 6D). In the CAR-enhancer treated group, all mice completely cleared OPM2 tumor cells from the bone marrow, as no signal was detected by imaging. One mouse in this group had significant OPM2 cell growth, due to formation of a solid tumor close to the eye and reached a humane end point on day 42. The remaining two mice completely cleared 0PM2 tumor cells, as no signal was detected by imaging and survived the experiment.

[000242] Next, the in vivo persistence of OPM2 and CAR T cells were analyzed in these mice by flow cytometry. One control mouse (OPM2 cells with no-CAR T cell infusion) was sacrificed on day 46, two CAR-only mice were sacrificed on day 46, and one CAR T cell and CAR-enhancer treated mouse was sacrificed on day 42. Sacrificed mice were analyzed for GFP+ OPM2 cells (FIGs. 7A-7C) and CD45⁺ CAR⁺ T cells (FIGs. 8A-8C) in the blood, spleen, lymph node, bone marrow and lung. The CAR T cell and CAR-enhancer treated mouse was also analyzed for GFP+ OPM2 cells and CD45+ CAR⁺ T cells in the eye tumor site.

[000243] FIGs. 7A - 7C show flow cytometry with GFP on the y-axis. GFP⁺ OPM2 cells were detected at similar levels in the bone marrow, lung (FIG. 7B), and liver (FIG. 7C) of the no-CAR control mouse and the CAR-only mice. One CAR-only mouse had significant levels of OPM2 cells in the blood and spleen (FIG. 7A).

[000244] The one mouse that received CAR T cell and CAR-enhancer treatment that developed an eye tumor had no to little OPM2 cells in the blood or spleen (FIG. 7A), bone marrow or lung (FIG 7B), and liver (FIG. 7C). This mouse had more OPM2 cells in the kidney (3.18% of GFP⁺ cells), and the majority of cells in the eye tumor site were OPM2 cells (96.5% of GFP⁺ cells).

[000245] FIGs. 8A - 8C show flow cytometry with anti-CD45 on the y-axis and BCMA⁺-CH3 tagged with Alexa Flour™ 647 (AF647) on the x-axis. CD45⁺ CAR⁺ T cells only persisted in CAR T cell and CAR-enhancer treated mice. CAR-only treated mice had little to no CD45⁺ cells in all organs tested (FIGs. 8A-8C). However, CAR T cell + CAR-enhancer treated mice had CD45⁺ cells that also stained positive for the BCMA cancer antigen (which is also the CAR binding target) tagged with AF647, as shown on the x-axis. CD45⁺ AF647⁺ double positive CAR T cells were detected in the blood, spleen, and lymph node (FIG. 8A), bone marrow and lung (FIG. 8B), and liver and kidney (FIG. 8C). CD45⁺ single positive cells were only detected in large numbers in liver and kidney (FIG. 8C). Little to no CD45⁺ AF647⁺ double positive CAR T cells were detected in the eye tumor site (FIG. 8C). These results indicated that CAR-enhancers reduce tumor burden, extend CAR T cell in vivo persistence, and extend survival.

Example 7: CAR-enhancer fate

[000246] The CAR-enhancers bind CAR T cells at the cell surface at 4 °C, and slowly internalize at 37 °C. Internalization of CAR-enhancer was assessed using fluorescently labeled BCMA-muIL2 CAR -enhancer. BCMA CAR T cells were exposed to AlexaFluor647-labeled BCMA-muIL2, BCMA-CH3, or VHH-muIL2 at a concentration of 2 nM. The cells were incubated at either 4 °C or 37 °C for various time intervals, followed by fixation and subsequent microscopy imaging. It was observed that the control VHH-muIL2 underwent rapid internalization within 30 minutes at 37 °C, whereas the internalization of BCMA-muIL2 CAR-enhancer was significantly slower (FIGs. 14A - 14C). The internalization rate ofBCMA-CH3 was similarly slow, even slower than that ofBCMA- muIL2 CAR-enhancer. In FIG. 2C, the CAR and dsRed transcripts were encoded within the transgene, and thus the dsRed signal reflects the expression level of CAR. All imaged cells with mean intensities higher than background were reported. For the dsRed channel, the cytoplasm mean intensity was reported, as the dsRed is expressed inside the cell, whereas for the AlexaFluor 647 channel, the mean intensity for the entire cell was measured.

[000247] The CAR-enhancer rapidly clears from the circulation. Pulsing CAR T cells with the CAR-enhancer treatment, where pulsing involves periods of stimulation followed by periods of resting, is superior to prolonged exposure to CAR-enhancers as extended exposure can lead to exhaustion or the generation of terminally differentiated CAR T cells. A CAR-enhancer with a short circulation half-life can be more effective at expanding CAR T cells, driving generation of memory CAR T cells, decrease potential competition with tumor antigen for CAR binding, and enhanced safety profile in patients. Therefore, the CH3 domain of IgGl was used in the CAR-enhancer platform. Pharmacokinetic studies illustrated that the circulatory half-life of the CAR-enhancer was short (1-1.5 hours) (FIG. 9A). NSG mice were administered 8 mg/kg of BCMA-muIL2 CAR- enhancer (delivered i.p., N=3 mice). Blood samples were collected via tail-vein puncture at five different time points (30 min, 2 h, 8 h, 24 h, 48 h) post-administration. The sera were then obtained by centrifugation and used for the subsequent analysis. An ELISA was performed to determine the concentration of the treatments in the sera. The ELISA plates were coated with 5 pg/ml anti-His6 antibody overnight, followed by incubation with the sera for 2 h at room temperature. An anti-FLAG HRP antibody was next used for the detection; the CAR-enhancer was engineered to have FLAG and His6 tags at the C-terminus. Based on the collected five time points, the initial concentration of the treatment in the sera was estimated to be 20% higher than the first (30 min) collected time point. The BCMA CAR-E was >90% and >99% cleared from the circulation in 8 h and 24 h, respectively. The circulating half-life was estimated to be about 1.5 hours for the BCMA CAR-enhancer. Error bars represent mean with standard deviation. [000248] The BCMA CAR-enhancer enhances activity and persistence of CAR T cells in a multiple myeloma (MM) model. An MM xenograft mouse model with OPM2 cells engrafted in immunocompromised NOD-SCID IL-2Ry^nu11 (NSG) mice was utilized. Accordingly, NSG mice were intravenously injected with OPM2 cells (human MM, 1 million cells) via the tail vein. Two weeks after OPM2 cell injection, freshly prepared BCMA CAR T cells (0.5 million CAR⁺ cells containing an 41BB-CD3^ CAR construct) were intravenously administered. A cohort of mice received the BCMA-muIL2 CAR-enhancer treatment (FIG. 9B). NSG mice (n=5) were injected with OPM2 (human MM) cells followed by BCMA CAR (human) T cell administration according to the schedule. BCMA-muIL2 CAR-E treatment (200 pg) was administered twice per week for two weeks, followed by once per week until the endpoint. After one month or longer, mice were euthanized, and flow cytometric analyses were performed on the harvested organs. These results revealed a significant expansion of CAR T cells in the spleen and bone marrow of the BCMA- muIL2 CAR-enhancer-treated group, demonstrating over a 100-fold selective expansion of CAR T cells compared to non-treated animals that only received CAR T cells in the spleen (FIG. 9C, left panel) and bone marrow (FIG. 9C, right panel). These experiments were replicated multiple times with similar outcomes (FIG. 9D and FIGs. 15A - 15C; n=12 for CAR T cells only, n=22 for CAR T cells plus CAR-enhancer treatment).

