Development of a CAR engager platform to enhance CAR T cell functionality and / or persistence

Abstract

Disclosed are chimeric antigen receptor (CAR) engagers and proteinaceous entities and dimers comprising the ectodomain of an antigen present on a cancer cell and a first immune cell effector domain, and their use in concert with CAR immune cells to treat cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 441,253, filed January 26, 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. The XML copy, created on January 22, 2024, is named 52095_774001WO_ST.xml and is 109 KB in size. [Background technology]

[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 approved by the FDA. CD19 is a B-cell coreceptor expressed on B cells and a wide variety of blood-borne malignancies. CD19 CAR T cells were first approved for the treatment of acute lymphoblastic leukemia (ALL), and subsequently for Burkitt lymphoma and mantle cell lymphoma. BCMA is a receptor expressed on the surface of B-cell lineage cells and is a key marker for multiple myeloma (MM). MM is associated with the uncontrolled proliferation of plasma cells in the bone marrow, which can progress to extramedullary lesions elsewhere in the body. BCMA CAR T-cell therapy has shown great promise for MM, with studies demonstrating an 80% overall response rate, even in patients with extramedullary disease (Gagelmann et al., Eur. J. Haematol. 104(4):318-327(2020)).

[0004] However, challenges remain with CAR T-cell therapy. A recent meta-analysis of 22 CAR T-cell clinical trials highlighted its ineffectiveness in solid tumors, with a low mean overall response rate of 9% (Hou et al., Dis. Markers 2019:3425291(2019)). Maintenance of response duration remains a challenge even in hematological cancers, with nearly all BCMA CAR-treated MM patients eventually 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)). Treatment can have serious side effects, including cytokine release syndrome (CRS) and neurotoxicity.

[0005] CAR T cells must home to tumor sites, proliferate, and persist in the circulation at least until they neutralize and kill the last remaining cancer cells. Therefore, approaches to modulate CAR T cell activity in a controlled manner are greatly needed. Summary of the Invention [Means for solving the problem]

[0006] The disclosed chimeric antigen receptor (CAR) engagers and methods of use thereof are expected to address the above-mentioned needs. CAR engagers increase the functionality and persistence of CAR immune cells in vivo. They can also reduce the cell dose required for CAR immune cell therapy, which can result in a reduction in adverse side effects (e.g., cytokine release syndrome) caused by the larger doses typically used in clinical trials. Therefore, CAR engagers are also referred to herein as CAR enhancers. Furthermore, because the binding of CAR engagers to CARs is reversible, CAR engagers do not induce immune synapse formation of CARs on the surface of CAR immune cells. Therefore, CAR engagers do not block CAR-mediated cancer cell killing. 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 engagers can support the persistence, proliferation, and efficacy of CAR T cells during MRD conditions.

[0007] A first aspect of the present disclosure relates to a chimeric antigen receptor (CAR) engager comprising an ectodomain of an antigen present on a cancer cell linked to an immune cell effector domain. The connection between the ectodomain-containing portion and the immune effector domain can be peptidic or non-peptidic (covalent), such that the CAR engager can be a contiguous protein or polypeptide, or a portion comprising two proteinaceous entities connected by a covalent bond. The ectodomain of the CAR engager is designed to bind to the extracellular domain of the CAR, which targets the ectodomain of the antigen on the cancer cell. In some embodiments, the binding between the CAR and the cancer antigen is direct. In some embodiments, the binding between the CAR and the cancer antigen is indirect. In these embodiments, the ectodomain of the CAR engager is a cancer-irrelevant antigen present in a protein therapeutic. The cancer-irrelevant antigen in the protein therapeutic and the ectodomain of the CAR engager binds to the CAR. The protein therapeutic functions to redirect the CAR immune cell from the cancer-irrelevant antigen to the cancer antigen via the cancer antigen-binding domain. Such CAR immune cells are called universal CARs or binary activated CARs (BAT-CARs). A representative cancer-irrelevant antigen is the small molecule fluorescein isothiocyanate (FITC), which targets universal anti-FITC CAR immune cells.

[0008] Another aspect of the present disclosure relates to a heterodimeric CAR engager comprising a first entity comprising an ectodomain of an antigen present on a cancer cell connected to a first dimerization domain, and a second entity comprising a first immune cell effector domain connected to a second dimerization domain, wherein the first and second dimerization domains bind to form the heterodimeric CAR engager. In embodiments where the linkage is a peptide bond, the first and second entities are referred to as first and second proteins or polypeptides.

[0009] Still other aspects of the present disclosure relate to a nucleic acid encoding a CAR engager protein, a nucleic acid encoding a first entity of a heterodimeric CAR engager protein, and a nucleic acid encoding a second entity of a heterodimeric CAR engager protein.

[0010] Yet other aspects of the present disclosure relate to vectors comprising a nucleic acid encoding a CAR engager protein, and vectors comprising the first and / or second protein of a heterodimeric CAR engager.

[0011] Yet another aspect of the present disclosure relates to a cell transformed with the vector.

[0012] Another aspect of the present disclosure relates to a pharmaceutical composition comprising a CAR engager and a pharmaceutically acceptable carrier.

[0013] Another aspect of the present disclosure relates to a method of making a CAR engager protein, which involves culturing cells transformed with a vector containing a nucleic acid encoding the CAR engager protein or a proteinaceous entity thereof in a medium under conditions in which the nucleic acid is expressed, and isolating the CAR engager protein or proteinaceous entity thereof from the cells and / or medium. In embodiments in which the nucleic acid encodes the proteinaceous entity, the proteinaceous entity is linked via click chemistry.

[0014] Another aspect of the present disclosure relates to a method for treating cancer, the method comprising administering to a subject an effective amount of a CAR engager. In some embodiments, the subject has previously received immune cells expressing a CAR comprising an extracellular domain that binds to an antigen present on a cancer cell, the extracellular domain comprising an ectodomain, and an intracellular domain comprising the ectodomain, transmembrane domain, and stimulatory domain of the CAR engager.

[0015] The examples disclosed herein demonstrate that CAR engagers enhance BCMA-targeting CAR immune cells, direct them towards the production of memory cells, and prevent CAR immune cell exhaustion. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 shows a schematic representation of the domains of a CAR engager according to some embodiments, including an ectodomain (Ag) of an antigen on the surface of a cancer cell, a CH3 dimerization domain, and an immune cell effector domain (ICE). The CAR engager can be a monomer, dimer, or multimer.

[0017] [Figure 2] Figures 2A-2I are a set of diagrams and line plots depicting three CAR engagers. Figure 2A shows a schematic representation of a CAR engager containing a BCMA ectodomain and a Neo2 / 15 synthetic cytokine immune cell effector domain. Figure 2B shows a schematic representation of a CAR engager containing a BCMA ectodomain and two weak-affinity mutant IL-2 (mIL2) synthetic cytokine immune cell effector domains. Figure 2C shows a schematic representation of a CAR engager containing a BCMA ectodomain and a 4-1BBL immune cell effector domain. Figure 2D shows a line plot depicting dose-dependent staining of CAR T cells or non-transduced T cells (NT T cells) binding to CD19 using CAR engagers or control proteins. Figure 2E shows a line plot depicting dose-dependent staining of CAR T cells or non-transduced T cells (NT T cells) binding to BCMA using CAR engagers or control proteins. Figure 2F and Figure 2G are line and bar plots, respectively, showing the dose-dependent activation of CAR T cells after CAR engager treatment. Figure 2H is a line plot showing that the BCMA-muIL2 CAR engager does not block the killing efficacy of CAR T cells. Figure 2I is a line plot showing the phosphorylation of signal transducer and activator of transcription (STAT5) in BCMA CAR T cells.

[0018] [Figure 3] Figures 3A-3B are a set of diagrams and line plots showing the effects of CAR engagers on untransduced T cells. Figure 3A shows the schematic experimental design. Figure 3B is a set of line plots showing T cell counts and carboxyfluorescein succinimidyl ester (CFSE) staining of untransduced activated T cells treated with teceleukin, a CAR engager containing a BCMA ectodomain and two weak-affinity mutant IL-2 (muIL2), or a CAR engager containing a BCMA ectodomain and a neoleukin domain.

[0019] [Figure 4] Figures 4A-4C are a set of diagrams and line plots showing that CAR engagers specifically activate CAR T cells. Figure 4A shows a schematic of the experimental design. Figure 4B is a bar plot showing the percentage of CD69+ anti-BCMA CAR-transduced activated T cells after treatment with a BCMA CAR engager, a BCMA CAR engager without an immune cell effector domain control, or a non-antigen-specific CAR engager control. Figure 4C is a bar plot showing the percentage of CD69+ anti-CD19 CAR-transduced activated T cells after treatment with a CD19 CAR engager or a non-antigen-specific CAR engager control.

[0020] [Figure 5] Figure 5 is a line plot showing that CAR engagers do not inhibit killing by BCMA CAR T cells and the percentage of OPM2 target cell survival after incubation with CAR T cells and CAR engagers (red) or non-transduced T cells (blue).

[0021] [Figure 6]Figures 6A-6C are a set of diagrams and photographs showing that CAR engagers reduce tumor burden in vivo. Figure 6A shows a schematic of the experimental design. Figures 6B-6C are a set of photographs showing tumor burden in mice before and after CAR T cell infusion and CAR engager treatment.

[0022] [Figure 7] Figures 7A-7C are a set of flow cytometry plots showing tumor burden in mice after CAR T cell infusion and CAR engager treatment. Figure 7A is a set of flow cytometry plots showing OPM2 tumor burden in the blood, spleen, and lymph nodes. Figure 7B is a set of flow cytometry plots showing OPM2 tumor burden in the bone marrow and lungs. Figure 7C is a set of flow cytometry plots showing OPM2 tumor burden in the liver, kidney, and eye tumor sites. eGFP (OPM2 cells) is shown on the Y-axis, and PERCP signal control is shown on the X-axis.

[0023] [Figure 8] Figures 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 engager treatment. Figure 8A is a set of flow cytometry plots showing CAR T cells in the blood, spleen, and lymph nodes. Figure 8B is a set of flow cytometry plots showing CAR T cells in the bone marrow and lungs. Figure 8C is a set of flow cytometry plots showing CAR T cells in the liver, kidney, and eye tumor sites. CD45 staining is shown on the y-axis, and CAR engager labeled with AF647 staining is shown on the x-axis.

[0024] [Figure 9]Figures 9A-9E are a set of schematics, line plots, and box plots showing that CAR engager treatment results in enhanced CAR T cell activity and persistence in vivo. Figure 9A is a line plot showing the circulating half-life of BCMA CAR engagers. Figure 9B schematically shows the experimental design. Figures 9C and 9D are a set of flow cytometry plots and box plots showing selective expansion and persistence of BCMA CAR T cells. Figure 9E is a box plot showing the percentage of CD8+ CAR T cells after CAR engager treatment.

[0025] [Figure 10] Figures 10A-10J are a set of schematics, survival plots, line plots, bar plots, and t-distributed stochastic neighbor embedding (tSNE) plots and photographs showing that CAR engager treatment reduces the required dose of CAR T cells. Figure 10A outlines the experimental design. Figure 10B is a set of photographs showing tumor burden in mice before and after CAR T cell infusion and CAR engager treatment. Figure 10C is a Kaplan-Meier plot showing survival analysis. Figure 10D is a line plot showing flow cytometry analysis of CAR T cells in blood samples. Figure 10E is a set of bar plots showing the generation of memory CAR T cells. Figures 10F and 10G are a set of flow cytometry plots and bar plots showing substantial numbers of CAR T cells 2 months after CAR T cell injection. Figure 10H is a line plot showing that mice maintained consistent weight throughout the experiment. Figure 10I is a set of flow cytometry plots showing that CAR T cells from CAR engager-treated mice have a stem cell memory phenotype. Figure 10J is a set of tSNE plots showing FlowSOM-defined clusters among persistent BCMA CAR T cells.

[0026] [Figure 11]Figures 11A-11E are a set of schematics, photographs, and line, bar, and tSNE plots showing that CAR engager treatment results in CAR T cell persistence in vivo. Figure 11A outlines the experimental design. Figure 11B is a set of photographs showing tumor burden in mice before and after CAR T cell infusion and CAR engager treatment. Figure 11C is a set of flow cytometry plots showing CAR T cell persistence. Figure 11D is a set of bar plots showing an in vitro killing assay of persisting T cells. Figure 11E is a set of t-SNE plots showing immune cell markers from CD8+ T cells.

[0027] [Figure 12] Figures 12A-12B are a set of schematics and bar plots showing that CAR-E treatment expands CAR T cells in vivo in the absence of tumor cells. Figure 12A shows the experimental design in outline. Figure 12B is a set of bar plots showing the number of CAR T cells 30 days after injection.

[0028] [Figure 13] Figures 13A-13B are a set of flow cytometry plots showing that neither BCMA-muIL2 nor VHH-muIL2 treatments exhibited binding to any specific population within human PBMCs. PBMCs were labeled with various markers to pregate on B cells (CD20), T cells (CD3), or myeloid cells (CD11b). Cells were stained with different concentrations of treatments followed by anti-FLAG-Alexa647 secondary staining. Figure 13A is a set of flow cytometry plots showing that BCMA-muIL2 does not bind to human PBMCs. Figure 13B is a set of flow cytometry plots showing that VHH-muIL2 does not bind to human PBMCs.

[0029] [Figure 14]Figures 14A-14C are a set of photomicrographs and dot plots showing the specific binding and gradual internalization of BCMA-muIL2 in CAR T cells. Figure 14A is a set of photomicrographs showing cells stained with CellTracker Blue CMAC, incubated with the indicated treatments for 1-5 hours, and imaged after labeling each treatment with Alexa647 (BCMA-muIL2) or dsRed (VHH-muIL2). Photomicrographs are representative of over 100 cell images. Figure 14B is a dot plot showing quantitative analysis of the imaged cells. Figure 14C is a dot plot showing the correlation between Alexa647 mean fluorescence intensity (BCMA-muIL2) and dsRed mean fluorescence intensity (VHH-muIL2).

[0030] [Figure 15] Figures 15A-15C are sets of flow cytometry plots showing individual flow cytometry data corresponding to the pooled data shown in Figure 9D. Figure 15A is a set of flow cytometry results from mice treated with CAR T cells only. Figure 15B is a set of flow cytometry results from mice treated with CAR T cells and VHH-muIL2. Figure 15C is a set of flow cytometry results from mice treated with CAR T cells and BCMA-muIL2.

[0031] [Figure 16] Figures 16A-16C are sets of flow cytometry plots showing individual flow cytometry data for the mice shown in Figures 10A-10J. Figure 16A is a set of flow cytometry results from a mouse treated with CAR T cells only. Figure 16B is a set of flow cytometry results from a mouse treated with CAR T cells and VHH-muIL2. Figure 16C is a set of flow cytometry results from a mouse treated with CAR T cells and BCMA-muIL2.