[000249] BCMA CAR T cells were detected by co-staining with an anti-human CD45 antibody and Alexa647-labeled BCMA antigen. Similar results were obtained in repeated experiments. Additional control cohorts received VHH-muIL2 treatment with the same dose and schedule as BCMA-muIL2. FIG. 9D shows pooled data from these experiments. Data were analyzed by group mean comparisons using one-way ANOVA and subsequent Tukey post-hoc analysis. Individual flow graphs for the pooled data are shown in FIGs. 15A - 15C. Error bars represent mean with standard deviation.

[000250] The control group that received CAR T cells plus VHH-muIL2 treatment (n=7) did not exhibit significant expansion or persistence of CAR T cells compared to the control group that received only CAR T cells without treatment. These results show that the BCMA-muIL2 CAR- enhancer can expand CAR T cells in vivo. Further analysis revealed that the BCMA-muIL2 treatment had a more pronounced effect on CD8⁺ CAR T cells specifically, resulting in an unexpected significant increase in their proportion from the initial -30% to -70% of the total CD4⁺ and CD8⁺ CAR T cell population (FIG. 9E). The cohorts receiving only CAR T cells or CAR T cells with the VHH-muIL2 control treatment did not yield a sufficient number of persisting CAR cells for a similar analysis. Data were analyzed by group mean comparisons using one-way ANOVA and subsequent Tukey post-hoc analysis. Error bars represent mean with standard deviation.

[000251] The BCMA CAR-enhancer enhances CAR T cell trafficking in an MM model. An MM xenograft mouse model with OPM2 cells engrafted in immunocompromised NSG mice was utilized. Accordingly, NSG mice were intravenously injected with OPM2 cells (human MM, 1 million cells) via the tail vein. Ten days after OPM2 cell injection, freshly prepared BCMA CAR T cells (0.5 million CAR⁺ cells containing a 41BB-CD3^ CAR construct) were intravenously administered. A cohort of mice received the BCMA-muIL2 CAR-enhancer treatment (FIG. 20A). NSG mice (n=5) were injected with OPM2 (human MM) cells followed by BCMA CAR (human) T cell administration according to the schedule. BCMA-muIL2 CAR-E treatment (200 pg) was administered twice per week for two weeks, followed by once per week until the endpoint. After one month or longer, mice were euthanized, and flow cytometric analyses were performed on the harvested organs. These results (Fig. 20A) revealed a significant CAR T cell trafficking to the spleen, bone marrow, liver, kidney, and lung of the BCMA-muIL2 CAR-enhancer-treated group, demonstrating the presence and persistence of CAR T cells in all major organs tested as compared to non-treated animals that only received CAR T cells (FIG. 20B).

Example 8: BCMA CAR-enhancer treatment enables CAR T therapy with low-dose of CAR T cells

[000252] To further demonstrate the effectiveness of CAR-enhancer treatment and clearance of tumor cells by CAR T cells, a similar protocol as described above was conducted. However, in this study, a lower dose of only 100,000 CAR T cells was utilized (FIG. 10A). All mice treated with BCMA-muIL2 CAR-enhancer achieved complete tumor clearance (5/5), whereas not a single control mouse receiving either only CAR T cells (n=4) or CAR T cells combined with VHH-muIL2 treatment (n=4) were able to eliminate the tumors (FIGs. 10B-10C).

[000253] Analysis of blood samples collected at various time points revealed a substantial expansion of CAR T cells in the circulation following CAR-enhancer treatment, with the peak expansion observed at week 4 (FIG. 10D). Flow cytometric analyses of blood samples revealed robust expansion of CAR T cells in the treatment group compared to PBS or VHH-muIL2 cohorts. In contrast, the VHH-muIL2 treatment, despite slightly enhancing the initial response, did not induce a significant expansion of CAR T cells. Data was analyzed with two-way ANOVA for day 7, 14 and 21. Once all mice in PBS cohort were euthanized, BCMA-muIL2 and VHH-muIL2 comparisons were performed with multiple Mann-Whitney tests on days 28 and 35. Error bars represent mean with S.E.M. This expansion correlated to the levels of IFN-y detected in the circulation (FIGs. 17A - 17C). Additionally, the treatment facilitated the generation of memory CAR T cells, demonstrating long-lasting effects (FIG. 10E and FIGs. 17A - 17C). *P<0.05, **P<0.01. Error bars represent mean with S.E.M.

[000254] The mice treated with CAR-enhancer exhibited no signs of toxicity based on clinical observations and weight measurements (FIG. 10H). Subsequent analysis conducted two months after CAR T cell injection demonstrated a substantial presence of CAR T cells, including memory CAR T cells, in the CAR-enhancer treated mice (FIGs. 10F-10J, FIGs. 16A - 16C, and FIGs. 17A - 17C). In the CAR + PBS group, CAR T cells were detected in the spleen; however, these mice succumbed to tumor growth at around 20 days post-CAR T cell injection. The BCMA-muIL2 treatment had also increased the presence of CAR T cells in bone marrow compared to PBS or VHH-muIL2 cohorts, but the difference was less significant than spleen. Data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test. *P<0.05, ***P<0.001, ****P<0.0001. Individual flow data are shown in FIGs. 16A - 16C. Error bars represent mean with S.E.M. Data in FIG. 101 show that persisting CAR T cells persist in the spleens of mice that received the BCMA- muIL2 CAR-enhancer and exhibited a CCR7⁺CD45RA⁺CD62L⁺ stem-cell memory phenotype, which was absent in the CAR + VHH-muIL2 or CAR + PBS cohorts.

[000255] tSNE analysis was based on surface marker expression of CD8a, CD4, CD45, CD45RA, CD45RO, CD62L, CD69, PD-1, HLA-DR, CCR7, and BCMA-CAR and revealed the presence of distinct memory T cell populations. CAR T cells were detected in the bone marrow of the VHH- muIL2 -treated group but not the spleen. In the BCMA-muIL2 group, persisting CAR T cells were predominantly CD8 T cells, while the majority of bone marrow CAR T cells in the VHH-muIL2 group were CD4 T cells. Further analyses are shown in FIG. 17A - 17C. The Flt-SNE mapping shown in FIG. 17C is of CAR T cells derived from the PBS, BCMA-muIL2 and VHH-muIL2 treated mice as shown in FIG. 10A. The expression of ten immune cell markers (CD45-Pacific Blue, CD8-FITC, CD4-PE Dazzle594, BCMA-CAR (antigen)-AlexaFluor647, CD69-BV421, PD- 1-BV605, CD45RA-APC-Cy7, CD45RO-PerCP-Cy5.5, CD62L-PE, CCR7-AlexaFluor700) on splenocytes and bone marrow from 3 PBS mice, 3 BCMA-muIL2 mice and 4 VHH-muIL2 mice were analyzed by flow cytometry. CD45⁺, a-BCMA-CAR⁺ immune cells from the mice were concatenated to form a total of -9800 (PBS spleen), -8100 (BCMA-muIL2 spleen), -7600 (PBS bone marrow), -14200 (BCMA-muIL2 bone marrow), -1420 (VHH-muIL2 Bone Marrow). The entire high dimensional dataset was merged to create a single Flt-SNE map for each condition with the signal strength of various phenotypic markers defining specific immune phenotypes expressed with a blue-green-yellow-red continuous color scale. FltSNE was conducted with the following parameters: max iterations: 1000, theta: 0.5, learning rate: 200, perplexity: 20. There were inadequate numbers of CAR T cells in VHH-muIL2 cohort spleen to conduct Flt-SNE. To enhance visibility, the dots representing the VHH-muIL2 bone marrow samples were enlarged, as fewer cells were detectable in these mice. In the BCMA-muIL2 treated mice, the majority of CAR⁺ cells were CD8⁺ cells, while in the VHH-muIL2 samples, CD4⁺ cells constituted the majority of CAR⁺ cells. Notably, CAR⁺ cells in the CAR+PBS cohort exhibited low or no expression of CD45RA, CD45RO, or CD62L, whereas the BCMA-muIL2 treated mice showed a CAR⁺ population with elevated expression levels of these memory markers.