[0032] [Figure 17]Figures 17A-17C are a set of bar plots, line plots, and tSNE plots showing human T cell-derived cytokines in the serum of mice receiving OPM2 cancer cells followed by a low dose of CAR T cells. Figure 17A is a bar plot showing the levels of IFNγ, GM-CSF, and TNFα. Serum samples were diluted 1:40. The same plate was used to incubate both standard and serum samples, and standard curves were plotted for each cytokine. Figure 17B is a set of line plots showing IFNγ levels between the BCMA-muIL2 and VHH-muIL2 groups (error bars represent the mean and standard deviation). Figure 17C is a set of Flt-SNE mapping of CAR T cells from mice treated with PBS, BCMA-muIL2, and VHH-muIL2, showing the expression of 10 immune cell markers.

[0033] [Figure 18] Figure 18 is a set of t-SNE maps showing the expression of nine immune cell markers in CD4+CAR+ T cells from five BCMA-muIL2 CAR-E treated mice.

[0034] [Figure 19]Figures 19A-19G are a set of flow cytometry plots, tSNE plots, bar plots, violin plots, and pipe plots, as well as heat maps, showing single-cell RNA-seq analysis revealing the effect of BCMA-muIL2 on CAR T cells. Figure 19A is a set of flow cytometry plots showing CAR+ cells analyzed 89 days after CAR-T administration. Figure 19B is a tSNE plot showing data after the Harmony algorithm, showing the percentage of CD4, CD8, and proliferating (CD4 and CD8) cells. Figure 19C is a tSNE plot showing separation between groups treated with BCMA-muIL2 or VHH-muIL2 treatment. Figure 19D is a set of heat maps of significantly differentially expressed genes in CD4+ CAR T cells and CD8+ CAT T cells after the indicated treatments. Figure 19E is a set of violin plots of gene scores between CD8 and CD4 cells, where the scores are constructed using the normalized expression of the different genes for each phenotype in Figure 19D. Figure 19F is a set of pipe plots showing T cell receptor (TCR) clonotype diversity. Figure 19G is a bar plot showing clonotype diversity within the total cell population of the sample.

[0035] [Figure 20] Figures 20A-20B are a set of schematics and flow cytometry plots showing that CAR engager treatment results in enhanced organ trafficking of CAR T cells in vivo. Figure 20A shows the experimental design in brief. Figure 20B is a set of flow cytometry plots showing selective trafficking, proliferation, and persistence of BCMA CAR T cells.

[0036] [Figure 21]Figures 21A-21C are a set of line and bar plots showing the effect of CAR engagers on non-transduced T cells and CAR T cells. Figures 21A and 21B are line and bar plots, respectively, showing the dose-dependent activation of CAR T cells after CAR engager treatment (Figure 21A) and that CAR engagers do not activate non-transduced T cells (Figure 21B). Figure 21C is a line plot showing that the CD19-muIL2 CAR engager does not block the killing efficacy of CD19 CAR T cells or non-transduced T cells (NT T cells). DETAILED DESCRIPTION OF THE INVENTION

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this specification belongs. As used in this specification and the appended claims, unless specified to the contrary, the following terms have the meanings set forth to facilitate understanding of this disclosure.

[0038] As used in this specification 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, etc.

[0039] Unless otherwise specified, the term "about" is understood to be within normal tolerances in the art, e.g., within two standard deviations of the mean. "About" may 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 the context, all numerical values ​​provided herein are modified by the term "about."

[0040] The term "about," as used herein, unless otherwise stated or clear from the context (except where such number exceeds 100% of possible values), refers to a range of values ​​that falls 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 or less) of the stated reference value.

[0041] The transitional term "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element or method step not expressly recited in the claim (or the particular element or method step with which the phrase "consisting of" is associated). The transitional phrase "consisting essentially of" limits the claim to the particular elements and methods or steps of the claimed disclosure as well as "unrecited elements and method steps that do not materially affect the basic and novel characteristics."

[0042] CAR Engager In one aspect, the present disclosure provides a CAR engager, also referred to herein as a CAR enhancer, comprising a first proteinaceous entity and a second proteinaceous entity. The first proteinaceous entity comprises an ectodomain of an antigen (also referred to herein as a cancer antigen) present on a cancer cell, and the second proteinaceous entity comprises an immune cell effector domain linked to the ectodomain. The term "antigen" as used herein refers to a target protein expressed by a cancer (e.g., tumor) cell. The ectodomain of the antigen is at least a portion of the antigen exposed on the cancer cell surface. The ectodomain binds to the extracellular domain of a CAR expressed on an immune cell, which is used in conjunction with the CAR engager used in the method. The immune cell effector domain binds to a cognate receptor on the same immune cell.

[0043] In some embodiments, the CAR engager is a contiguous protein, and the first proteinaceous entity and the second proteinaceous entity are linked by a peptide bond. In some embodiments, the first proteinaceous entity and the second proteinaceous entity are linked by click chemistry.

[0044] In some embodiments, the CAR engager is formulated and administered as a monomeric protein or proteinaceous entity, hi other embodiments, the CAR engager is formulated and administered in a dimeric form, either as a homodimeric or heterodimeric protein or proteinaceous entity.

[0045] Ectodomain The ectodomain of the CAR engager binds to the extracellular domain of the CAR expressed on immune cells. As 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 to a T cell receptor on an immune cell or a CAR. The binding between the CAR and the cancer antigen can 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 the cancer antigen, or may be an antibody or antibody fragment (including nanobodies and nanobody fragments) that binds to a CAR expressed on an immune cell. In some embodiments, the CAR engager may comprise the entire extracellular domain of the cancer antigen. The ectodomain may be derived from the cancer antigen (e.g., identified in the cancer antigen) according to standard techniques. See, for example, Gershoni et al., Biodrugs 21(3):145-156(2007) and Francino-Urdaniz and Whitehead, RSC Chem. Biol. 2(6):1580-1589(2021). The term "derived" as used herein when referring to proteins and nucleic acids refers to a sequence that is derived from and identified from a parent (e.g., wild-type or endogenous) protein and nucleic acid sequence, 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. A variant may include substitutions, insertions, or deletions. Thus, for example, an amino acid sequence derived from a parent sequence may be identical to a particular range of amino acids of the parent, but may not include amino acids outside that particular region.

[0046] The amino acid sequences of representative cancer antigens from which ectodomains can be derived are provided under the NCBI accession numbers set forth in Table 1, which are incorporated herein by reference.

[0047] Table 1: Gene names, symbols, and NCBI accession numbers of representative cancer antigens [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0048] The ectodomain is not limited to known cancer antigens. Unique cancer antigens (neoantigens) can be identified by known methods. For example, neoantigens can be identified by comparing the cancer genome with the normal cell genome. In some embodiments, the cancer transcriptome is compared with the normal cell transcriptome. Computational methods can then be used to identify a suitable binding site for the CAR. In most cases, the CAR binds to a portion of the extracellular domain of the antigen. In some embodiments, the CAR and the corresponding cancer antigen are known in the art.

[0049] In some embodiments, the ectodomain of the CAR engager comprises the entire extracellular domain of a cancer antigen, hi some embodiments, the CAR engager comprises a portion of the extracellular domain of the cancer antigen targeted by the CAR.

[0050] In some embodiments, the ectodomain of a CAR engager comprises the extracellular domain of BCMA. The amino acid sequence of a representative CAR engager comprising the BCMA extracellular domain is MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA (SEQ ID NO: 1).

[0051] In some embodiments, the ectodomain of a CAR engager comprises two repeats of the extracellular domain of BCMA. The amino acid sequence of a representative CAR engager comprising two repeats of the BCMA extracellular domain is shown below (SEQ ID NO: 2): JPEG2026503523000010.jpg19169

[0052] In some embodiments, the ectodomain of the CAR engager comprises a variant of the extracellular domain of CD 19. The amino acid sequence of a representative CAR engager comprising a variant of the CD19 extracellular domain is shown below (SEQ ID NO: 3): JPEG2026503523000011.jpg27169

[0053] 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, or at least 99% sequence identity to SEQ ID NO:3.

[0054] The amino acid sequence of a representative CAR engager comprising a second variant of the CD19 extracellular domain is shown below (SEQ ID NO: 4): JPEG2026503523000012.jpg27169

[0055] In some embodiments, the ectodomain of the CAR engager comprises the extracellular domain of CD 19. The amino acid sequence of a representative CAR engager comprising the CD19 extracellular domain is shown below (SEQ ID NO: 5): JPEG2026503523000013.jpg26169

[0056] In some embodiments, the ectodomain of the CAR engager comprises a portion of the extracellular domain of CD 19. In some embodiments, the ectodomain of the CAR engager is KDRPEIWEGEPP (SEQ ID NO: 103), which corresponds to positions 142-153 of SEQ ID NO: 5.

[0057] In some embodiments, the ectodomain of a CAR engager comprises the extracellular domain of CD20. The amino acid sequence of a representative CAR engager comprising the CD20 extracellular domain is KISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQS (SEQ ID NO: 6).

[0058] In some embodiments, the ectodomain of the CAR engager comprises the extracellular domain of CD22. The amino acid sequence of a representative CAR engager comprising the CD22 extracellular domain is shown below (SEQ ID NO: 7): JPEG2026503523000014.jpg58169

[0059] In some embodiments, the ectodomain of a CAR engager comprises a portion of the extracellular domain of CD22 (SEQ ID NO: 7). In some embodiments, the ectodomain of a CAR engager comprises Ig domains 2-3 of CD22. The amino acid sequence of a representative CAR engager comprising Ig domains 2-3 of CD22 is shown below (SEQ ID NO: 104): JPEG2026503523000015.jpg18169

[0060] In some embodiments, the ectodomain of the CAR engager comprises Ig domain 3 of CD22. The amino acid sequence of a representative CAR engager comprising Ig domain 3 of CD22 is shown below (SEQ ID NO: 105): JPEG2026503523000016.jpg14169

[0061] In some embodiments, the ectodomain of the CAR engager comprises Ig domains 5-7 of CD22. The amino acid sequence of a representative CAR engager comprising Ig domains 5-7 of CD22 is shown below (SEQ ID NO: 106): JPEG2026503523000017.jpg26169

[0062] In some embodiments, the ectodomain of the CAR engager comprises Ig domains 5-7 of CD22. The amino acid sequence of a representative CAR engager comprising Ig domains 6-7 of CD22 is shown below (SEQ ID NO: 107): JPEG2026503523000018.jpg22169

[0063] In some embodiments, the ectodomain of a CAR engager comprises the extracellular domain of claudin-18.2. The amino acid sequence of a representative CAR engager comprising the first extracellular domain of claudin-18.2 is shown below (SEQ ID NO: 8): JPEG2026503523000019.jpg10169

[0064] The amino acid sequence of a representative CAR engager comprising the second extracellular domain of claudin-18.2 is shown below (SEQ ID NO: 9): JPEG2026503523000020.jpg8100

[0065] In some embodiments, the ectodomain of a CAR engager comprises the extracellular domain of SLAMF7. The amino acid sequence of a representative CAR engager comprising the SLAMF7 extracellular domain is shown below (SEQ ID NO: 10): JPEG2026503523000021.jpg21169

[0066] In some embodiments, the ectodomain of a CAR engager comprises the extracellular domain of PD-1. The amino acid sequence of a representative CAR engager comprising the PD-1 extracellular domain is shown below (SEQ ID NO: 11): JPEG2026503523000022.jpg18169

[0067] In some embodiments, the ectodomain of a CAR engager comprises a variant of the extracellular domain of PD-1. In some embodiments, the ectodomain of a CAR engager comprises the N-loop of PD-1. The amino acid sequence of a representative CAR engager comprising 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.

[0068] In some embodiments, the ectodomain of a CAR engager comprises the CD loop of PD-1. The amino acid sequence of a representative CAR engager comprising 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.

[0069] In some embodiments, the ectodomain of a CAR engager comprises the extracellular domain of KIT. The amino acid sequence of a representative CAR engager comprising the KIT extracellular domain is shown below (SEQ ID NO: 12): JPEG2026503523000023.jpg45169

[0070] In some embodiments, the ectodomain of a CAR engager comprises the extracellular domain of TROP2. The amino acid sequence of a representative CAR engager comprising the TROP2 extracellular domain is shown below (SEQ ID NO: 13): JPEG2026503523000024.jpg26169

[0071] In some embodiments, the ectodomain of the CAR engager comprises the extracellular domain of CD38. The amino acid sequence of a representative CAR engager comprising the CD38 extracellular domain is shown below (SEQ ID NO: 14): JPEG2026503523000025.jpg26169

[0072] In some embodiments, the ectodomain of the CAR engager is derived from mesothelin (MSLN). MSLN is a GPI-anchored protein, and therefore the entire MSLN protein is extracellular. The amino acid sequence of a representative MSLN is shown below (SEQ ID NO: 15): JPEG2026503523000026.jpg53169

[0073] In some embodiments, the ectodomain of a CAR engager comprises a portion of the extracellular domain of a cancer antigen. In some embodiments, the ectodomain of a CAR engager comprises a portion of the MSLN protein. In some embodiments, the ectodomain of a CAR engager is IPNGYLVLDLSMQEALS (SEQ ID NO: 16). In some embodiments, the ectodomain of a CAR engager is YNVNDLSMQEL (SEQ ID NO: 17), where N is any amino acid.

[0074] In some embodiments, the CAR engager ectodomain is an antibody or antibody fragment that binds to a CAR expressed on an immune cell. In these embodiments, the CAR engager ectodomain can be an antibody fragment, including a nanobody fragment. In some embodiments, the CAR engager ectodomain is an antibody or fragment thereof directed against a CAR extracellular domain. In some embodiments, the CAR engager ectodomain is an anti-mouse antibody that binds to a mouse-derived antibody fragment that is incorporated into the CAR extracellular domain. See Kochenderfer et al., J. Immunother. 32(7):689-702(2009) and Cheng et al., Cytometry A. 103(1):16-26(2023). In some embodiments, the CAR engager ectodomain is an anti-idiotypic antibody fragment that binds to the variable region of a CAR antibody fragment. In some embodiments, the CAR engager ectodomain is an antibody or fragment thereof described in U.S. Patent No. 9,701,758 and U.S. Patent Application Publication No. 2005 / 0287148, both of which are incorporated by reference in their entireties. In some embodiments, the CAR engager ectodomain is Peptostreptococcus magnus protein L (NCBI Accession No. Q51918), which binds to antibodies and scFv VL regions of the kappa light chain.