[000256] Thus, the treatment not only facilitates robust proliferation and eradication of tumor cells using low doses of CAR T cells but also promotes the development of long-lasting memory cells, demonstrating the efficacy of BCMA-muIL2 CAR-enhancer treatment in enhancing the clearance of tumor cells by CAR T cells, and generation of long-lasting memory cells.

Example 9: Persisting CAR T cells treated with CAR-enhancer treatment remain functional three months post infusion

[000257] Mice received 1 million OPM2 cells followed by 0.5 million BCMA CAR T cells (FIG. HA). One group of mice received CAR-enhancer treatment, administered twice per week for two weeks, followed by once per week for an additional two weeks (6 doses, 200 pg per dose on days 4, 10, 14, 17, 21, and 28; n=5). The control group received VHH-muIL2 treatment at the same dosage and schedule (n=5), while an additional control cohort received only tumor cells (n=3). All mice receiving CAR T cells exhibited an initial response compared to control mice without CAR T cells (FIG. 1 IB). All CAR-enhancer treated mice (5 out of 5) and 3 out of 5 mice in the VHH-muIL2 group survived for over three months, which encompassed the duration of the experiment. One VHH-muIL2 mouse died in about a month, and a second mouse succumbed to cancer cell relapse with liver metastasis (FIG. 1 IB, day 77). The surviving mice were euthanized three months postinjection of CAR T cells, and the splenocytes and bone marrow cells were analyzed to assess the presence of CAR T cells. Remarkably, CAR-enhancer-treated mice exhibited a significant abundance of CAR T cells homing and persisting in the bone marrow and spleen compared to mice receiving CAR T cells with VHH-muIL2 treatment (FIG. 11C). Given the two-month period of no treatment before the mice were sacrificed, these results further suggest that the treatment facilitated the generation of memory cells among CAR T cells.

[000258] To demonstrate the functionality of the persisting CAR T cells in CAR-enhancer-treated mice, an in vitro killing assay was performed using the BCMA CAR T cells harvested from bone marrow and spleen. Bone marrow and splenocytes were analyzed via flow cytometry to detect and quantify CAR-expressing T cells and the bone marrow cells or splenocytes were co-incubated with OPM2 target cells at various E:T ratios (1 : 1 and 2:1) based on CAR-expressing cells. Survival was determined 24, 48, and 72 hours later using flow cytometric analysis. The three-month-old CAR T cells demonstrated efficient killing of tumor cells and long-term functionality (FIG. 1 ID). Only one of the VHH-muIL2 treated mice exhibited sufficient CAR T cells to perform a similar killing assay, and while it exhibited tumor cell killing, the efficiency was lower than that observed in CAR- enhancer treated CAR T cells (FIG. 1 ID) (error bars represent mean with standard deviation). Therefore, CAR-enhancer treatment robustly expands and drives the persistence of CAR T cells while maintaining their killing potential.

[000259] To further characterize the phenotype of these persistent CAR T cells, flow cytometric analyses were conducted to evaluate the expression of a series of T cell markers (CD45, BCMA CAR, CD4, CD8, CD62L, CD45RO, CD45RA, CD69, and PD-1). In order to facilitate interpretation, a t-SNE mapping of splenocytes, and bone marrow cells was generated (FIG. 1 IE). anti-CD45-Pacific Blue, anti-CD8-FITC, anti-CD4-PE Dazzle594, BCMA (antigen)- AlexaFluor647, anti-CD69-BV421, anti-PD-l-BV605, anti-CD45RA-APC-Cy7, anti-CD45RO- PerCP-Cy5.5, anti-CD62L-PE, and CCR7-AlexaFluor700) on splenocytes and bone marrow from the five BCMA-muIL2 treated mice were analyzed by flow cytometry. CD45⁺, CD8⁺, a-BCMA- CAR⁺ cells from the five mice were concatenated to form a total of -17600 (spleen) and -10800 (bone marrow) cells. The entire high dimensional dataset (excluding the CD45, CD8, and CD4 parameters) was merged to create a single tSNE map with the signal strength of six phenotypic markers defining specific immune phenotypes expressed with a blue-green-yellow-red continuous color scale. tSNE analysis was performed using 1000 iterations, a perplexity of 30 and a learning rate of 1237 and 756 for spleen and bone marrow respectively. Population labeled as 1 appears to display a memory-like phenotype, expressing higher levels of CD45RO, CD62L and CD45RA. Population labeled as 2 appears to display an effector-like phenotype, expressing low levels of CD45RO, CD62L and CD45RA.

[000260] The flow cytometric analyses revealed that the persisting CAR T cells consisted of both CD4⁺ and CD8⁺ populations. The CD8 CAR cells appeared to exhibit two distinct populations: effector cells and CD45RA⁺CD62L⁺ memory cells. The memory population exhibited higher expression levels of BCMA CAR and CD45 (FIG. 1 IE). Similarly, CD4⁺ CAR T cells demonstrated two populations of effector and memory cells (FIG. 18). An insufficient number of CAR T cells could be detected from the VHH-muIL2 treated mice to perform a similar flow cytometric analysis. Therefore, the CAR-enhancer treatment leads to generation of long-lasting memory CAR T cells.

[000261] FIG. 18 shows t-SNE mapping of CD4⁺ CAR⁺ T cells derived from the five BCMA- muIL2 CAR-E treated mice as shown in FIGs. 11A - HE. The expression of nine immune cell markers (aCD45-PacificBlue, aCD8-FITC, aCD4-PE Dazzle594, BCMA (antigen)- Al exaFluor647, aCD69-BV421, aPD-l-BV605, aCD45RA-APC-Cy7, aCD45RO-PerCP-Cy5.5, aCD62L-PE) on splenocytes and bone marrow from the five BCMA-muIL2 treated mice were analyzed by flow cytometry. CD45⁺, CD4⁺, a-BCMA-CAR⁺ immune cells from the five mice were concatenated to form a total of -9000 (spleen) and -6600 (bone marrow) cells. The entire high dimensional dataset (excluding the CD45, CD8, and CD4 parameters) was merged to create a single t-SNE map with the signal strength of six phenotypic markers defining specific immune phenotypes expressed with a blue-green-yellow-red continuous color scale. tSNE analysis was performed using 1000 iterations, a perplexity of 30 and a learning rate of 630 and 466 for spleen and bone marrow, respectively. The population labeled as “1” appears to display a memory-like phenotype, expressing higher levels of CD45RO, CD62L and CD45RA. The population labeled as “2” appears to display an effector-like phenotype, expressing low levels of CD45RO, CD62L and CD45RA.