[0075] Indirect Coupling Embodiments Embodiments that rely on indirect binding of CARs to cancer antigens include an ectodomain containing a non-cancer-associated antigen. The non-cancer-associated antigen is not an antigen on the surface of cancer cells, but an exogenous antigen contained in the protein therapeutic. The protein therapeutic typically contains at least a non-cancer-associated antigen and a cancer antigen-binding domain (e.g., an antibody or antibody fragment), and serves to redirect CAR immune cells from the non-cancer-associated antigen to the cancer antigen. CARs that bind to non-cancer-associated antigens are called universal CARs or binary activated CARs (BAT-CARs). In some embodiments, the cancer-non-associated antigen is a fluorescent molecule (e.g., fluorescein, fluorescein isothiocyanate (FITC), anthracene, alexafluor, rhodamine, rhodol, acridine, or xanthene), 4-[(6-methylpyrazin-2-yl)oxy]benzoic acid (MPOB), anthraquinone-2-carboxylate (AQ), anthraquinone-2-oic acid, tetraxetane (DOTA), amphetamine, benzodiazepine, benzoylecgonine, buprenorphine, opioid, cannabinoid, phencyclidine, tricyclic antidepressant, dextromethorphan, fentanyl, meprobamate, methadone, methamphetamine, oxycodone, THC, tramadol, zolpidem, ketamine, LSD, MDMA, methaqualone, propoxyphene, norketamine, biotin, or leucine zipper. Additional non-cancer-associated antigens are described, for example, in U.S. Patent No. 11,225,520, U.S. Patent Application Publication Nos. 2020 / 0306376, 2021 / 0137987, and 2021 / 0228699, and International Patent Application Publication No. WO2023 / 060126, each of which is incorporated herein by reference.

[0076] Immune Cell Effector Domains The immune cell effector domain of a CAR engager binds to a cognate receptor on an immune cell expressing a nucleic acid encoding the CAR. This binding event regulates the activity of the CAR immune cell. As used herein, the terms "modulate" and "modulation" encompass both activation and inhibition of the CAR immune cell. Thus, the immune cell effector domain may be a cytokine, an immune cell activating moiety, or an immune cell inhibitory moiety, as well as variants and fragments thereof that bind to their cognate targets. As known in the art, the term "cytokine" includes low-molecular-weight extracellular polypeptides / glycoproteins that promote, regulate, and control immune responses (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). As used herein, the term "immune cell activating variant" of a cytokine refers to a non-naturally occurring variant of a cytokine that can bind to a cytokine receptor on an immune cell and initiate signaling through that receptor to achieve substantially the same effect as a naturally occurring cytokine.

[0077] In some embodiments, the CAR engager comprises multiple (i.e., two or more) immune cell effector domains, any two or more of which may be the same or different from one another. In some embodiments, the CAR engager comprises two immune cell effector domains. In some embodiments, the CAR engager comprises three immune cell effector domains.

[0078] In some embodiments, the immune cell effector domain is an immune cell-activating moiety, such as an immune cell-activating cytokine and immune cell-activating variants and fragments thereof, which activate, promote, or maintain the activity of an immune cell.

[0079] In some embodiments, the immune cell effector domain is selected from the group consisting of CD40, CD48, CD58, CD70, CD80, CD86, CD112, glucocorticoid-inducible TNFR-related protein ligand (GITRL; TNFSF18), herpesvirus entry mediator (HVEM; TNFSF14), semaphorin 3B (SEMAA; SEMA3B), signaling lymphocyte activation molecule family member 1 (SLAM; SLAMF1; CD150), T cell immunoglobulin G (T cell immunoglobulin G), and / or T cell immunoglobulin G (T cell immunoglobulin G). derived from phospho- 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 immune cell-activating variants thereof.

[0080] The amino acid sequences of representative immune cell activating moieties (e.g., cytokines) from which immune cell effector domains can be derived are provided under the NCBI accession numbers set forth in Table 2, which are incorporated herein by reference.

[0081] Table 2. Gene names, symbols, and NCBI accession numbers of representative immune cell activation proteins [Table 2-1] [Table 2-2]

[0082] In some embodiments, the immune cell effector domain is wild-type IL-2 having the amino acid sequence shown below (SEQ ID NO: 102; NCBI Accession No. NP_000577): JPEG2026503523000029.jpg19169

[0083] In some embodiments, the immune cell effector domain is a synthetic, i.e., non-naturally occurring, IL-2 that is a variant of wild-type IL-2 (SEQ ID NO: 102) in that it has amino acid substitutions at positions 16 and / or 42.

[0084] In some embodiments, the immune cell effector domain has an H16A substitution (i.e., alanine (A) at position 16 instead of histidine (H)) relative to SEQ ID NO: 102, and / or an F42A substitution (i.e., alanine (A) at position 42 instead of 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 a weak affinity variant of IL-2 (muIL2) having the amino acid sequence shown below (SEQ ID NO: 19), comprising the H16A and F42A substitutions as follows: JPEG2026503523000030.jpg18169

[0085] By "weak affinity" is meant that the native (wild-type) sequence of human IL-2 (SEQ ID NO: 102) has a higher affinity for the IL-2 receptor (IL-2R) than SEQ ID NO:19.

[0086] More specifically, the muIL2 (SEQ ID NO: 19) immune cell effector domain has a dissociation constant (K) of approximately 1200 nM for IL-2Rα (CD25). D ), which represents a 110-fold decrease compared to wild-type IL-2, and has a K of approximately 610 nM for IL-2Rβ. D which represents a three-fold reduction compared to wild-type IL-2.

[0087] In some embodiments, a CAR engager comprises more than one immune cell effector domain. The immune cell effector domains in a CAR engager can be the same or different.

[0088] In some embodiments, the CAR engager comprises two immune cell effector domains, for example, a first and a second immune cell effector domain that is a low affinity IL-2 variant that together has the amino acid sequence shown below (SEQ ID NO: 20): JPEG2026503523000031.jpg26169

[0089] In some embodiments, the immune cell effector domain may be derived from IL-7. A representative IL-7 amino acid sequence is shown below (SEQ ID NO: 21): JPEG2026503523000032.jpg17169

[0090] In some embodiments, the immune cell effector domain may be derived from IL-15. A representative IL-15 amino acid sequence is shown below (SEQ ID NO: 22): JPEG2026503523000033.jpg18169

[0091] In some embodiments, the immune cell effector domain may be derived from IL-18. A representative IL-18 amino acid sequence is shown below (SEQ ID NO: 23): JPEG2026503523000034.jpg22169

[0092] In some embodiments, the immune cell effector domain may be derived from IL-21. A representative IL-21 amino acid sequence is shown below (SEQ ID NO: 24): JPEG2026503523000035.jpg18169

[0093] In some embodiments, the immune cell effector domain may be derived from IL-27. A representative IL-27 amino acid sequence is shown below (SEQ ID NO: 25): JPEG2026503523000036.jpg27169

[0094] In some embodiments, the immune cell effector domain is an immune cell-activating variant of a cytokine, ie, neoleukin-2 / 15 (Neo-2 / 15), which binds to IL-2R-β and has the amino acid sequence set forth below (SEQ ID NO:26). JPEG2026503523000037.jpg13169

[0095] In some embodiments, the CAR engager contains two immune cell effector domains of Neo-2 / 15, each having the amino acid sequence of SEQ ID NO:26.

[0096] 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). A representative 4-1BBL amino acid sequence is provided in NCBI accession number NP_003802, which is 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 comprises a portion of the extracellular domain of 4-1BBL having the amino acid sequence shown below (SEQ ID NO: 27): JPEG2026503523000038.jpg18169

[0097] In some embodiments, the CAR engager contains three immune cell effector domains, for example, the first, second, and third immune cell effector domains are all the extracellular domain of 4-1BBL, each having the amino acid sequence of SEQ ID NO: 27.

[0098] In some embodiments, the immune cell effector domain can be a fragment that binds to and activates the CAR immune cell, such as a single-chain variable antibody fragment (scFv). In some embodiments, the immune cell effector domain is an scFv that binds to 4-1BB, CD2, CD27, CD28, CD30 (TNFRSF8), CD40L, CD226, CTLA4, GITR, IL-2R, LIGHT, OX40, PD-1, TIM2, SLAM, or TIM1.

[0099] In some embodiments, the immune cell effector domain is an scFv that binds to CTLA4. In some embodiments, the immune cell effector domain is derived from a commercially available anti-CTLA4 antibody, antibody fragment, or derivative, such as 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 to CTLA4 are shown in Table 3.

[0100] Table 3. Amino acid sequences of the heavy and light chains of representative anti-CTLA antibodies [Table 3-1] [Table 3-2]

[0101] In some embodiments, the immune cell effector domain comprises a VL having the amino acid sequence shown below (SEQ ID NO: 36): JPEG2026503523000041.jpg13169

[0102] In some embodiments, the immune cell effector domain comprises a VH having the amino acid sequence shown below (SEQ ID NO: 37): JPEG2026503523000042.jpg12169

[0103] In some embodiments, the immune cell effector domain binds to OX40. In some embodiments, the immune effector domain is derived from a commercially available anti-OX40 antibody, antibody fragment (e.g., scFv), or derivative thereof, such as tavolimab or vonlerolizumab (Pogalizumab; MOXR 0916). The amino acid sequences of representative heavy and light chains are shown in Table 4.

[0104] Table 4. Amino acid sequences of the heavy and light chains of representative anti-OX40 antibodies [Table 4]

[0105] In some embodiments, the immune cell effector domain comprises a VL having the amino acid sequence shown below (SEQ ID NO: 42): JPEG2026503523000044.jpg13169

[0106] In some embodiments, the immune cell effector domain comprises a VH having the amino acid sequence shown below (SEQ ID NO: 43): JPEG2026503523000045.jpg12169

[0107] In some embodiments, the immune cell effector domain binds to 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, such as atezolizumab, avelumab, bintrafusp alfa, cosibelimab, danburstotug, durvalumab (Imfinzi®), inbakicept, lodapolimab, pimivalimab, or socazolimab. The amino acid sequences of representative heavy and light chains are shown in Table 5.

[0108] Table 5. Amino acid sequences of the heavy and light chains of representative anti-PD-1 antibodies [Table 5-1] [Table 5-2]

[0109] In some embodiments, the immune cell effector domain comprises a VL having the amino acid sequence shown below (SEQ ID NO: 52): JPEG2026503523000048.jpg14169

[0110] In some embodiments, the immune cell effector domain comprises a VH having the amino acid sequence shown below (SEQ ID NO: 53): JPEG2026503523000049.jpg13169

[0111] In some embodiments, the immune cell effector domain is an immune cell inhibitory moiety, representative types of which include immune cell inhibitory cytokines and immune cell inhibitory variants and fragments thereof. Immune cell inhibitory moieties suppress or block the activity and function of immune cells. In some embodiments, the immune cell inhibitory 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 inhibitory proteins from which the immune cell effector domain may be derived are provided under the NCBI accession numbers set forth in Table 6 and are incorporated herein by reference.

[0112] Table 6. Gene names, symbols, and NCBI accession numbers of immune cell inhibitory proteins [Table 6-1] [Table 6-2]

[0113] 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 shown below (SEQ ID NO: 54): JPEG2026503523000052.jpg22169

[0114] 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 shown below (SEQ ID NO: 55): JPEG2026503523000053.jpg22169

[0115] 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 shown below (SEQ ID NO: 56): JPEG2026503523000054.jpg31169

[0116] 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 shown below (SEQ ID NO: 57): JPEG2026503523000055.jpg39169

[0117] In some embodiments, the immune cell effector domain is the extracellular domain of Ceacam-1. The amino acid sequence of a representative Ceacam-1 extracellular domain is shown below (SEQ ID NO: 58): JPEG2026503523000056.jpg16169

[0118] 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 shown below (SEQ ID NO: 59): JPEG2026503523000057.jpg29169

[0119] In some embodiments, the immune cell effector domain is the extracellular domain of galectin-3. The amino acid sequence of a representative galectin-3 extracellular domain is shown below (SEQ ID NO: 60): JPEG2026503523000058.jpg25169

[0120] In some embodiments, the immune cell effector domain is the extracellular domain of HLA-E. The amino acid sequence of a representative HLA-E extracellular domain is shown below (SEQ ID NO: 61): JPEG2026503523000059.jpg26169

[0121] In some embodiments, the immune cell effector domain is the extracellular domain of HVEM (CD270). A representative set of amino acid sequences for the HVEM extracellular domain is shown below (SEQ ID NO: 62): JPEG2026503523000060.jpg18169

[0122] In some embodiments, the immune cell effector domain is the extracellular domain of Nectin-2 (CD112, HVEB). The amino acid sequence of a representative Nectin-2 extracellular domain is shown below (SEQ ID NO: 63): JPEG2026503523000061.jpg32169

[0123] In some embodiments, the immune cell effector domain is the extracellular domain of PD-L1. The amino acid sequence of a representative PD-L1 extracellular domain is shown below (SEQ ID NO: 64): JPEG2026503523000062.jpg22169

[0124] In some embodiments, the immune cell effector domain is the extracellular domain of PD-L2. A representative set of amino acid sequences for the PD-L2 extracellular domain is shown below (SEQ ID NO: 65): JPEG2026503523000063.jpg21169

[0125] In some embodiments, the immune cell effector domain is the extracellular domain of VTCN1 (B7-H4). A representative amino acid sequence of VTCN1 is shown below (SEQ ID NO: 66): JPEG2026503523000064.jpg22169

[0126] Dimerization domain In some embodiments, the CAR engager further comprises a dimerization domain. In these cases, the CAR engager forms a homodimer or homomultimer and is administered in this form. Thus, the homodimer comprises two CAR engager entities. The order of the ectodomain, immune effector domain, and dimerization domain is not important. In some embodiments, the dimerization domain is located between the ectodomain and the immune cell effector domain.

[0127] In some embodiments, the CAR engager is in the form of a heterodimer comprising a first entity comprising an ectodomain of an antigen present on a cancer cell connected to a first dimerization domain, and a second entity comprising 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 the heterodimer.

[0128] In some embodiments, the first and second dimerization domains comprise a knob-in-hole configuration. One of the dimerization domains comprises a protuberance (knob), and the other dimerization domain comprises a cavity (hole) that is sterically compensatory for the protuberance, such that the tertiary structure of the protuberance can be positioned within the tertiary structure of the cavity. Dimerization domains with a knob-in-hole configuration can have directed amino acid mutations where the protuberance is an amino acid with a larger side chain volume than present on the dimerization domain derived from a natural source (e.g., IgA, IgD, IgG, IgM, or IgE) and the cavity is an amino acid with a smaller side chain volume than present on the dimerization domain derived from a natural source.

[0129] In some embodiments, the protuberance is a threonine (T) to lysine (K) amino acid change, and the corresponding cavity is a leucine (L) to aspartic acid (D) or lysine (K) amino acid change. In some embodiments, the first dimerization domain contains two amino acid substitutions, e.g., a threonine (T) to lysine (K) substitution and a leucine (L) to lysine (K) substitution, while the second dimerization domain includes a leucine (L) to aspartic acid (D) or glutamic acid (E) substitution and a tyrosine (Y) to glutamic acid (E) or aspartic acid (D) substitution.

[0130] In some embodiments, the knobs-in-hole dimerization domains are based on opposite charges, i.e., a first dimerization domain contains a positively charged amino acid and a second dimerization domain contains a negatively charged amino acid that sterically opposes the positively charged amino acid on the first dimerization domain.

[0131] Additional ridge and cavity arrangements are known in the art, see, e.g., U.S. Patent Nos. 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 Publication Nos. 2005 / 0079170, 2006 / 0025576, 2013 / 0089554, and 2014 / 0024111.

[0132] In some embodiments, the dimerization domain may be derived from IgA, IgD, IgG, IgM, or IgE. The first and second dimerization domains may contain the same or different amino acid sequences, provided that they bind to each other. In some embodiments, the first and second dimerization domains are IgG1 constant heavy chain (CH)3 domains. The amino acid sequence of a representative IgG1 CH3 domain is shown below (SEQ ID NO: 67): JPEG2026503523000065.jpg17169

[0133] In some embodiments, the first and second dimerization domains are IgG1 constant heavy chain CH2 domains. The amino acid sequence of a representative IgG1 CH2 domain is shown below (SEQ ID NO: 68): JPEG2026503523000066.jpg14169

[0134] In some embodiments, the first and second dimerization domains are IgG1 CH2 and CH3 domains. The CH2 and CH3 domains may be interconnected by a linker.

[0135] Linker In some embodiments, the CAR engager comprises one or more linkers that allow the ectodomain and immune cell effector domain to bind to their respective cognate receptors on the CAR-expressing immune cell or that can provide flexibility regarding steric spacing (i.e., spacer) between the ectodomain and immune cell effector domain.

[0136] A linker can be placed between any two CAR engager components (also referred to herein as domains, entities or portions or parts) (e.g., an ectodomain and an immune cell effector domain).

[0137] The linker may be disposed between the dimerization domain and the adjacent domain. In some embodiments, the linker may be disposed between the dimerization domain and the immune cell effector domain. In some embodiments, the CAR engager contains two linkers, a first linker disposed between the ectodomain and the dimerization domain, and a second linker disposed between the dimerization domain and the immune cell effector domain.

[0138] In some embodiments, the linker comprises an amino acid sequence 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 repeats 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), GGGGSGGGGGSGGGGS (SEQ ID NO:74), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:75), GSPRGGGGSGGGGSGGGGGS (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).

[0139] 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 CD3ζ, CD4, CD8α, CD28, IgG1, IgG2, or IgG4. The amino acid sequences of representative linkers are shown in Table 7.

[0140] Table 7. Amino acid sequences of representative linkers [Table 7]

[0141] In some embodiments, the CAR engager is in the form of a fusion protein, and the components are linked by peptide bonds. In other embodiments, the CAR engager comprises proteinaceous entities linked together by click chemistry, chemical linkages formed by methods of controlled protein ligation. The linkage may be an azide-alkyne bond, an oxime or hydrazine bond, a tetrazine-transcyclooctene bond, an azide-nitrone bond, a thiol-alkene bond, an alkene-tetrazole bond, an alkene-tetrazine bond, an alkene-azide bond, a conjugated diene-alkene bond, or an isonitrile-tetrazine bond.

[0142] Further control protein ligation chemistries, systems and methods are known in the art.See, for example, U.S. Patent Nos. 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 No. 2009 / 0069561.

[0143] nucleic acid In another aspect, the present disclosure provides a nucleic acid encoding a CAR engager protein. As used herein, the term "nucleic acid" refers to a polymer of nucleotides, each of which is an organic molecule composed of a nucleoside (a nucleic acid base and a pentose sugar) and a phosphate. The term nucleotide, unless otherwise specified or clear from the context, includes nucleosides with a ribose sugar (i.e., ribonucleotides that form ribonucleic acids, RNA) or a 2'-deoxyribose sugar (i.e., deoxyribonucleotides that form deoxyribonucleic acids, DNA). Nucleotides serve as monomer units of nucleic acid polymers or polynucleotides. The four nucleic acid bases in DNA are guanine (G), adenine (A), cytosine (C), and thymine (T). The four nucleic acid bases in RNA are guanine (G), adenine (A), cytosine (C), and uracil (U). A nucleic acid is a linear chain of nucleotides (e.g., at least three nucleotides) chemically joined by a series of ester bonds between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar (i.e., ribose or 2'-deoxyribose) of an adjacent nucleotide.

[0144] In some embodiments, the CAR engager is encoded by two nucleic acids, e.g., the ectodomain is encoded by a first nucleic acid and the immune cell effector domain is encoded by a second nucleic acid.

[0145] In some embodiments, the nucleic acid encoding the CAR engager includes a nucleic acid encoding a signal peptide located 5' to the nucleic acid encoding the ectodomain. As used herein, the term "signal peptide" refers to a short stretch of amino acids (e.g., 5-30 or 10-100 amino acids in length) that directs protein transport during translation. CAR engagers containing a signal peptide are secreted from cells. Typically, the signal peptide is cleaved from the CAR engager before secretion. The signal peptide can be linked to the nucleic acid encoding the ectodomain or the nucleic acid encoding the immune cell effector domain.

[0146] In some embodiments, the signal peptide can be derived from Ig-gamma-3 heavy chain (IGHG3), albumin, CD8α, CD33, erythropoietin (EPO), IL-2, human or mouse Ig-kappa chain V-III (IgK VIII), tissue plasminogen activator (tPA), or secreted alkaline phosphatase (SEAP). The signal peptide can also be synthetic (i.e., non-naturally occurring). The amino acid sequences of representative signal peptides are shown in Table 8.

[0147] Table 8. Amino acid sequences of representative signal peptides [Table 8]

[0148] vector The nucleic acid encoding the CAR engager can be introduced into cells by a suitable vector. In embodiments where the ectodomain and immune effector domain are chemically linked, for example, via click chemistry, the nucleic acid encoding the CAR can be introduced into one or more cells by a separate vector. The vector is configured to contain the elements necessary for transport into immune cells and for expression of the nucleic acid after transformation. Such elements include an origin of replication, a polyA tail sequence, a selection marker, and one or more suitable sites for inserting nucleic acid sequences (e.g., multiple cloning sites (MCS)), one or more suitable promoters (each promoter operably linked to the insertion site of the nucleic acid sequence and the selection marker), and any additional regulatory elements.

[0149] As used herein, the term "promoter" refers to a nucleic acid sequence that directly or indirectly regulates transcription of a corresponding nucleic acid coding sequence to which it is operably linked, which in the context of this disclosure is a CAR engager protein. A promoter may function alone to regulate transcription or may act in concert with one or more other regulatory sequences (e.g., enhancers or silencers, or regulatory elements that may be present in a nucleic acid sequence or vector). Promoters are located near the transcription start site of a gene, on the same strand of DNA, and upstream (toward the 5' region of the sense strand). Promoters typically range in length from about 100 to 1,000 base pairs.

[0150] As used herein, the term "operably linked" should be understood to mean that a nucleic acid sequence is spatially positioned or arranged in a vector relative to another nucleic acid sequence, e.g., a promoter is operably linked to drive expression of a nucleic acid coding sequence (e.g., a nucleic acid sequence encoding a CAR engager).

[0151] In some embodiments, the single vector comprises a single promoter operably linked to a nucleic acid encoding a CAR engager. In some embodiments, the single vector comprises a single promoter operably linked to a nucleic acid encoding an ectodomain and a nucleic acid encoding an immune cell effector domain. 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 comprises a first promoter operably linked to a nucleic acid encoding an ectodomain and a second promoter operably linked to a nucleic acid encoding an immune cell effector domain.

[0152] In some embodiments, two vectors are provided, in which a first vector comprises a promoter operably linked to a nucleic acid encoding an ectodomain, and a second vector comprises a promoter operably linked to a nucleic acid encoding an immune cell effector domain.

[0153] In some embodiments, the vector comprises a strong mammalian promoter, such as the cytomegalovirus (CMV) promoter, the simian virus 40 (SV40) early promoter, a synthetic promoter (e.g., RPBSA (synthetic from Sleeping Beauty), or CAG (synthetic, CMV early enhancer element, chicken β-actin, and rabbit β-globin splice acceptor)), or a promoter derived from the β-actin, phosphoglycerate kinase (PGK), or EF1α factor genes. In some embodiments, the promoter may contain a core region located near the nucleic acid coding sequence. In some embodiments, the promoter is modified to remove methylation-sensitive motifs (e.g., a cytosine nucleotide followed by a guanine nucleotide, or "CpG") or by the addition of regulatory sequences that bind transcription factors that repress DNA methylation. In some embodiments, the vector comprises an A / T-rich nuclear matrix-interacting sequence known as a scaffold matrix-attaching region (S / MAR), which enhances transformation efficiency and improves the stability of transgene expression.

[0154] In some embodiments, the vector is a viral vector, such as 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 is described in, for example, U.S. Patent Nos. 5,665,577, 5,981,276, 6,013,516, 7,090,837, 8,119,119, and 10,954,530.

[0155] 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 still other embodiments, the vector may contain both viral and non-viral elements.

[0156] In some embodiments, the vector is a plasmid. In addition to a promoter operably linked to the nucleic acid, the plasmid may also contain other elements that facilitate the transport and expression of the nucleic acid, for example, in immune cells. The plasmid may be linearized with a restriction enzyme, transcribed in vitro to produce mRNA, and then modified with a 5' cap and a 3' polyA tail. In some embodiments, the vector comprises multiple plasmids, a first plasmid encoding a first proteinaceous entity (e.g., an ectodomain of a CAR engager) and a second plasmid encoding a second proteinaceous entity (e.g., an immune effector domain of a CAR engager).

[0157] cell One aspect of the present disclosure is a genetically modified (or transformed) cell harboring a vector containing a nucleic acid encoding a CAR engager or a component of a CAR engager for the purpose of making and purifying the CAR engager protein.

[0158] Cells useful for cloning and other manipulation of these vectors are conventional. Cells from various strains of E. coli can be used for vector replication and other steps in the construction of the CAR engagers of the present disclosure.

[0159] Suitable host cells or cell lines for expression of CAR engager encoding nucleic acids include eukaryotic cells. In some embodiments, the cells are mammalian cell lines. In some embodiments, the cells are mammalian cells such as CHO (e.g., DG44, CHO-S), fibroblasts (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, e.g., myeloma cells (e.g., NS0 (NS zero)). In some embodiments, the nucleic acid encoding the CAR engager is expressed in CHO or myeloma cells. Human cells may be used, thus allowing the expressed CAR engager to be modified with a human glycosylation pattern. Selection of appropriate mammalian cells and methods for transformation, culture, amplification, screening, and product production and purification are known in the art. See, for example, Green et al., eds., Molecular Cloning: A Laboratory Manual, 5 th ed., Cold Spring Harbor Laboratory Press, New York, 2012.

[0160] In some embodiments, the cell is a prokaryotic cell. Prokaryotic (i.e., bacterial) cells may prove useful as suitable host cells for expressing nucleic acids encoding CAR engagers (see, e.g., Pluckthun, Immunol. Rev. 130:151-188 (1992)). However, because proteins expressed in bacterial cells tend to be unfolded or improperly folded, or non-glycosylated, any CAR engagers produced in bacterial cells are screened for retention of function (e.g., CAR binding ability). If the CAR engager expressed by the bacterial cell is produced in a properly folded form, the bacterial cell is a desirable host, or in an alternative embodiment, the CAR engager can be expressed in a bacterial host and then refolded. 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 can also be used.

[0161] After expression in the cells, the CAR engager is isolated from the cells (e.g., cell lysate) or from the medium in which the cells are cultured. Protein isolation techniques are known in the art. Representative isolation techniques include chromatography, affinity chromatography, nickel nitrilotriacetic 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, for example, U.S. Patent Nos. 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 Publication Nos. 2008 / 0090995, 2012 / 0244075, 2017 / 0158760, 2019 / 0276492, and 2021 / 0206815, and Traunecker et al., Embo J. 10(12):3655-9 (1991).

[0162] In some embodiments, the CAR engager is encoded by two or more nucleic acids, e.g., the ectodomain-containing portion is encoded by one nucleic acid and the immune cell effector domain is encoded by a second nucleic acid. In these embodiments, after expression and purification in a suitable cell, the purified ectodomain and purified immune cell effector can be linked by a suitable chemical ligation reaction as described above.

[0163] Pharmaceutical Composition The pharmaceutical composition of the present disclosure comprises an effective amount of a CAR engager and a pharmaceutically acceptable carrier. As used herein, the term "effective amount" refers to an amount of a CAR engager sufficient to provide a desired effect, e.g., the amount of a CAR engager that binds to CAR-expressing immune cells. The amount of a CAR engager administered to a subject will vary widely depending on the location, type, and severity of the cancer, the age, weight, and condition of the individual being treated, and the like. The physician will ultimately determine the appropriate dose to be used. The CAR engager in the pharmaceutical composition may be in the form of a monomer (in embodiments lacking a dimerization domain), a homodimer, or a heterodimer, as described herein.

[0164] The amount of CAR engager administered to a subject can vary within wide limits, depending on numerous factors, such as the location, type, and severity of the cancer, as well as the age, weight, and condition of the individual being treated. A physician ultimately determines the appropriate amount and dosage of CAR engager to be used. Typically, the CAR engager is administered in a series of doses. In some embodiments, an effective amount of CAR engager is approximately 50 mg to approximately 180 mg per subject per dose. In some embodiments, an effective amount of CAR engager is approximately 1 mg to approximately 18 mg per kg of subject body weight.

[0165] The composition may be provided as a sterile solid or liquid preparation. Solid preparations may be reconstituted or diluted into a liquid preparation before use, for example, with a carrier, including an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous solution, which may be buffered to a selected pH. Liquid carriers may also include aqueous or non-aqueous carriers. Representative examples of liquid carriers include sterile water for injection, saline, lactated Ringer's injection, phosphate-buffered saline, soluble proteins, soluble sugars (e.g., dextrose), dimethyl sulfoxide (DMSO), polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), ethanol, and suitable mixtures thereof. In some embodiments, the liquid carrier contains a protein dissolved or dispersed therein, representative examples of which include serum albumin (e.g., human serum albumin, recombinant human albumin), gelatin, and casein. The composition is typically isotonic, i.e., has the same osmotic pressure as blood. Citric acid, sodium chloride, sugars, polyalcohols, and isotonic electrolyte solutions (e.g., Plasma-Lyte®) can be used to achieve the desired isotonicity. Depending on the carrier, other excipients may be added, such as wetting agents, dispersing or emulsifying agents, gelling and viscosity enhancing agents, preservatives, etc., as known in the art. In some embodiments, the composition comprises citric acid, ethylenediaminetetraacetic acid (EDTA), and polysorbate 20, and has a pH range of about 6.8 to about 7.2.

[0166] cancer In some embodiments, the present disclosure is directed to treating cancer in a subject. The method involves administering to a subject in need thereof a pharmaceutical composition comprising a CAR engager described herein. As used herein, the term "cancer" refers to a disease or disorder characterized by excessive proliferation or decreased apoptosis in a subject. Cancers that can be treated with the CAR engagers disclosed herein include both hematopoietic cancers and cancers characterized by the presence of solid tumors.