[000262] Single-cell RNA-sequencing (scRNAseq) analysis was performed on CAR⁺ T cells isolated from mice treated with either the BCMA-muIL2 or the VHH-muIL2 control. Despite the limited presence of CAR T cells in the VHH-muIL2-treated mice, a sufficient number of cells were obtained from one of the VHH-treated mice for the experiment (FIG. 19A). CAR⁺ cells were sorted after staining with BCMA-AlexaFluor647 and Total Seq-C hashing antibodies from BCMA-muIL2 or VHH-muIL2 treated mice as shown in the red and green boxes, respectively in FIG. 19A. 5000 CAR⁺ cells from BCMA-muIL2 mice bone marrow and spleen and 2500 CAR⁺ cells from VHH- muIL2 mice bone marrow and spleen were loaded onto the 10X channel. The scRNAseq analysis revealed that the predominant population of persistent CAR T cells in the BCMA-muIL2-treated mouse consisted of CD8⁺ T cells (FIGs. 19B - 19C), which exhibited an enrichment of genes associated with an activated T cell state (FIGs. 19D - 19E). Heatmaps in FIG. 19D show significantly differentially expressed genes between CAR-enhancer treatment and VHH conditions in CD8 and CD4 CAR T cells, split among different relevant conditions. Genes marked with an * are the significantly differentially expressed genes between BCMA-muIL2 and VHH-muIL2 treated mice in the subset of interest. This was evidenced by elevated expression levels of granzyme family genes, other cytotoxicity-associated genes, and MHC class II genes. No significant differences in activation markers were observed between CAR T cells obtained from the BCMA-muIL2 or VHH- muIL2 treated mice, as the mice had already cleared the tumors over 60 days prior. The BCMA- muIL2 treatment did not induce upregulation of exhaustion markers, showing the treatment did not induce exhaustion in the persisting CAR T cells.

[000263] Next, the diversity of T cell receptor (TCR) clonotypes was evaluated in the BCMA- muIL2 and VHH-muIL2 treated mice (FIGs. 19F - 19G). Both groups displayed similar diversity in clonotypes present, showing that the BCMA-muIL2 CAR-enhancer treatment could effectively facilitate the generation of a diverse TCR repertoire in persisting CAR T cells, as opposed to promoting the dominance of a restricted set of TCR clones. Pie plots in FIG. 19F show the diversity of TCR clonotypes, with each slice of the pie chart representing the proportion of a different TCR clonotype present; colors were randomly assigned to different clonotypes. The clonotype diversity within each sample’s total cell count was visualized with a stacked bar plot (FIG. 19G), where similar clonotypes with counts below 50 were combined. To evaluate the diversity within each sample, the Simpson index was calculated, with higher values indicating greater diversity. Overall, the results showed that the BCMA CAR-enhancer could not only help CAR T cells to fully clear tumor cells, but also robustly induce generation of long-lasting and functional memory CAR T cells.

Example 10: CAR-enhancer expands CAR T cells in the absence of tumor antigens

[000264] CAR T cell expansion typically occurs following infusion in patients, with peak expansion observed around 10-14 days post-infusion (Rodriguez-Otero et al., N. Engl. J. Med. 355(77/1002-1014 (2023)).

[000265] Eradication of minimal residual disease (MRD) facilitates long-lasting and complete responses. However, the limited presence of the corresponding antigen associated with MRD may not adequately support the proliferation and efficacy of conventional CAR T cells. To demonstrate efficacy in the absence of tumor antigen, NSG mice were solely injected with 0.25 million BCMA CAR T cells in the absence of tumor cells. These mice received two 25 pg doses of BCMA-muIL2 on days 1 and 8 post-injection of CAR T cells. The control group received VHH-muIL2 treatment (n=4 for each group). On day 30, the mice were euthanized, and their spleen and bone marrow were assessed for the presence of CAR T cells. The BCMA-muIL2 treated mice exhibited higher numbers of CAR⁺ T cells in the spleen (approximately 6.8-fold higher) and bone marrow (approximately 5.5- fold higher), showing that BCMA CAR-enhancer expanded CAR T cells in vivo, even without the presence of tumor cells (FIGs. 12A-12B); error bars represent mean with standard deviation. Overall, these findings demonstrate that the CAR-enhancer can expand CAR T cells, even in the absence of tumor antigen. Additionally, the effectiveness of the treatment was evident even at lower doses and frequencies.

Example 11 : CD19 CAR-E does not inhibit killing efficacy of CD 19 CAR T cells

[000266] CAR-enhancers that bind BCMA were observed to bind but not inhibit killing efficacy of BCMA CAR T cells (FIGs. 2D - 2H). To investigate the impact of CAR-E containing other cancer antigens on antigen-specific CAR T cells, a killing assay was conducted using CD19 CAR T cells and patient-derived CD19⁺ leukemia cells in the presence of varying concentrations of the CD 19 CAR-enhancer. Remarkably, the results demonstrated no inhibition of killing even at the highest tested concentration (1000 nM of the CAR-enhancer) (FIG. 21C). Nalm6 cells were co-incubated with CD19 CAR T cells (filled) or non -transduced T cells (open) (E:T ratio 1 : 1 ; 30,000 cells of each) in the presence of varying concentrations of the CD19-muIL2 CAR-E treatment. Live (PT) Nalm6 cells were counted 48 hours later, with an N of 3 for each of the experiments. The experimental findings disclosed herein with CD 19 are consistent with the findings in BCMA cancer models and BCMA CAR-E, above.

Example 12: CAR-enhancers effectively enhance CAR immune cell efficacy and persistence at low doses on initial treatment and tumor rechallenge

[000267] Next, an experiment to investigate the efficacy of a reduced frequency of CAR-E treatment initiated two weeks after CAR T cell injection was conducted. Mice were intravenously injected with 0PM2 cells (l* 10⁶, i.v ). A week later, CAR T cells (0.5* 10⁶, i.v.) were administered. Two weeks post-injection of CAR T cells, the mice were divided into two cohorts, with mice receiving the CAR-E treatment or not. The treatment group received four doses of CAR-E treatment (4 mg/kg per dose) on days 14, 18, 21, and 28. Surviving mice underwent re-challenge with 1 x l O⁶ of 0PM2 cells on day 60, followed by 4 mg/kg CAR-enhancer treatment on days 68, 70, 74, 77, and 80 (FIG. 22A).

[000268] Remarkably, bioluminescence imaging (BLI) analyses demonstrated that all mice (5/5) receiving CAR-E cleared tumors, while none of the control mice (0/4) achieved tumor clearance; the same BLI quantification scale is used for all images (photons/sec) (FIGs. 22B - 22D). Blood analyses indicated that the four doses of CAR-E treatment were sufficient to robustly expand CAR T cells in all the treated mice compared to the control cohort (FIGs. 22E and 25). Cytokine assessments also revealed elevated levels of IFN-y in the CAR-E treatment cohort by sandwich ELISA on 1 :40 diluted serum samples collected the same days when CAR-T counts were assessed as shown FIG. 22E (FIG. 22F).