[0167] As used herein, the term "subject" (or "patient") includes all members of the animal kingdom that are susceptible to (or prone to) or afflicted with the indicated cancers. In some embodiments, the subject is a human. Thus, a subject "having" or "in need of" treatment according to the present disclosure broadly encompasses positively diagnosed subjects, including subjects with active disease who may have been previously treated with one or more therapies, and subjects who are currently untreated (e.g., in remission) but may still be at risk of recurrence, as well as subjects who have not been positively diagnosed but who are predisposed to cancer (e.g., based on previous medical history and / or family history, or who otherwise present one or more risk factors such that a medical professional may reasonably suspect that the subject is predisposed to cancer).

[0168] As used herein, the terms "treat," "treating," and "treatment" (or "treat," "treating," and "treatment") include any type of intervention, process, or administration of an active agent performed on a subject in need thereof for therapeutic purposes (a "therapeutic effect") to reverse, alleviate, ameliorate, inhibit, reduce, slow, arrest, stabilize, or prevent the onset, progression, development, severity, or recurrence of a symptom, complication, or condition, or biochemical manifestation, associated with cancer.

[0169] In some embodiments, cancer is hematopoietic cancer.Representative blood cancers include plasma cell neoplasms (e.g., myeloma, multiple myeloma, relapsed or refractory multiple myeloma, plasma cell myeloma, extramedullary multiple myeloma, monoclonal gammopathy of undetermined significance (MUG), asymptomatic smoldering multiple myeloma or solitary plasmacytoma), lymphomas (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, plasmablastic lymphoma, plasmacytoid lymphoma or diffuse large B-cell lymphoma), leukemias (e.g., relapsed or refractory acute B-lymphocytic leukemia or relapsed or refractory acute lymphoblastic leukemia), and carcinomas (e.g., Waldenstrom's macroglobulinemia or glioblastoma (astrocytoma)). In these embodiments, the therapeutic effect may include one or more art-recognized indicia of therapeutic efficacy, representative examples of which include prevention or prolongation of metastasis, improved survival, and total / complete or partial remission of the cancer, e.g., no detectable cancer cells and fewer or smaller tumor cells, or a reduction in tumor cell count, respectively. In some embodiments, the hematopoietic cancer is multiple myeloma, lymphoma, or leukemia.

[0170] In some embodiments, the cancer is characterized by the presence of a solid tumor. In some embodiments, the cancer is selected from the group consisting of bladder cancer (e.g., transitional cell carcinoma, also known as urothelial carcinoma), kidney cancer (e.g., renal cell carcinoma (RCC), kidney renal clear cell carcinoma (KIRC), transitional cell carcinoma, or Wilms' tumor), skin cancer (e.g., melanoma, cutaneous melanoma (SKCM), basal cell carcinoma, and squamous cell carcinoma of the skin), lung cancer (e.g., non-small cell lung cancer, including small cell lung cancer, lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC)), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCC), also known as head and neck squamous cell carcinoma (HNSC)). HN), laryngeal and hypopharyngeal cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, oral cavity and oropharyngeal cancer, and salivary gland cancer), colon or rectal cancer (e.g., colorectal carcinoma (CRC), colorectal adenocarcinoma (COAD), rectal adenocarcinoma (READ)), ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma, epithelial ovarian carcinoma, fallopian tube carcinoma, and primary peritoneal carcinoma), endometrial cancer, cervical cancer (e.g., cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC)), prostate cancer (e.g., prostate adenocarcinoma (PRAD)), and gastric cancer (e.g., gastric adenocarcinoma (STAD)).

[0171] In some embodiments, the cancer is characterized as being in a state of minimal residual disease (MRD). MRD is a state in which a cancer patient has a small number of cancer cells remaining 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 cancer and are often undetectable by traditional methods, such as looking at cells under a microscope and / or tracking abnormal serum proteins in the blood.

[0172] The amount of cancer antigens present in subjects with MRD is limited. And this limited presence of cancer antigens may not adequately support the proliferation and efficacy of CAR immune cells. The additional presence of CAR engager not only presents CAR immune cells with additional cancer antigens, but also presents supporting immune cell effector domains that can regulate the activity of CAR immune cells to promote proliferation, efficacy, and / or persistence.

[0173] In some embodiments, the subject receiving the CAR engager is in a state of MRD. In some embodiments, the method of treating cancer comprises treating a state of minimal residual disease (MRD) in the subject. In some embodiments, the method of treating cancer comprises eliminating MRD in the subject.

[0174] To test MRD, the sample from either blood collection or bone marrow aspiration can be used.The most widely used test for measuring MRD is flow cytometry, polymerase chain reaction (PCR) and next-generation sequencing.The method that may be suitable for measuring MRD is described in, for example, United States Patent No. 8,124,353, United States Patent No. 9,528,160, United States Patent No. 10,280,462, United States Patent No. 11,618,787 and United States Patent No. 11,633,426, and United States Patent Application Publication No. 2011 / 0294148 and United States Patent Application Publication No. 2022 / 0380852.

[0175] Administration In some embodiments, the methods of the present disclosure involve administering an effective amount of a CAR engager to a cancer patient who has previously received immune cells comprising a CAR that includes an extracellular domain that binds to the ectodomain of the CAR engager, a transmembrane domain, and an intracellular domain that includes a stimulatory domain. In some embodiments, the CAR engager is administered to the subject after determining that the CAR-immune cells have lost vitality or persistence in the subject. This determination may be made according to known techniques. In some embodiments, for example, a sample is obtained from the subject after administration of the immune cells. The concentration of immune cells present in the sample can be used to calculate the difference between the concentration of immune cells administered to the subject and the concentration of immune cells measured in the sample. The CAR engager can be administered after the measured concentration of immune cells is lower than the administered concentration of immune cells. In some embodiments, the CAR engager is administered after the measured concentration of immune cells is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 25%, less than 10%, or less than 5% of the concentration of immune cells administered.

[0176] In some embodiments, administration of the CAR engager occurs at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, or at least about 1 year after administration of the CAR-immune cells.

[0177] In some embodiments, the CAR engager is administered as an infusion over about 30 to about 90 minutes once every three weeks (a 21-day cycle). In some embodiments, the CAR engager is administered for five consecutive days every 21 days, repeated for eight cycles.

[0178] In other embodiments, the method further involves co-administering an effective number of immune cells expressing a CAR (also referred to as CAR immune cells) in the same course of treatment as the administration of the CAR engager. As used herein, the term "effective number of CAR immune cells" (which indirectly includes a corresponding amount of a CAR) refers to a number of CAR immune cells sufficient to provide the desired effect.

[0179] More broadly, the order in which a CAR engager and CAR immune cells are administered during the same course of treatment may not be important, so long as they are able to interact and cause the desired effect in vivo. In some embodiments, the CAR engager is co-administered to a subject with the CAR immune cells substantially simultaneously. In some embodiments, the CAR engager is contacted with the CAR immune cells in vitro prior to co-administration to the subject. In some embodiments, the CAR engager is administered to a subject after administration of the CAR immune cells. In some embodiments, the CAR engager is administered to a subject before administration of the CAR immune cells.

[0180] The extracellular domain of the CAR that binds to the ectodomain of the cancer antigen may contain an antibody fragment. In some embodiments, the CAR binds to BCMA. CAR extracellular domains that bind to BCMA are known in the art. For example, the FDA-approved CAR-expressing immune cells ciltacabtagene autoleucel (Carvykti®) and idecabtagene See vicleucel (Abecma®), U.S. Pat. Nos. 10,072,088, 10,683,369, 11,084,880, and 10,174,095, and U.S. Patent Application Publication Nos. 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, such as belantamab (Blenrep®), linvoseltamab (REGN5458), pacanalotamab (AMG420), pavurutamab (AMG701), and teclistamab (Tecvayli®). In some embodiments, the CAR extracellular domain binds to the BCMA ectodomain of a CAR engager having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0181] In some embodiments, the CAR binds to CD19. CAR extracellular domains that bind to CD19 are known in the art. For example, see FDA-approved CAR-expressing immune cells lysocabtagene malareucel (Breyanzi®), tisagenlecleucel (Kymriah®), brexcabtagene autolucel (Tecartus®), and axicabtagene siloleucel (Yescarta®), U.S. Patent Nos. 9,629,877, 10,273,300, and 10,533,055, and U.S. Patent Application Publication Nos. 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, such as loncastuximab (Zynlonta®), tafasitamab (Monjuvi®), denintuzumab (SGN-CD19A), and inabilizumab (Uplizna®). In some embodiments, the CAR extracellular domain binds to the CD19 ectodomain of a CAR engager having the amino acid sequence of any one of SEQ ID NOs: 3-5, or 103.

[0182] In some embodiments, CAR binds to CD20. CAR extracellular domains that bind to CD20 are known in the art. For example, see U.S. Patent Nos. 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 No. 2018 / 0187149. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-CD20 antibody, anti-CD20 binding fragment, or derivative thereof, such as ofatumumab (Arzerra®, Kesimpta®), veltuzumab (IMMU-106), tositumomab (Bexxar®), and rituximab (Rituxan®, Riabni®, Truximab®). In some embodiments, the extracellular domain of the CAR binds to the CD20 ectodomain of a CAR engager having the amino acid sequence of SEQ ID NO:6.

[0183] In some embodiments, the CAR binds to CD22. In these embodiments, the extracellular domain of the CAR binds to the CD22 ectodomain of the CAR engager. CAR extracellular domains that bind to CD22 are known in the art. See, for example, U.S. Patent Nos. 9,139,649, 9,181,343, and 10,494,435, U.S. Patent Application Publication Nos. 2015 / 0175711, 2018 / 0086843, 2021 / 0047402, 2021 / 0095022, 2022 / 0220198, and 2022 / 0273710, and Fry et al., Nat. Med. 24(1):20-28 (2018). In some embodiments, the CAR extracellular domain is derived from a commercially available anti-CD22 antibody, anti-CD22 binding fragment, or derivative thereof, such as bectumomab, epratuzumab, inotuzumab, moxetumomab, and epratuzumab. In some embodiments, the CAR engager comprises a CD22 ectodomain having the amino acid sequence of SEQ ID NO: 7. In some embodiments, the CAR engager comprises a CD22 ectodomain having any one of the amino acid sequences of SEQ ID NOs: 104-107.

[0184] In some embodiments, the CAR binds to SLAMF7. CAR extracellular domains that bind to SLAMF7 are known in the art. See, for example, U.S. Patent No. 10,799,536, and U.S. Patent Application Publication Nos. 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, such as elotuzumab (Empliciti®). In some embodiments, the CAR extracellular domain binds to the SLAMF7 ectodomain of a CAR engager having the amino acid sequence of SEQ ID NO: 10.

[0185] In some embodiments, CAR binds to PD-1. CAR extracellular domains that bind to PD-1 are known in the art. For example, see U.S. Patent Nos. 10,124,023 and 11,136,392, and U.S. Patent Application Publication Nos. 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, such as 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. Thus, in some embodiments, the extracellular domain of the CAR binds to the PD-1 ectodomain of a CAR engager having the amino acid sequence of SEQ ID NO: 11.

[0186] In some embodiments, the CAR binds to the receptor tyrosine kinase KIT proto-oncogene (KIT). CAR extracellular domains that bind to KIT are known in the art. See, for example, U.S. Patent Application Publication Nos. 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, such as barzolvolimab. In some embodiments, the CAR extracellular domain binds to the KIT ectodomain of a CAR engager having the amino acid sequence of SEQ ID NO: 12.

[0187] In some embodiments, CAR binds to CD38.The CAR extracellular domain that binds to CD38 is known in the art.For example, see US Patent No. 10,709,775, US Patent No. 10,799,536, US Patent No. 10,836,998 and US Patent No. 11,365,394 and US Patent Application Publication No. 2017 / 0296623, US Patent No. 2019 / 0135894, US Patent No. 2019 / 0135937, US Patent No. 2020 / 0308541, US Patent No. 2021 / 0046118 and US Patent No. 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, such as daratumumab (Darzalex®), isatuximab (Sarclisa®), and mezagitamab. In some embodiments, the extracellular domain of the CAR binds to the CD38 ectodomain of a CAR engager having the amino acid sequence of SEQ ID NO: 14.

[0188] The intracellular domain of CAR comprises a signal transduction domain that enables intracellular signal transduction and immune cell function. The signal transduction domain can comprise a primary signal transduction domain and / or a costimulatory signal transduction domain. In some embodiments, the intracellular domain can deliver a signal similar to the signal of natural ligation of ITAM-containing molecules or receptor complexes, such as TCR receptor complexes.

[0189] In some embodiments, the signaling domain comprises multiple, e.g., two or three, costimulatory signaling domains selected from, e.g., 4-1BB, CD3ζ, CD28, CD27, ICOS, and OX40. In some embodiments, the signaling domain can comprise a CD3ζ domain as the primary signaling domain and any of the following pairs of costimulatory signaling domains from the extracellular to intracellular direction: 4-1BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28-4-1BB; OX40-CD28; CD28-OX40; 4-1BB-CD3ζ; CD3ζ-4-1BB; CD28-CD3ζ; CD3ζ-CD28; CD28-4-1BB, and 4-1BB-CD28. In some embodiments, the primary signaling domain is derived from CD3ζ, CD27, CD28, CD40, KIR2DS2, MyD88, or OX40. In some embodiments, the costimulatory signaling domain is derived from one or more of CD3γ, CD3δ, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD68, CD72, CD80, CD86, CD137 (4-1BB; TNFRSF9), CD154, CLEC-1, 4-1BB, DAP10 (hematopoietic cell signaling substance (HCST)), DAP12 (TYROBP), Dectin-1, FcαRI, FcγRI, FcγRII, FcγRIII, IL-2RB, ICOS, KIR2DS2, MyD88, OX40, and ZAP70.

[0190] A representative CAR with a CD3ζ stimulatory signaling domain is the FDA-approved CAR-expressing immune cell tisagenleucel (Kymriah®). Representative CARs with CD3ζ and 4-1BB costimulatory signaling domains are the FDA-approved CAR-expressing immune cell idecabtagene vicleucel (Abecma®), lysocatagene malaleucel (Breyanzi®), and ciltacabtagene autoleucel (Carvykti®). Representative CARs with CD28 and CD3ζ costimulatory signaling domains are the FDA-approved CAR-expressing immune cell brexucabtagene autoleucel (Tecartus®) and axibatagene ciloleucel (Yescarta®).

[0191] 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, for example, U.S. Patent Nos. 5,906,936, 7,446,190, 7,741,465, 8,389,282, 8,399,645, 9,422,351, 9,790,267, 9,885,298, Nos. 10,124,023, 10,815,301, and 11,433,100, and U.S. Patent Application Publication Nos. 2019 / 0375815, 2020 / 0281973, 2021 / 0300986, 2022 / 0056101, and 2022 / 0193138.

[0192] Without being bound by theory, the interaction between CAR engager and CAR does not cause clustering effect and synapse formation.In contrast, cancer antigen causes clustering effect and synapse formation between CAR and cancer antigen on the surface of CAR immune cells.Therefore, the binding of CAR engager to CAR immune cells is short-term and reversible, which ensures that CAR immune cells can bind to cancer cells via CAR in the presence of CAR engager.

[0193] The number of CAR immune cells administered to the subject varies within a wide range, depending on the location, type and severity of cancer, the age, weight and condition of the individual being treated, etc. Doctors ultimately decide the appropriate number of cells and dosage to be used.Usually, CAR immune cells are given in a single dose.

[0194] In some embodiments, the effective number of CAR immune cells is approximately 1 x 10 per subject. 5 ~approximately 1×10 10 In some embodiments, the effective number of CAR immune cells is about 1 x 10 cells per kg of subject body weight. 5 ~Approx. 6×10 8 Each cell is an individual cell.

[0195] Because CAR engagers enhance the functionality and persistence of CAR immune cells in vivo, they can reduce the cell dose required for CAR immune cell therapy, which in turn can reduce adverse side effects (e.g., cytokine release syndrome) caused by the larger doses typically used in clinical practice. Thus, in some embodiments, the effective number of CAR immune cells is approximately 1 x 10 per subject. 4 ~approximately 1×10 7 In some embodiments, the effective number of CAR immune cells is about 1 x 10 cells per kg of subject body weight. 4 ~Approx. 6×10 5 Each cell is an individual cell.

[0196] A composition comprising an effective amount of a CAR engager and an effective number of CAR immune cells can be administered to a subject for the treatment of cancer by any medically acceptable route. The CAR engager and CAR immune cells are typically delivered intravenously, but may also be introduced at other convenient sites (e.g., diseased organs or tissues) or in other ways, as determined by the attending physician.

[0197] Growth and differentiation agents can be provided before, during, or after administration of the cells to increase the differentiation, proliferation, and / or persistence of CAR immune cells (e.g., T cells and NK cells).

[0198] The administration of CAR immune cells can be autologous or allogeneic. For example, immune cells or their precursors can be isolated from the tissue of a body fluid from a subject before administration to the same subject (autologous) or a different compatible subject (allogeneic).

[0199] In some embodiments, the CAR engager is administered periodically, for example, once a week, once every two weeks, or once every three weeks. This cycle is repeated, for example, for two, three, five, or eight cycles. In some embodiments, the CAR engager is administered for several consecutive days prior to the periodic administration, for example, once daily for five days, and then once every three weeks. In some embodiments, the CAR engager is administered as an intravenous infusion over a period of time. Typical infusion times are 30, 60, and 90 minutes. In some embodiments, the infusion time is between 30 and 60 minutes. In some embodiments, the first administration is infused into the patient over 90 minutes, and subsequent administrations are infused into the patient over 30 minutes.

[0200] Combination therapy In some embodiments, the method may involve co-administration of a CAR engager with another anti-cancer agent, or a CAR engager and a CAR immune cell. The term "co-administration" includes sequential, substantially simultaneous administration, either in the same or separate dosage forms, or as part of the same treatment regimen or sequential treatment regimens, for example. Thus, when administered sequentially, at the start of administration of the second therapy, the first of the two therapies may still be detectable at effective concentrations at the treatment site, as the case may be. The order and time intervals may be determined so that they can act together (e.g., synergistically to provide increased benefit over their alternative administration). For example, therapeutic agents may be administered sequentially in any order, at the same time or at different times, but if not administered simultaneously, they may be administered sufficiently closely to provide the desired therapeutic effect, which may be in a synergistic manner. Thus, the term is not limited to administering the active agents exactly at the same time.

[0201] Anti-cancer agents that can be used in combination with the cells of the present invention are known in the art. See, for example, U.S. Patent No. 9,101,622 (Section 5.2). An "anti-cancer" agent can negatively affect cancer in a subject by, for example, killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to tumors or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting the progression of cancer, or extending the lifespan of a subject with cancer. More generally, these other compositions are provided in a combined amount effective to kill or inhibit the growth of cancerous cells. This process can include simultaneously contacting the cancer cells with the recipient cells and the agent or multiple factors. This can be achieved by contacting the cancer cells with a single composition or pharmacological formulation containing both agents, or by simultaneously contacting the cancer cells with two different compositions or formulations, where one composition contains the recipient cells and the other contains the second agent.

[0202] In some embodiments, the CAR engagers and CAR immune cells of the present disclosure are used in conjunction with or following a prior therapy such as chemotherapy, radiation therapy, immunotherapy intervention, targeted therapy, pro-apoptotic therapy, or cell cycle modulating therapy.

[0203] In some embodiments, the CAR engagers and CAR immune cells of the present disclosure are combined with high-dose chemotherapy prior to administration of the genetically modified immune cells, hi some embodiments, bone marrow cells or peripheral blood stem cells are administered after the high-dose chemotherapy.

[0204] In some embodiments, the CAR engagers and CAR immune cells of the present disclosure are combined with an effective amount of thalidomide, lenalidomide, bortezomib, or a combination thereof.

[0205] Additional enhancement therapies that can be used in conjunction with the genetically modified immune cells of the present disclosure include melphalan. Melphalan (Alkeran®, Evomela®), an alkylating anti-neoplastic agent, is used for high-dose conditioning prior to hematopoietic stem cell transplantation in patients with multiple myeloma, as well as for palliative treatment of multiple myeloma and for the palliation of unresectable epithelial carcinoma of the ovary. Melphalan is also used to treat AL amyloidosis, neuroblastoma, rhabdomyosarcoma, breast cancer, ocular retinoblastoma, some conditioning regimens prior to bone marrow transplantation, and in some cases, malignant melanoma. Melphalan can be administered orally in pill form. Typically, a 2 mg dose is taken on an empty stomach. In some cases, melphalan can be administered as an injection or intravenous infusion. Dosage depends on weight, height, disease and disease state, and the subject's overall health.

[0206] immunotherapy Immunotherapy, including immune checkpoint inhibitors, can be used to treat diagnosed cancers. Examples of immune checkpoint molecules include PD-1, PDL1, CTLA4, KIR, TIGIT, TIM-3, LAG-3, BTLA, VISTA, CD47, and NKG2A. Clinically available examples of immune checkpoint inhibitors include durvalumab (Imfinzi®), atezolizumab (Tecentriq®), and avelumab (Bavencio®). Clinically available examples of PD-1 inhibitors include nivolumab (Opdivo®), pembrolizumab (Keytruda®), and cemiplimab (Libtayo®). Additional inhibitors that may be useful in the practice of the present disclosure are known in the art. See, for example, U.S. Patent Application Publication Nos. 2012 / 0321637, 2014 / 0194442, and 2020 / 0155520.

[0207] chemotherapy Anti-cancer therapy also includes a variety of combination therapies with both chemo- and radiation-based treatments, including, for example, Abraxane®, altretamine, docetaxel, Herceptin®, methotrexate, Novantrone®, Zoladex®, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, dactinomycin, daunorubicin, doxorubicin, bromide, thiazolinone ... These include leomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, Taxol®, gemcitabine, Navelbine®, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, or any analog or derivative variant of the foregoing, and combinations thereof.

[0208] Radiation therapy Anticancer therapy also includes radiation-based DNA damage therapy. Combined radiation therapy includes gamma rays, commonly known as X-rays, and / or the directed delivery of radioisotopes to tumor cells, which cause widespread damage to DNA, DNA replication and repair, and chromosome assembly and maintenance. The dose range of radioisotopes varies widely and depends on the half-life of the isotope, the strength and type of radiation emitted, and uptake by tumor cells, and is determined by the attending physician.

[0209] Radiation therapy can include external or internal radiation therapy. External radiation therapy involves a radiation source outside the subject's body that sends radiation toward the area of ​​the cancer inside the body. Internal radiation therapy uses radioactive material enclosed in needles, seeds, wires, or catheters that are placed directly into or near the cancer.

[0210] These and other aspects of the present application will be further understood in light of the following examples, which are intended to illustrate particular embodiments of the present application but are not intended to limit its scope, which is defined by the claims. [Example]

[0211] Example 1: Materials and Methods Protein cloning and expression were performed according to standard approaches. Other procedures, including flow cytometry analysis, BLI imaging, CAR T cell production, cell culture, and animal handling, were performed according to standard protocols, as briefly described below.

[0212] Generation of CAR engagers. 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 product production, a stable HEK293 cell line was generated. HEK293 cells were transfected with pPAX2, pVSVG (packaging vector), and a lentiviral plasmid containing the sequence of interest. Lentivirus was harvested at 48, 72, and 96 hours posttransfection, sedimented at 20,000 x g for 2 hours, and resuspended in optiMEM medium. A new batch of HEK293 cells was then subjected to three rounds of transduction with the virus. Cells were recovered in DMEM complete medium and subjected to puromycin selection to retain only cells that had integrated the lentiviral plasmid. Cells were then grown to confluence in four 15 cm culture dishes, carefully washed with PBS, and incubated in serum-free DMEM for 24–48 hours. The supernatant was collected, and protein expression was confirmed by SDS-PAGE and immunoblotting. The protein was purified by adsorption onto a nickel nitriloacetic acid (Ni-NTA) metal affinity column. Nonspecifically bound proteins were removed by washing with 40 mM imidazole. The imidazole concentration was increased to 250 mM, allowing for recovery of the target protein. The protein was further purified by size-exclusion chromatography and stored at -80°C in 50 mM HEPES buffer, pH 7.5, until use.

[0213] Some CAR engagers were isolated by passing them 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 having a pH of about 3 to about 4, thereby washing the CAR engagers off the affinity chromatography resin. A basic buffer was used to neutralize the acidic buffer, and then tangential flow filtration of the neutralized buffer with a formulation buffer could be performed to isolate a concentrated, purified solution containing the CAR engagers.

[0214] Generation of CAR T cells. The CAR construct binding to human CD19 contains an scFv derived from the anti-human CD19 antibody clone FMC69, followed by human CD28 and CD3ζ intracellular signaling domains. The CAR construct binding to human BCMA contains an scFv derived from the anti-human BCMA antibody clone MSK54, followed by human 41BB and CD3ζ intracellular signaling domains. The human signaling CAR construct was transduced into HeLa cells stably producing a gammaretrovirus pseudotyped with the envelope of feline endogenous virus (RD114), which has been shown to transduce human hematopoietic cells (HSCs) with high efficiency (Ward et al., Mol. Ther. 8(5):804-12 (2003)). High-titer clones were isolated by limiting dilution. High-expressing clones were seeded and grown to 80% confluence in DMEM complete medium containing 10% FBS. The medium was replaced with RPMI complete medium containing 10% FBS. After 24 hours, the virus-containing medium was collected, sterile filtered using a 0.45 μm PES filter, and used for the production of CAR T cells.

[0215] CAR T cell production was adapted from previous studies. See, e.g., Li et al., Methods Mol. Biol. 1514:111-118 (2017). Briefly, whole blood was obtained from apheresis leukapheresis collars of platelet-healthy donors due to the high number of viable leukocytes. PBMCs were isolated by centrifugation through a Ficoll gradient. CD8 + Whole PBMCs were used without T cell selection. PBMCs were cultured in RPMI medium containing 10% fetal bovine serum (FBS), 200 IU / mL IL-2, 60 ng / mL IL-7, 10 ng / mL IL-15, 2 μg / mL anti-human CD3 (OKT3 clone), and 0.5 μg / mL anti-human CD28 (CD28.1 clone) at 4 × 10 in 3 mL of medium per well in a 6-well plate. 6The cells were resuspended at a cell concentration of 4 x 10 cells / mL. After 24 hours, the cells were harvested, spun down, and resuspended in the same volume of fresh medium containing the medium harvested from the anti-human BCMA CAR gammaretrovirus-producing cells plus FBS, IL-2, IL-15, and IL-7, resulting in PBMC inoculation with gammaretrovirus. The PBMCs were then plated at a concentration of 4 x 10 cells in 3 mL into 6-well plates coated with 20 μg retronectin. 6 PBMCs were plated at 2000 x g / mL (coated with 1 mL of 20 μg / mL retronectin in PBS for 24 hours at 4°C). PBMCs were centrifuged at 2000 x g for 1 hour at 30°C and cultured at 37°C. The transduction process was repeated with fresh gamma retrovirus containing medium, cytokines, and spinoculation. Flow cytometry analysis was used to assess the transduction efficiency of the CAR transgene using recombinant BCMA labeled with a dsRed reporter gene and AlexaFlour-647.

[0216] In vivo experiments. For all experiments, NOD / SCID / gamma (NSG; NOD.Cg-Prkdc scid Il2rg tm1Wjl NSG mice (SzJ / SzJ) were used due to their immunodeficient state and ability to effectively engraft human cancer cell lines. Cells derived from the human multiple myeloma OPM2 cell line were used to establish a multiple myeloma mouse model in NSG mice. In vivo experiments were performed using 1 x 10 cells expressing GFP and firefly luciferase. 6 Treatment was initiated by intravenous injection of OPM2 cells via the tail vein, followed by biweekly bioluminescence imaging (BLI). Three weeks later, upon effective engraftment, mice were injected intravenously with CAR T cells via the tail vein. BLI was then performed biweekly to assess tumor burden. Quantitation was measured in photons / second using Aura software.

[0217] Organ Analysis. At the end of the experiment, surviving mice were sacrificed, and the spleen, bone marrow, blood, liver, kidneys, and lungs were collected and weighed. The liver, kidneys, and lungs were minced and digested with collagenase (final concentration 1 μg / mL collagenase) and incubated at 37°C for 1 hour. The spleen was crushed, and the bone marrow was aspirated using a 30-gauge insulin needle. All treated cells were extruded through a 70 μm strainer to generate a single-cell suspension. The cells were resuspended in 1 mL of ammonium chloride-potassium (ACK) lysis buffer, and the sample was depleted of red blood cells for 2 minutes at room temperature. The resulting single-cell suspension was washed with fluorescence-activated single-cell sorting (FACS) buffer (PBS and 0.5% BSA), stained, and analyzed using flow cytometry.

[0218] Cell lines and cultures. The OPM2 cell line, which endogenously expresses BCMA, was engineered to express green fluorescent protein (GFP) and firefly luciferase. Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll gradient leukapheresis 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 RPMI-1640 (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 at 37°C in a 5% CO2, 95% air humidified incubator.