[000269] One of the CAR-E treated mice (M5) showed liver relapse on day 60 (FIG. 22B). Regardless, all the five CAR-E treated mice underwent re-challenge, using 1 million of the liver metastasis-derived OPM2 cells, on day 60 to assess the generation of memory CAR-T cells. All mice showed considerably less signal compared to the naive control mice (FIG. 22B; see day 66). While the mice exhibited liver signals, none showed bone marrow signals, suggesting the presence of functional memory CAR T cells in the bone marrow inhibiting tumor growth. To explore whether CAR-E treatment could contribute to re-expanding CAR T cells and controlling tumor growth in liver metastasis, the mice were re-treated with CAR-E (4 mg/kg) on days 68, 70, 74, 77, and 80. Impressively, all mice successfully cleared the liver metastasis, indicating that CAR-E could facilitate the re-expansion and trafficking of CAR T cells to eliminate tumor cells (FIG. 22B; see day 83). The mouse that showed relapse on day 60 (M5, FIG. 22B) also cleared the tumor from liver after re-challenge, although it exhibited some signal on the last day of the experiment. Blood analyses confirmed that the CAR-E had resulted in a robust re-expansion of the CAR T cells in the circulation in all mice (FIG. 22E).

[000270] On day 90 post-injection of CAR T cells, the mice were euthanized, and organ analysis via flow cytometry revealed that all CAR-E treated mice harbored a significant amount of persisting CAR-T cells with diverse memory phenotypes (FIGs. 22G, 26A, and 26B). For analysis, the human CD45⁺ BCMA-CAR⁺ cells from the bone marrow and spleen of the mice were gated and concatenated, then FLOWSOM analysis was conducted on the pooled populations to identify eight major phenotypic metaclusters. A heatmap representing the Mean Fluorescence Intensity (MFI) of each marker within each metacluster was used to qualitatively describe each cluster (FIGs. 27A - 27D). In FIG. 22G, the proportion of each metacluster within the bone marrow and spleen of each mouse is depicted. The no-treatment “CAR-T only” cohort did not have enough persisting CAR T cells in the bone marrow or spleen to allow for a similar analysis. This experiment utilized PBMCs from one donor.

[000271] In summary, these results indicate that BCMA CAR-E treatment not only facilitated the complete clearance of tumor cells by the CAR T cells but also significantly promoted the formation of functional memory CAR T cells. Notably, even a few doses of CAR-E administered two weeks after CAR T cell administration was effective in expanding CAR T cells and fostering the development of functional memory CAR T cells. The persisting CAR T cells retained the capacity to re-expand in response to CAR-E treatment.

Example 13: CAR T cell expansion in the absence of tumor antigens is dose-dependent.

[000272] Typically, CAR T cell expansion occurs post-infusion in patients, with peak expansion observed around 10-14 days post-infusion (Rodriguez-Otero et al. , N. Engl. J. Med. 388(17/1002- 1014 (2023)). This expansion is driven by antigen availability and the tumor-killing process, which promotes CAR T cell proliferation (Turtle etal., J. Clin. Invest. 126(6):2\23-38 (2016), Gardner et al., Blood 729(25/3322-3331 (2017), Lee et al., Leukemia 35(7/255-258 (2021), Hossain et al., Blood 132(Suppl 7/490-490 (2018)). However, patients who exhibit limited CART cell expansion post-infusion show poor responses (Fraietta et al., Nat. Med. 24(5/563-571 (2018)). Additionally, achieving long-lasting complete responses necessitates the eradication of minimal residual disease. Yet, the limited presence of the corresponding antigen associated with minimal residual disease may not sufficiently support the proliferation and efficacy of CAR T cells.

[000273] Without being bound by theory, the CAR-E mechanism of action may be independent of tumor cells and antigens presented thereon and may thus expand CAR T cells in the absence of tumor cells (and therefore tumor antigen), addressing the critical clinical challenge of limited in vivo CAR T cell expansion post-infusion. To test this hypothesis, as well as simultaneously assessing the dose-dependent nature of CAR-E treatment, mice were injected solely with 0.25 million BCMA CAR T cells in the absence of tumor cells. As illustrated in FIG. 23 A, NSG-DKO mice treated with 0.25>< 10⁶ BCMA CAR T cells and were assigned to different cohorts receiving varying doses of BCMA-muIL2 CAR-E treatment (2 mg/kg, 4 mg/kg, 8 mg/kg, or no CAR-E treatment; twice per week for four weeks; n=5 for each cohort). Organs were collected one-month post-injection of CAR T cells, and flow cytometric analyses were conducted to assess the presence of CAR T cells.

[000274] CAR-E treatment resulted in expansion and persistence of the CAR T cells in spleen and bone marrow (FIGs. 23B - 23C and 28 A - 28D). These results revealed that CAR-enhancer treatment lead to dose-dependent expansion of the CAR T cells. Error bars shown in FIGs. 23B - 23C are mean ± standard deviation and displays column bars indicating the absolute number of detected CAR T cells in each condition and statistical analyses that demonstrate that CAR-enhancer treatment leads to dose-dependent expansion of CAR T cells. The significance between the group of mice that received PBS and the group of mice received the lowest concentration CAR-enhancer treatment (2 mg/kg) was measured using the Mann-Whitney test. Additionally, a simple linear regression was conducted to demonstrate the dose-dependent effect of the treatment; the error bars on the graph represent a 95% confidence interval.

[000275] Further, the results demonstrated that CAR-E impact on CAR T cells is dose-dependent (FIGs. 23B - 23C). Interestingly, even at the lowest tested dose of 2 mg/kg, there was substantial persistence of CAR T cells compared to the no-treatment cohort, where very few to nearly no CAR T cells were detected one month post CAR-T injection (FIGs. 23B - 23C). Additional analyses indicated that CAR-E induced the generation of memory CAR T cells (FIGs. 23D - 23E). TEM cells are CD45RA' CD45RO⁺ CCR7'; TEMRA cells are CD45RA⁺ CD45RO⁺ CCR7'; TSCM cells are CD45RA⁺ CD45RO⁺ CCR7⁺; TCM cells are CD45RA' CD45RO CCR7⁺; T_Naive cells are CD45RA⁺ CD45RO' CCR7⁺. The experiment in panels FIGs. 23A - 23E utilized PBMCs from one donor. Overall, these findings demonstrate that CAR-E treatment induces the expansion of CAR T cells and enables the CAR T cells develop diverse memory phenotypes, regardless of the presence of tumor cells.

[000276] To further investigate CAR-E’ s capacity to expand CAR T cells and to confirm the essential role of the low-affinity IL-2 component of BCMA-muIL-2 CAR-E in influencing CAR T cells, a similar experiment, as described above, was repeated using the 4 mg/kg dose and 0.25* 10⁶ CAR T cells. Mice received either the BCMA-muIL2 CAR-E treatment, treatment with the control BCMA-CH3 molecule, which contains only the BCMA antigen and not the low-affinity mutated IL-2 component, or no treatment. As anticipated, the antigen-only, BCMA-CH3 treatment cohort did not result in the expansion or persistence of CAR T cells compared to the CAR-E treatment cohort, further validating that both the ectodomain (e.g, BCMA antigen) component and the immune cell effector domain (e.g., low-affinity IL-2) component of the CAR-E molecule are necessary for its impact on CAR T cells (FIG. 23F - 23G and 29A - 29E). Mice received CAR T cells and different treatments (4 mg/kg) following a schedule similar to that shown in FIG. 23 A. The BCMA-CH3 antigen, VHH-muIL2, or the low-dose wild-type IL-2 treatments did not result in expansion or persistence of CAR T cells compared to the CAR-enhancer-treated cohort. Error bars represent mean with standard deviation. The experiment in panels FIGs. 23F - 23G utilized PBMCs from two donors. The Kruskal-Wallis test was used for each subset of CAR T cells and total T cells. Subsequently, post-hoc Dunn’s analysis was conducted to compare each group with the treatment group. The table in FIG. 23G displays the adjusted p-values. This aligns with the in vitro and in vivo analyses disclosed herein.

[000277] To compare the efficacy of CAR-E treatment with a low-dose wild-type IL-2, which is used in the clinic in combination with CAR T therapy, an additional cohort was included that received low-dose wild-type IL -2 (SEQ ID NO: 102) (4.5 pg per mouse, for 14 days starting from day 1, and then twice per week for an additional two weeks). The low-dose IL-2 group failed to result in substantial persisting CAR-T cells compared to the CAR-E treatment cohort (FIGs. 23F - 23G). Clinical studies that have used low-dose IL-2 in combination with CAR-T have not resulted in significant benefits, and in some cases, low-dose IL-2 was stopped due to IL-2-associated toxi cities (Katz etal., Clin. Cancer Res. 27(74/3149-59 (2015)). Furthermore, as expected, the nontargeted, low-affinity IL-2 (VHH-muIL2) CAR-E did not lead to the expansion or persistence of CAR T cells (FIGs. 5F - 5G), consistent with the prior findings disclosed herein.

[000278] In summary, these findings demonstrate that the CAR-E molecule can expand CAR T cells and robustly promote the development of diverse memory phenotypes, regardless of the presence of tumor cells. Both the ectodomain (e.g., BCMA antigen) component and the immune cell effector domain (e.g., low-affinity IL-2) component of the CAR-E molecule are necessary for the observed impact on CAR T cells. Additionally, the effectiveness of the CAR-E treatment was evident even at lower doses.

Example 14: The efficacy of the CAR-E requires signaling through both the CAR and the IL-2R intracellular signaling domains

[000279] To better understand the mechanism of action of CAR-E, it was investigated whether the effects on CAR T cells are solely mediated by anchoring the low-affinity IL-2 onto CAR T cells through BCMA-to-CAR binding or if it involves simultaneous engagement of both IL-2R and CAR intracellular signaling domains. For this purpose, BCMA CAR T cells were made using a BCMA CAR construct lacking the 41BB-CD3^ intracellular signaling domain but retaining an identical extracellular ectodomain (referred to as CAR-Intracellular domain deletion, or CAR-ICD-A). In vitro analyses demonstrated that the BCMA-muIL2 CAR-E molecule induced pSTAT5 in CAR- ICD-A T cells after a 30-minute incubation with CAR-E (n=3), similar to full CAR T cells (FIG. 24A), suggesting that the effects of low-affinity IL-2 are similar in both CAR constructs and are mediated via antigen-to-CAR binding.

[000280] However, interestingly, while CAR-E robustly activated full CAR T cells, as evidenced by elevated CD69 expression (FIG. 24B) and increased production of ZFN-y (FIG. 24C) and TNF- a (FIG. 24D), its impact on CAR-ICD-A CAR T cells was negligible. Additional control conditions included the non-targeted VHH-muIL2 and the antigen-only BCMA-CH3 (n=3 for each condition), error bars represent mean with standard deviation and the experiment utilized PBMCs from one donor. Dasatinib, a lymphocyte cell-specific protein-tyrosine kinase (LCK) inhibitor, and Ruxolitinib, a Janus kinase (JAK) inhibitor, both individually and in combination, significantly inhibited the impact of the CAR-E molecule on CAR T cells (FIGs. 24E - 24G), further suggesting that the CAR-E molecules engages both CAR and IL-2R receptors and activating their intracellular signaling pathways. CAR T cells were treated with varying doses of CAR-E treatment and the individual inhibitor or their combination, with assessments conducted 24 hours later (n=3 for each condition); error bars represent mean with standard deviation. Subsequently, the in vivo impact of CAR-E on full CAR T cells and CAR-ICD-A T cells was compared. Mice injected with 0.25* 10⁶ CAR T cells in the absence of tumor cells received CAR-E treatments (4 mg/kg twice per week for four weeks; FIG. 24H). After a month, animals were euthanized, and organs were analyzed via flow cytometry. Remarkably, while CAR-E robustly expanded CAR T cells with the full CAR construct consistent with the previous results, it did not result in expansion or persistence of CAR-ICD-A T cells (FIG. 241). The experiment utilized PBMCs from one donor; statistical analyses were performed using an unpaired T-test.

[000281] To gain additional insights into the mechanism of action of CAR-E, their impact on the transcriptome of CAR T cells was investigated. Various treatments, including the BCMA-muIL2 CAR-E molecule, the non-targeted low-affinity IL-2 (VHH-muIL2), BCMA-CH3 antigen, and wild-type IL-2, were added to CAR T cells. As an additional control, CAR-ICD-A T cells were treated with the BCMA-muIL2 CAR-E molecule. Treatments were removed after 2 hours to mimic in vivo conditions, and cells were subjected to bulk RNA-sequencing either 2 or 22 hours later.

[000282] Remarkably, the CAR-E induced substantial transcriptomic changes, demonstrating quantitatively larger fold-changes compared to all other conditions, including wild-type IL-2 (FIGs. 24J - 24N). The differences in gene upregulation of the BCMA-muIL2 compared to wildtype IL-2, VEIH-muIL2, and BCMA-CH3, as well as GSEA of these different conditions (FIG. 30) show that while these control treatments have an effect on their own, the stimulation induced by the CAR-E molecule is greatly superior (FIG. 24L - 24M). The impact of CAR-E on CAR-ICD-A T cells was mild, further emphasizing the significant role of the CAR-intracellular signaling domain in the mechanism of action of the CAR-E molecule (FIGs. 24J - 24K). CAR-T cells underwent a 2- hour incubation with 10 nM of the BCMA-muIL2 CAR-E molecule or control molecules, followed by treatment removal through washing. Subsequently, RNA-sequencing is performed 2 and 24 hours later.

[000283] In summary, these findings indicate that the mechanism of action of the CAR-E molecule goes beyond merely delivering low-affinity IL-2 to CAR T cells. Instead, it operates by engaging and, importantly, bridging the intracellular signaling domains of the CAR and IL-2R receptors, inducing significant T cell activation and substantial transcriptomic changes.

[000284] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[000285] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.

Claims

What is claimed is:

1. A method of enhancing activity of chimeric antigen receptor (CAR) immune cells, comprising: administering to a subject having had CAR immune cell therapy that targets an antigen present on a cancer cell, a first course of an effective amount of CAR-enhancer therapy, wherein the CAR-enhancer comprises a first moiety that binds an epitope on an extracellular domain of the CAR connected to a second moiety comprising a first immune cell effector domain; wherein administration of the first course of CAR-enhancer therapy is initiated at any time up to about 6 months after the subject received the CAR immune cell therapy.

2. The method of claim 1, wherein the first course of the CAR-enhancer therapy is initiated at any time up to about 5 months after the CAR immune cell therapy.

3. The method of claim 1 or 2, wherein the first course of the CAR-enhancer therapy is initiated at any time CAR-enhancer up to about 4 months after the CAR immune cell therapy.

4. The method of any one of claims 1-3, wherein the first course of the CAR-enhancer therapy is initiated at any time CAR-enhancer up to about 3 months after the CAR immune cell therapy.