[0219] Mouse studies. All experiments followed appropriate ethical and safety protocols. The study was conducted under the supervision of the Dana-Farber Cancer Institute Institutional Animal Care and Use Committee (Protocol No. 20-006). The xenograft model utilized herein is described in Smith et al., Mol. Ther. 26(6):1447-1456 (2018). Briefly, 8- to 12-week-old NOD-scidIL2Rγ mice were transplanted. null (NSG) and NOD-scidH2-K1 null H2-Ab1 null H2-D1 null IL2Rg null NSG-MHC I / II double knockout (DKO) mice were purchased from Jackson Laboratory or bred in-house. All mice were gender- and age-matched to groups. Xenograft models were performed using 1 × 10 OPM2 or Nalm6 cells expressing GFP and luciferase in 200 mL of PBS. 6Tumor burden was established by intravenous injection of cells. Mice received the indicated treatments in 300 mL of PBS via intraperitoneal injection. Tumor burden was assessed at the indicated times after intraperitoneal injection of D-luciferin (150 mg / kg, from a 15 mg / ml solution) using an IVIS® Lumina Series III (Perkin Elmer). Each mouse was imaged in groups of up to five mice in a supine position at the same time point (5 minutes). BLI intensity was analyzed using Aura imaging analysis software (Spectral Instruments Imaging). Peripheral blood from mice was obtained by submandibular bleeding in EDTA-coated tubes and analyzed for CAR T cell detection and proliferation. Briefly, blood volume was measured and absolute values ​​were calculated. The samples were then centrifuged, and serum was collected. The cell pellet was resuspended in 500-1000 mL of ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 1 min. 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-PD1-BV605 (1:50, Biolegend), anti-HLA-DR-PE / Cy7 (1:50, Biolegend), anti-CD69-BV421 (1:50, Biolegend), and recombinant BCMA-AlexaFluor647 (produced in-house). Samples were analyzed using a Sony SP6800 spectrum analyzer. Flow rates and acquisition times were recorded, and absolute values ​​were calculated. All experiments were performed in a blinded and randomized manner. Animals were euthanized at the end of the experiment or when a pre-specified endpoint was met according to IACUC protocol.At the endpoint, major immune organs (spleen and bone marrow) and essential organs where metastatic lesions may form (liver, lungs, and kidneys) were collected, weighed, and analyzed. Briefly, the spleen was disrupted using a plunger and passed through a 40 μm strainer to obtain a single-cell suspension. Bone marrow was aspirated using a 30-gauge insulin needle. The liver, lungs, and kidneys were diced using surgical scissors in 3 mL of digestion buffer (1 mL RPMI + 2 mL PBS). Collagenase type 1 (Worthington) was added to a final concentration of 100 mg / mL and incubated at 37°C for 1 hour. The resulting sample was passed through a 40 μm strainer to obtain a single-cell suspension. The samples were then stained with the same antibodies used to stain the blood samples and analyzed using a Sony SP6800 Spectral Analyzer. Flow rates and acquisition times were recorded, and absolute values ​​were calculated.

[0220] Microscopy. Cells were first stained with CellTracker Blue CMAC and plated on poly-d-lysine-coated coverslips. Subsequently, samples were incubated with the indicated treatments labeled with Alexa647 for the indicated times and temperatures. After fixation with BD Cytofix buffer, cells were imaged using a Leica THUNDER Imager. The intensity cutoff for the AlexaFluor647 channel was set to 2000 for all images, except for the VHH-muIL2 sample (condition 4) shown on the right, for which the image sensitivity was increased 25-fold (intensity cutoff 80) to visualize the Alexa647 signal. Quantitative analysis of fluorescence intensity was performed by aligning the base-2 logarithm of the ratio of the integrated intensity of the membrane to the cell cytoplasm (see the y-axis in Figure 14B). For BCMA-muIL2 and BCMA-CH3 (conditions 1 and 2), only cells with a mean intensity-to-background ratio based on the dsRed channel greater than 4 were included to determine whether they were CAR. +These cells were identified as T cells and analyzed accordingly. For VHH-muIL2, cells with a mean intensity-to-background ratio of greater than 2 based on the Alexa647 channel were selected to eliminate background artifacts. Image quantification was performed using ImageJ software.

[0221] ELISA. ELISA analysis was performed to measure CAR T cell-derived cytokines in the serum of mice receiving OPM2 cancer cells followed by a low dose of CAR T cells, as shown in Figure 17A. Cytokines analyzed with the ELISA MAX™ Standard Set (Biolegend) included IFN-γ, GM-CSF, and TNF-α according to the manufacturer's protein specifications, but only IFN-γ was detectable in the collected samples. Serum samples were diluted at a ratio of 1:40. Both standard samples and serum samples were incubated on the same plate, and standard curves were plotted for each cytokine.

[0222] Example 2: In vitro characterization of BCMA-containing CAR engagers. A fusion protein consisting of the human BCMA ectodomain was fused to two low-affinity mutant human IL-2 (muIL2) domains. To improve pharmacokinetics and enhance stability, the CH3 domain of human IgG1 (approximately 14 kDa in size) (Feige et al., Trends Biochem. Sci. 35(4):189-198 (2010)) was incorporated between the antigen and muIL2 (Figure 2B) (Quayle et al., Clin. Cancer Res. 26(8):1953-1964 (2020)). As a result, the BCMA-muIL2 CAR engager binds to the surface of CAR T cells via low-affinity IL-2 antigen-CAR binding, minimizing effects on normal T cells, Tregs, or systemic toxicity.

[0223] IL-2 induces an alternative differentiation pathway in T cells, resulting in different, "better effector" CD8 +It has recently been demonstrated that IL-2Rα generates T cells (Hashimoto et al., Nature 610(7930):173-181 (2022)). This process may depend, at least in part, on IL-2 binding to IL-2Rα. Furthermore, IL-2Rβγ-biased agonists can direct T cells toward a terminally differentiated state (Codarri et al., Nature 610(7930):161-172 (2022)). CAR engagers may be able to overcome the requirement for IL-2Rα in alternative differentiation pathways by immobilizing low-affinity IL-2 on the surface of CAR T cells via antigen-CAR binding, thereby promoting the generation of memory CAR T cells. Potential synergistic effects between CAR signaling and IL-2 signaling may also exist.

[0224] To assess the binding affinity of BCMA-containing CAR engagers, flow cytometry analysis was performed by staining BCMA CAR T cells with various concentrations of BCMA CAR engager. EC 50was observed for the BCMA CAR engager, which was comparable to that of dimeric BCMA lacking muIL2 (BCMA-CH3) (Figure 2E), indicating that binding was primarily due to the BCMA ectodomain and not muIL2. Figure 2E shows dose-dependent staining of BCMA CAR T cells with the CAR engager using flow cytometry (n=3 per point). Untransduced T cells were used as a control, and a secondary Alexa647-labeled anti-FLAG antibody was used for staining. Error bars represent the mean with 95% confidence intervals. Minimal binding of the BCMA CAR engager to untransduced T cells was observed, as well as minimal binding of VHH-muIL2, a control construct in which the BCMA ectodomain is replaced with an irrelevant nanobody (VHH) in the CAR engager CH3-muIL2 construct. This observation further suggests that binding of CAR engagers to CAR T cells is primarily driven by the BCMA antigen, and that muIL2 exhibits weak binding to both CAR T cells and non-transduced T cells. The BCMA-muIL2 CAR engager did not exhibit binding to any immune cell populations in human peripheral blood mononuclear cells (PBMCs) (Figures 13A-13B).

[0225] Next, we evaluated the functional effect of BCMA CAR engagers on BCMA CAR T cells. After a 24-hour cytokine-free resting period, BCMA CAR T cells were incubated with various concentrations of BCMA-muIL2 CAR engager for 24 hours, and then flow cytometry analysis was used to assess the expression of the CD69 activation marker (Cibrian and Sanchez-Madrid, Eur. J. Immunol. 47(6):946-953 (2017)). Results showed a dose-dependent and selective increase in CD69 expression on CAR T cells (Figure 2F), whereas no effect was observed on untransduced T cells (Figure 2G). Furthermore, BCMA CAR engager treatment resulted in a significantly higher increase in CD69 expression compared with VHH-muIL2, BCMA-CH3, or their combination, suggesting that the observed effect was only evident when low-affinity IL-2 was fused to the antigen. An unpaired t-test showed a statistically significant (P ≤ 0.0001) increase in activation in the BCMA-muIL2-treated group compared with the VHH-muIL2, BCMA-CH3, or combination control groups at concentrations of 0.1 nM or higher (error bars represent the mean with 95% confidence intervals) (Figure 2F). Zero treatment and CD3 / CD28 activation in Figure 2G were used as negative and positive controls, respectively (error bars represent the mean with 95% confidence intervals).

[0226] BCMA CAR-E does not inhibit the killing efficacy of BCMA CAR T cells. Because both CAR engagers and cancer antigens bind to CARs, we investigated the potential inhibitory effect of BCMA CAR engagers on the killing activity of BCMA CAR T cells. To investigate, we used BCMA CAR T cells and patient-derived BCMA +A killing assay was performed using OPM2 cancer cells in the presence of various concentrations of BCMA CAR engager. Notably, the results showed no inhibition of killing even at the highest tested concentration (100 nM CAR engager) (Figure 2H). OPM2 cells were co-incubated with BCMA CAR T cells (shown in red) or non-transduced T cells (shown in gray) in the presence of various concentrations of BCMA-muIL2 CAR-E treatment (E:T ratio 1:1; 30,000 cells each). After 48 hours, viable (PI) cells were counted, with N=3 for each experiment. - ) OPM2 cells were counted. The error bars in Figure 2H represent the mean with standard deviation. Without being bound by theory, this finding may be due to the reversibility of CAR engager binding to the CAR, whereas the killing process involving clustering and synapse formation between the CAR and the cancer antigen is an irreversible event. Furthermore, the binding affinity of the CAR to membrane-bound BCMA exceeds that of the CAR to soluble antigen, which may contribute to this result. In particular, multiple myeloma patients exhibit high levels of soluble BCMA in their circulation due to shedding caused by γ-secretase (Laurent et al., Nat. Commun. 6:7333 1-12 (2015)). Despite this, BCMA CAR T cells generated a significant initial response in patients, suggesting that soluble BCMA antigen does not inhibit CAR T cell activity. The experimental findings disclosed herein are consistent with these previous findings.

[0227] BCMA CAR engagers selectively induce STAT5 activity in CAR T cells through in cis delivery of low-affinity IL-2 to the same target CAR T cells. IL-2 is known to exhibit potent activity against T cells, and STAT5 phosphorylation serves as a reliable indicator of IL-2 / IL-2R association 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 effect of BCMA CAR engagers on STAT5 activity, BCMA CAR T cells were exposed to various concentrations of BCMA CAR engagers. After 5 minutes of incubation at 37°C, cells were fixed and stained for pY694 STAT5. Results showed that the BCMA-muIL2 CAR engager induced an EC5 activity of approximately 0.014 nM in CAR T cells. 50 We found that VHH-muIL2 induced STAT5 phosphorylation at 1000 uM (Figure 2I). In contrast, the VHH-muIL2 control required a higher concentration to induce STAT5 phosphorylation (EC 50 =3.9 nM), indicating that BCMA-mediated delivery of low-affinity IL-2 to the surface of CAR T cells significantly enhances muIL-2 sensitivity by over 200-fold. Wild-type IL-2 exhibits a lower EC 50 (approximately 0.001 nM), suggesting differences in signaling kinetics. The two-step process involved in STAT5 activation mediated by the BCMA-muIL2 CAR engager involves (i) binding of the CAR to the antigen on the T cell surface and (ii) subsequent interaction of low-affinity IL-2 with the 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 the IL-2R, allowing it to more rapidly induce STAT5 activity. VHH-muIL2 can activate STAT5 only through low-affinity IL-2, which may explain the higher concentration required to induce STAT5 activity in T cells.

[0228] BCMA CAR T cells pre-blocked with BCMA-CH3 showed a significant reduction in pSTAT5 levels to the same extent as the control VHH-muIL2, verifying that CAR engager efficacy is mediated by antigen-CAR binding (Figure 2I). To determine whether CAR engager binding to target cells could result in STAT5 signaling (transactivation) in neighboring cells, unblocked and pre-blocked BCMA CAR T cells were co-cultured in the presence of various concentrations of CAR engager. Pre-blocked CAR T cells had lower pSTAT5 levels compared to their co-cultured unblocked CAR T cells, indicating that CAR engagers affect targeted CAR T cells (cisactivation) but not neighboring cells. BCMA CAR T cells were treated with the indicated treatments for 5 minutes at 37°C, followed by STAT5 phosphorylation assessment. For pre-blocking experiments, BCMA CAR T cells were treated with BCMA-CH3 (100 nM) for 20 min at 4°C before being exposed to CAR engager treatment for 5 min at 37°C (n=3 for each condition). Error bars in Figure 2I represent the mean with standard deviation. Taken together, analysis of STAT5 activity supports the notion that BCMA CAR engagers exert their influence on targeted CAR T cells via cis delivery of low-affinity IL-2, an effect mediated through antigen binding to the CAR.

[0229] Example 3: CAR engager immune cell effector domains stimulate T cells independently of the CAR. To demonstrate that CAR engager immune cell effector domains stimulate immune cells, the following experiment, the experimental setup of which is illustrated in Figure 3A, was performed in which peripheral blood mononuclear cells (PBMCs) were stimulated with anti-CD3, anti-CD28, IL-2, IL-7, and IL-15 to generate activated T cells that were not transduced with an exogenous transgene. Activated T cells were treated for 4 days with teceleukin (glycosyl-free recombinant human IL-2), a CAR engager containing an N-terminal ectodomain that binds BCMA (approximately 7 kDa), a CH3 domain (approximately 14 kDa), and two repeats of a weak-affinity mutant of the IL-2 immune cell effector domain, with an overall structure of BCMA-CH3-muIL2-muIL2, herein referred to as BCMA-muIL2, or a CAR engager containing an ectodomain that binds BCMA, a CH3 domain, and a Neo-2 / 15 immune cell effector domain, with an overall structure of BCMA-CH3-Neo-2 / 15, herein referred to as BCMA-Neo-2 / 15. After treatment, T cells were counted, stained with carboxyfluorescein succinimidyl ester (CFSE), and analyzed for mean fluorescence intensity (MFI) of CFSE to determine T cell division.

[0230] T cells treated with the CAR engagers BCMA-muIL2 or BCMA-Neo-2 / 15 resulted in T cell counts and division (CFSE staining) similar to those treated with teceleukin, which is known to activate T cells, but CAR engager treatment resulted in lower sensitivity compared to teceleukin treatment. Systemic administration of IL-2 is associated with severe side effects (see Rosenberg, J. Immunol. 192(12):5451-5458 (2014); Dutcher et al., J. Immunother. Cancer. 2(1):26 1-23 (2014); Pachella et al., J. Adv. Pract. Oncol. 6(3):212-221 (2015)), known to result in immunosuppression, including vascular leak syndrome and CD4 + CD25 +This includes preferential expansion of regulatory T (Treg) cells. The results of the present disclosure show that CAR engagers, when used at low concentrations, do not activate normal T cells, and that when stimulatory immune cell effector domains are bound to the ectodomain of the CAR engager to activate T cells, they retain their normal function.

[0231] Example 4: CAR engagers specifically activate CAR T cells via the ectodomain To demonstrate that the CAR engager immune cell effector domain stimulates immune cells, the following experiment was performed. As shown in Figure 4A, PBMCs were stimulated with anti-CD3, anti-CD28, IL-2, IL-7, and IL-15 to generate activated T cells, which were then transduced with a vector containing a CAR. Activated CAR-expressing T cells (CAR T cells) were rested for 24 hours and then treated with a CAR engager containing the immune cell effector domain or a CAR engager lacking the immune cell effector domain as an ectodomain control.