5. The method of any one of claims 1-4, wherein the first course of the CAR-enhancer therapy is initiated at any time CAR-enhancer up to about 2 months after the CAR immune cell therapy.

6. The method of any one of claims 1-5, wherein the first course of the CAR-enhancer therapy is initiated at any time CAR-enhancer up to about 1 month after the CAR immune cell therapy.

7. The method of any one of claims 1-6, wherein the first course of the CAR-enhancer therapy is initiated at any time CAR-enhancer up to about 4 weeks after the CAR immune cell therapy.

8. The method of any one of claims 1-7, wherein the first course of CAR-enhancer therapy is initiated about 2 weeks after the CAR immune cell therapy.

9. The method of any one of claims 1-8, wherein the first course of CAR-enhancer therapy is initiated 2 weeks after the CAR immune cell therapy.

10. The method of any one of claims 1-9, wherein the first course of the CAR-enhancer therapy is conducted over a period of time of about 1 to about 3 weeks.

11. The method of any one of claims 1-10, wherein the first course of the CAR-enhancer therapy is conducted over a period of time of about 2 weeks.

12. The method of claim 11, wherein the first course of the CAR-enhancer therapy comprises administering from about 1 to about 6 doses of the CAR-enhancer.

13. The method of claim 11, wherein the first course of the CAR-enhancer therapy comprises administering 4 doses of the CAR-enhancer.

14. The method of any one of claims 1-13, wherein the effective amount of the CAR-enhancer is from about 1 mg/kg to about 8 mg/kg per dose of the CAR-enhancer.

15. The method of any one of claims 1-14, further comprising administering a second course of an effective amount of a CAR-enhancer therapy to the subject, wherein subsequent to the administration of the first course of CAR-enhancer therapy, the subject has relapsed, or is at risk of relapse, and wherein the CAR-enhancer administered in the second course of CAR-enhancer therapy may be the same as or different from the CAR-enhancer administered in the first course of CAR- enhancer therapy.

16. The method of claim 15, wherein the CAR-enhancer administered in the first and the second courses of CAR-enhancer therapy are the same.

17. The method of any one of claims 15 or 16, wherein the second course of CAR-enhancer therapy is conducted over a period of time of about 1 to about 3 weeks.

18. The method of any one of claims claim 15-17, wherein the effective amount of the CAR- enhancer administered in the second course of CAR-enhancer therapy is from about 1 mg/kg to about 8 mg/kg per dose of CAR-enhancer.

19. The method of any one of claims 1-18, wherein the CAR immune cell therapy administered to the subject comprises about 1 * 10⁴ to about 1 x IO¹⁰ cells.

20. The method of claim 18, wherein the CAR immune cell therapy administered to the subject comprises about l * 10⁶ to about l *10¹⁰ cells per subject.

21. The method of claim 18, wherein the CAR immune cell therapy administered to the subject comprises about 1 * 10⁴ to about 1 * 10⁷ cells per subject.

22. The method of any one of claims 1-21, wherein the first moiety of the CAR-enhancer comprises an ectodomain of the antigen present on the cancer cell.

23. The method of claim 22, wherein the ectodomain is derived from AFP, AXL, B4GALNT1, B-cell maturation antigen (BCMA), CA9, CD5, CD7, CD19, CD20, CD22, CD23, CD33, CD34, CD38, CD44, CD52, CD70, CD80, CD86, CD123, CD133, CD174, CD274, CD276, CDS, Cancer/Testis Antigen IB (CTAG1B), carcinoembryonic antigen (CEA), CLEC12A, claudin 18.2 (CLDN 18.2), CSPG4, DLL3, EGFR, EPCAM, EPHA2, ERBB2, FAP, FOLH1, FOLR1, GD2, GPC3, GPRC5D, GPNMB, HER2, HPV E7, IL1RAP, IL3RA, IL13Ra2, KDR, KIT, KLRK1, L1CAM, MAGEA1, MAGEA4, MET, MME, MSLN, MUC1, MUC16, MS4A1, NCAM1, PD-1, PMEL, PROMI, PSCA, ROR1, ROR2, SDC1, SLAM7, TEM1, TROP2, TNF Receptor Superfamily Member (TNFRSF) 8, TNFRSF10B, TNFRSF13C, TNFRSF17, ULBP1, or ULBP2.

24. The method of claim 23, wherein the ectodomain comprises the amino acid sequence NO: 1).

25. The method of claim 23, wherein the ectodomain comprises the amino acid sequence

26. The method of claim 22, wherein the ectodomain is derived from CD 19.

27. The method of claim 26, wherein the ectodomain comprises an amino acid sequence having at least about 85% sequence identity with the amino acid sequence

28. The method of claim 27, wherein the ectodomain comprises the amino acid sequence SEQ ID NO: 3.

29. The method of claim 27, wherein the ectodomain comprises the amino acid sequence

30. The method of claim 22, wherein the ectodomain is derived from CD20.

31. The method of claim 30, wherein the ectodomain comprises the amino acid sequence

32. The method of claim 22, wherein the ectodomain is derived from SLAMF7.

33. The method of claim 32, wherein the ectodomain comprises the amino acid sequence

34. The method of claim 22, wherein the ectodomain is derived from PD-1.

35. The method of claim 34, wherein the ectodomain comprises the amino acid sequence

36. The method of claim 22, wherein the ectodomain is derived from KIT.

37. The method of claim 36, wherein the ectodomain comprises the amino acid sequence

38. The method of claim 22, wherein the ectodomain is derived from CD38.

39. The method of claim 38, wherein the ectodomain comprises the amino acid sequence VPRWRQQWSGPGTTI<RFPETVLARCVKYTEIHPEMRHVDCQSVWDAFI<GAFISI<HPCNI

40. The method of claim 22, wherein the ectodomain is derived from CD22.

41. The method of claim 40, wherein the ectodomain comprises the amino acid sequence

42. The method of claim 41, wherein the ectodomain comprises the amino acid sequence

43. The method of claim 1, wherein the first moiety is an antibody that binds an epitope on the extracellular domain (ED) of the CAR, or derivative thereof.

44. The method of claim 43, wherein the CAR present on the CAR immune cell binds AFP, AXL, B4GALNT1, BCMA, CA9, CD5, CD7, CD19, CD20, CD22, CD23, CD33, CD34, CD38, CD44, CD52, CD70, CD80, CD86, CD123, CD133, CD174, CD274, CD276, CDS, CTAG1B, CEA, CLEC12A, CLDN 18.2, CSPG4, DLL3, EGFR, EPCAM, EPHA2, ERBB2, FAP, F0LH1, FOLR1, GD2, GPC3, GPRC5D, GPNMB, HER2, HPVE7, IL1RAP, IL3RA, IL13Ra2, KDR, KIT, KLRK1, L1CAM, MAGEA1, MAGEA4, MET, MME, MSLN, MUC1, MUC16, MS4A1, NCAM1, PD-1, PMEL, PR0M1, PSCA, ROR1, ROR2, SDC1, SLAM7, TEM1, TROP2, TNFRSF8, TNFRSF10B, TNFRSF13C, TNFRSF17, ULBP1, or ULBP2]

45. The CAR-engager of claim 1, wherein the ED of the CAR further comprises a linker and wherein the first moiety binds an epitope on the linker.

46. The CAR-engager of claim 1, wherein the CAR binds CD 19 and wherein the first moiety has the amino acid sequence of SEQ ID NO: 124.

47. The method of any one of claims 1-46, wherein the CAR-enhancer further comprises a first linker that connects the first and second moieties.

48. The method of claim 47, wherein the CAR-enhancer further comprises a dimerization domain disposed between the first linker and the second moiety comprising the first immune cell effector domain.

49. The method of claim 48, wherein the CAR-enhancer further comprises a second linker that connects the dimerization domain and the second moiety, wherein the first and second linkers may be the same or different.

50. The method of claim 49, wherein the first linker and the second linker are flexible.

51. The method of claim 50, wherein the first linker and/or the second linker are derived from the hinge region of CD3(^, CD4, CD8a, CD28, IgGl, IgG2, or IgG4.

52. The method of claim 50, wherein the first linker and/or the second linker comprises the amino acid sequence GGGX, GGGGX (SEQ ID NO: 69), GSSGSX (SEQ ID NO: 70), GGGGS (SEQ ID NO: 71), or GSPRG (SEQ ID NO: 72), wherein X is either C or S.

53. The method of claim 50, wherein the first linker has the amino acid sequence of GGGGS (SEQ ID NO: 71), or GSPRG (SEQ ID NO: 72) and the second linker has the amino acid sequence of GSPRGGGGSGGGGSGGGGS (SEQ ID NO: 76).

54. The method of any one of claim 48-53, wherein the dimerization domain is derived from IgA, IgD, IgG, IgM, or IgE.

55. The method of claim 54, wherein the dimerization domain comprises an IgGl constant heavy (CH) 3 domain.

56. The method of claim 54, wherein the dimerization domain further comprises an IgG CH2 domain and an IgG CH3 domain.

57. The method of any one of claims 1-56, wherein the first immune cell effector domain comprises a cytokine or immune cell-activating moiety.

58. The method of claim 57, wherein the first immune cell effector domain is derived from CD30L, CD40, CD48, CD58, CD70, CD80, CD86, CD112, GITRL, HVEM, OX40L, SEMAA, SLAM, TIM4, interleukin-2 (IL-2), IL-7, IL-9, IL-10, IL-15, IL-18, IL-21, IL-27, 4-1BBL, or an immune cell-activating variant thereof.

59. The method of any one of claims 1-58, wherein the second moiety further comprises a plurality of immune cell effector domains.

60. The method of claim 59, wherein the second moiety further comprises a second immune cell effector domain, wherein the first and second immune cell effector domains may be the same or different.

61. The method of claim 60, wherein the second immune cell effector domain comprises a weak affinity variant of IL-2 which has the amino acid sequence APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLE

62. The method of claim 61, wherein the first immune cell effector domain and the second immune cell effector domain each has the amino acid sequence

63. The method of claim 60, wherein the first immune cell effector domain and the second immune cell effector domain each has the amino acid sequence of SEQ ID NO: 102 with a H16A substitution.

64. The method of claim 60, wherein the first immune cell effector domain and the second immune cell effector domain each has the amino acid sequence of SEQ ID NO: 102 with a F42A substitution.

65. The method of claim 60, wherein the first or the second immune cell effector domain comprises the amino acid sequence

66. The method of claim 65, wherein each of the first and the second immune cell effector domains comprises the amino acid sequence of SEQ ID NO: 26.

67. The method of claim 59, wherein the first immune cell effector domain comprises 4-1BBL or an immune cell-activating variant thereof.

68. The method of claim 67, wherein the first immune cell effector domain comprises the amino acid sequence

69. The method of claim 59, wherein the second moiety further comprises a third immune cell effector domain, wherein any two or more of the first, second, and third immune cell effector domains may be the same or different.

70. The method of claim 69, wherein each of the first, the second, and the third immune cell effector domains comprises the amino acid sequence of SEQ ID NO: 27.

71. The method of claim 57, wherein the first cell effector domain is a single-chain variable antibody fragment (scFv) that binds and activates immune cells.

72. The method of claim 71, wherein the first cell effector domain binds 4- IBB, CD2, CD27, CD28, CD30, CD40L, CD226, CTLA4, GITR, IL-2R, LIGHT, 0X40, PD-1, TIM2, SLAM, or TIME

73. The method of claim 72, wherein the first cell effector domain comprises a scFv that binds CTLA-4.

74. The method of claim 73, wherein the scFv comprises a VL domain with an amino acid sequence of domain with an amino acid sequence of

75. The method of claim 72, wherein the first cell effector domain binds 0X40.

76. The method of claim 75, wherein the scFv comprises a VL domain with an amino acid sequence of VH domain with an amino acid sequence of (SEQ ID NO: 43).

77. The method of claim 72, wherein the first cell effector domain binds PD-1 .

78. The method of claim 77, wherein the scFv comprises a VL domain with an amino acid sequence of VH domain with an amino acid sequence of (SEQ ID NO: 53).

79. The method of any one of claims 1-78, wherein the first immune cell effector domain comprises an immune cell-inhibiting moiety.

80. The method of claim 79, wherein the first immune cell effector domain is derived from CD80, CD86, CD112, CD155, CD276 (B7-H3), Ceacam-1, FGL1, galectin-3, HLA-E, HVEM, PD-L1, PD-L2, VISTA, or VTCN1 (B7-H4).

81. The method of claim 80, wherein the first immune cell effector domain is derived from PD- Ll.

82. The method of claim 81, wherein the first immune cell effector domain comprises the amino acid sequence

83. The method of claim 80, wherein the first immune cell effector domain is derived from CD80.

84. The method of claim 83, wherein the first immune cell effector domain comprises the amino acid sequence N

85. The method of claim 80, wherein the first immune cell effector domain is derived from CD276 (B7-H3).

86. The method of claim 85, wherein the first immune cell effector domain comprises the amino acid sequence

87. The method of claim 80, wherein the first immune cell effector domain is derived from VTCN1 (B7-H4).

88. The method of claim 87, wherein the first immune cell effector domain comprises the amino acid sequence NO: 66)

89. The method of any one of claims 1-89, wherein the CAR-enhancer is in the form of a fusion protein and the first and the second moi eties are connected by peptide bonds.

90. The method of any one of claims 1-89, wherein the first moiety comprising the CAR-binding domain is connected to the second moiety comprising the first immune cell effector domain, or the first linker by an azide-alkyne connection, an oxime or hydrazine connection, a tetrazine- transcyclooctene connection, an azide-nitrone connection, a thiol-alkene connection, an alkenetetrazole connection, an alkene-tetrazine connection, an alkene-azide connection, a conjugated diene-alkene connection, or an isonitrile-tetrazine connection.

91. The method of any one of claims 48-90, which is in the form of a homodimer comprising two of the CAR-enhancers.

92. The method of any one of claims 1-91, wherein the subject is a human.

93. The method of any one of claims 1-92, wherein the cancer is a hematopoietic cancer.

94. The method of claim 93, wherein the hematopoietic cancer is a leukemia, lymphoma, or multiple myeloma.

95. The method of claim 93 or 94, wherein the hematopoietic cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, or non-Hodgkin lymphoma.

96. The method of any one of claims 1-92, wherein the cancer is characterized by a solid tumor.

97. The method of claim 96, wherein the cancer is malignant mesothelioma, ovarian cancer, breast cancer, pancreatic cancer, lung cancer, liver cancer, glioblastoma, gastric cancer, endometrial cancer, cervical cancer, biliary cancer, uterine serous carcinoma, cholangiocarcinoma, neuroblastoma, sarcoma, lung cancer, or melanoma.