[0232] CAR engagers containing the BCMA ectodomain, CH3 domain, and either 4-1BBL (BCMA-41BBL), weak-affinity IL-2 (BCMA-muIL2), or Neo-2 / 15 (BCMA-Neo-2 / 15) immune cell effector domain were tested for T cell activation. To test the ectodomain specificity of the CAR engagers for CAR T cells, as a control, an irrelevant nanobody binding to the intracellular protein UBC6E (VHH6E) fused to the CH3 domain, and either 4-1BBL (VHH6E-41BBL) or weak-affinity IL-2 (VHH6E-muIL2) were tested for T cell activation. Additional ectodomain-specificity controls, including nanobodies binding to FN1 fused to the CH3 domain (clone NJB2, abbreviated as NJB2-VHH), and Neo-2 / 15 stimulatory (NJB2-VHH-Neo-2 / 15), were tested for T cell activation. The ectodomain-specificity controls have a similar overall structure to the CAR engagers used in this experiment (protein domain-CH3-muIL2-muIL2 or protein domain-CH3-Neo-2 / 15). The ectodomain-specificity controls and CAR engagers were incubated with CAR T cells for 10 hours, and cells were stained for CD69 as an activation marker and measured by flow cytometry.

[0233] All of the BCMA ectodomain CAR engagers induced CD69 expression in CAR T cells (Figure 4B). BCMA-CH3-Neo-2 / 15 had the lowest threshold for induced expression of CD69 in CAR T cells (0.01 nM CAR engager). BCMA-CH3 protein (lacking the immune cell effector domain) had the least effect on CAR T cell expression of CD69 at the highest concentration tested, 10 nM BCMA-CH3 protein. None of the ectodomain-specific control proteins induced CAR T cell expression of CD69. These results indicate that CAR engagers containing a cancer antigen ectodomain specifically activate CAR T cells expressing a CAR that recognizes the cancer antigen ectodomain of the CAR engager.

[0234] Example 5: CAR engagers stimulate CAR T cells to kill target cells To demonstrate that CAR engagers do not inhibit CAR T1 cell killing, the following experiment was performed. CAR T cells were generated as described above and treated with CAR engagers and BCMA + CAR T cells were co-incubated with multiple myeloma cancer cells. OPM2 BCMA CAR T cells were co-incubated with multiple myeloma cancer cells at an E:T ratio of 1:1. + The cells were incubated for 1 day and analyzed for target cell survival compared to target cells without T cell co-incubation (Figure 5A).

[0235] BCMA ectodomain CAR engagers bearing either the muIL2 (BCMA-CH3-muIL2) or 4-1BBL (BCMA-CH3-41BBL) immune cell effector domains did not inhibit killing of OPM2 cells (Figure 5B). These results indicate that CAR engagers 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 engager. Example 6: CAR engagers reduce tumor burden, extend survival, and prolong CAR T cell persistence in vivo

[0236] To demonstrate that CAR engagers reduce tumor burden, extend CAR T cell in vivo persistence, and prolong survival, the following experiment was performed. 6 OPM2 BCMA + Multiple myeloma cancer cells were transfected with NOD-scid IL2Rγ null (NSG) mice were intravenously (iv) injected with 5 × 10 mAb and, 10 days later, administered a suboptimal dose of 5 × 10 mAb by i.v. injection. 5anti-BCMA CAR T cells were injected into mice. After CAR T cell infusion, mice were treated twice weekly for 2 weeks, followed by weekly treatment with 200 μg / mouse of a CAR engager containing the BCMA ectodomain, CH3 domain, and two low-affinity IL-2 immune cell effector domains (BCMA-CH3-muIL2-muIL2) via intraperitoneal (ip) injection (Figure 6A).

[0237] Mice were subjected to bioluminescence imaging (BLI) for luciferase (indicating tumor burden of luciferase+ OPM2 cells) on the days indicated in Figures 6B-6D. Control mice administered OPM2 cells but not CAR T cell infusions developed progressively larger tumor burdens throughout the experiment, reaching the humane endpoint on days 39 and 46. Mice administered OPM2 cells and a suboptimal dose of CAR T cells controlled tumor growth until day 32, where they also developed progressively larger tumor burdens throughout the experiment, reaching the humane endpoint on day 46. Mice receiving OPM2 cells, CAR T cells, and CAR engager therapy exhibited reduced tumor burden (Figures 6B-6D). In the CAR engager-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 proliferation due to the formation of a solid tumor near the eye and reached the humane endpoint on day 42. The remaining two mice had complete elimination of OPM2 tumor cells and survived the experiment, as no signal was detected by imaging.

[0238] Next, the in vivo persistence of OPM2 and CAR T cells was analyzed in these mice by flow cytometry. One control mouse (OPM2 cells without 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 engager-treated mouse was sacrificed on day 42. Sacrificed mice were analyzed for GFP+ OPM2 cells in the blood, spleen, lymph nodes, bone marrow, and lungs (Figures 7A-7C) and CD45 + CAR +T cells (Figures 8A-8C). CAR T cell and CAR engager treated mice were analyzed for GFP+ OPM2 cells and CD45+ CAR T cells in the ocular tumor site. + T cells were also analyzed.

[0239] 7A-7C show flow cytometry with GFP on the y-axis. + OPM2 cells were detected at similar levels in the bone marrow, lungs (Figure 7B), and liver (Figure 7C) of control mice without CAR and CAR-only mice. One CAR-only mouse had significant levels of OPM2 cells in the blood and spleen (Figure 7A).

[0240] One mouse that received CAR T cell and CAR engager treatment and developed an eye tumor had no or few OPM2 cells in the blood or spleen (Figure 7A), bone marrow or lungs (Figure 7B), and liver (Figure 7C). This mouse had more OPM2 cells in the kidney (GFP + 3.18% of the cells, and the majority of the cells in the ocular tumor site were OPM2 cells (GFP + 96.5% of cells.

[0241] Figures 8A-8C show anti-CD45 on the y-axis and Alexa Flour on the x-axis. TM BCMA tagged with 647 (AF647) + Flow cytometry using CD45-CH3. + CAR + T cells persisted only in CAR T cell and CAR engager treated mice. Mice treated with CAR alone retained CD45 T cells in all organs examined. + However, CAR T cell + CAR engager treated mice also stained positive for CD45 cells tagged with AF647 (which is also the CAR binding target), as shown on the x-axis. + The cells had CD45 + AF647 +Double-positive CAR T cells were detected in the blood, spleen, and lymph nodes (Figure 8A), bone marrow and lungs (Figure 8B), and liver and kidneys (Figure 8C). + Single-positive cells were detected in large numbers only in the liver and kidney (Fig. 8C). + AF647 + Double-positive CAR T cells were barely detectable at the ocular tumor site (Figure 8C). These results indicated that CAR engagers reduced tumor burden, prolonged CAR T cell in vivo persistence, and prolonged survival.

[0242] Example 7: Fate of CAR Engagers CAR engagers bind to CAR T cells on the cell surface at 4°C and are slowly internalized at 37°C. CAR engager internalization was assessed using a fluorescently labeled BCMA-muIL2 CAR engager. BCMA CAR T cells were exposed to AlexaFluor647-labeled BCMA-muIL2, BCMA-CH3, or VHH-muIL2 at a concentration of 2 nM. Cells were incubated at either 4°C or 37°C for various time intervals, followed by fixation and subsequent microscopic imaging. While control VHH-muIL2 underwent rapid internalization within 30 minutes at 37°C, internalization of the BCMA-muIL2 CAR engager was observed to be significantly slower (Figures 14A-14C). The internalization rate of BCMA-CH3 was similarly slow, even slower than that of the BCMA-muIL2 CAR engager. In Figure 2C, the CAR and dsRed transcripts are encoded within a transgene; therefore, the dsRed signal reflects the expression level of CAR. All imaged cells with an average intensity higher than background were reported. For the dsRed channel, the cytoplasmic average intensity was reported because dsRed is expressed intracellularly, while for the AlexaFluor 647 channel, the average intensity for the whole cell was measured.

[0243] CAR engagers are rapidly cleared from the circulation. Pulsing CAR T cells with CAR engager treatment is superior to prolonged exposure to CAR engagers, as prolonged exposure can result in the generation of exhausted or terminally differentiated CAR T cells if the pulse includes a stimulation period followed by a resting period. CAR engagers with a short circulating half-life may be more effective in expanding CAR T cells, driving the generation of memory CAR T cells, reducing potential competition with tumor antigens for CAR binding, and enhancing the safety profile in patients. Therefore, the CH3 domain of IgG1 was used in the CAR engager platform. Pharmacokinetic studies showed that the circulating half-life of the CAR engager was short (1–1.5 h) (Figure 9A). NSG mice were administered 8 mg / kg of BCMA-muIL2 CAR engager (ip delivery, N = 3 mice). Blood samples were collected via tail vein puncture at five different time points after administration (30 min, 2 h, 8 h, 24 h, and 48 h). Serum was then obtained by centrifugation and used for subsequent analysis. ELISA was performed to determine the concentration of the treatment agent in the serum. ELISA plates were coated overnight with 5 μg / ml anti-His6 antibody and subsequently incubated with the serum for 2 hours at room temperature. Anti-FLAG HRP antibody was then used for detection; the CAR engager was engineered to have FLAG and His6 tags at the C-terminus. Based on the five time points collected, the initial concentration of treatment in the serum was estimated to be 20% higher than the first (30 min) collection time point. BCMA CAR-E was cleared from the circulation by >90% and >99% at 8 and 24 hours, respectively. The circulating half-life was estimated to be approximately 1.5 hours for the BCMA CAR engager. Error bars represent the mean with standard deviation.

[0244] BCMA CAR engagers enhance CAR T cell activity and persistence in a multiple myeloma (MM) model: immunodeficient NOD-SCID IL-2Rγ nullWe utilized a MM xenograft mouse model with OPM2 cells implanted into nonsteroidal anti-cancer (NSG) mice. Therefore, NSG mice were intravenously injected with OPM2 cells (human MM, 1 million cells) via the tail vein. Two weeks after OPM2 cell injection, we injected 0.5 million CAR T cells containing freshly prepared BCMA CAR T cells (41BB-CD3ζ CAR construct). + BCMA-muIL2 CAR-E cells were administered intravenously. Cohorts of mice received BCMA-muIL2 CAR engager treatment (Figure 9B). NSG mice (n = 5) were injected with OPM2 (human MM) cells, followed by BCMA CAR (human) T cells according to the schedule. BCMA-muIL2 CAR-E treatment (200 μg) was administered twice weekly for 2 weeks, then once weekly until endpoint. After more than one month, mice were euthanized, and flow cytometry analysis was performed on harvested organs. These results revealed significant expansion of CAR T cells in the spleen and bone marrow of the BCMA-muIL2 CAR engager-treated group, demonstrating a more than 100-fold selective expansion of CAR T cells in the spleen (Figure 9C, left panel) and bone marrow (Figure 9C, right panel) compared to untreated animals that received CAR T cells alone. These experiments were repeated multiple times with similar results (Figure 9D and Figures 15A-15C; n=12 for CAR T cells only, n=22 for CAR T cells + CAR engager treatment).

[0245] BCMA CAR T cells were detected by co-staining with anti-human CD45 antibody and Alexa647-labeled BCMA antigen. Similar results were obtained in repeated experiments. An additional control cohort received VHH-muIL2 treatment at the same dose and schedule as BCMA-muIL2. Figure 9D shows pooled data from these experiments. Data were analyzed by group mean comparison using one-way ANOVA followed by Tukey's post-hoc analysis. Individual flow graphs for the pooled data are shown in Figures 15A-15C. Error bars represent the mean with standard deviation.

[0246] The control group (n=7) receiving CAR T cell + VHH-muIL2 treatment did not demonstrate significant proliferation or persistence of CAR T cells compared to the control group receiving CAR T cells alone without treatment. These results indicate that the BCMA-muIL2 CAR engager can expand CAR T cells in vivo. Further analysis revealed that BCMA-muIL2 treatment enhanced the proliferation of CD8 + It has a more pronounced effect specifically on CAR T cells, resulting in their proportion being higher than the total CD4 + and CD8 + An unexpectedly significant increase in the CAR T cell population from an initial ∼30% to ∼70% was revealed (Figure 9E). Cohorts receiving CAR T cells alone or CAR T cells with VHH-muIL2 control treatment did not yield sufficient numbers of persistent CAR cells for similar analysis. Data were analyzed by group mean comparison using one-way ANOVA followed by Tukey's post hoc analysis. Error bars represent the mean with standard deviation.

[0247] BCMA CAR engagers enhance CAR T cell delivery in MM models. We utilized an MM xenograft mouse model with OPM2 cells implanted into immunodeficient NSG mice. Therefore, 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 T cells containing the 41BB-CD3ζ CAR construct) were injected into the mice. +Cohorts of mice received BCMA-muIL2 CAR engager treatment (Figure 20A). NSG mice (n=5) were injected with OPM2 (human MM) cells, followed by BCMA CAR (human) T cells according to the schedule. BCMA-muIL2 CAR-E treatment (200 μg) was administered twice weekly for 2 weeks, then once weekly until endpoint. After more than a month, mice were euthanized, and flow cytometry analysis was performed on harvested organs. These results (Figure 20A) revealed significant CAR T cell trafficking to the spleen, bone marrow, liver, kidney, and lung in the BCMA-muIL2 CAR engager-treated group, demonstrating the presence and persistence of CAR T cells in all major organs examined compared to untreated animals receiving CAR T cells alone (Figure 20B).

[0248] Example 8: BCMA CAR engager treatment enables CAR T therapy with low doses of CAR T cells To further demonstrate the efficacy of CAR engager treatment and tumor cell clearance by CAR T cells, we performed a similar protocol to that described above. However, this study utilized a lower dose of only 100,000 CAR T cells (Figure 10A). While all mice treated with the BCMA-muIL2 CAR engager achieved complete tumor clearance (5 / 5), a single control mouse receiving either CAR T cells alone (n=4) or CAR T cells combined with VHH-muIL2 treatment (n=4) failed to eliminate tumors (Figures 10B-10C).

[0249] Analysis of blood samples collected at various time points revealed substantial expansion of CAR T cells in the circulation after CAR engager treatment, with peak expansion observed at week 4 (Figure 10D). Flow cytometry analysis of blood samples revealed robust expansion of CAR T cells in the treatment group compared with the PBS or VHH-muIL2 cohorts. In contrast, VHH-muIL2 treatment, despite slightly enhancing the initial response, did not induce significant expansion of CAR T cells. Data were analyzed by two-way ANOVA on days 7, 14, and 21. After all mice in the PBS cohort were euthanized, comparisons of BCMA-muIL2 and VHH-muIL2 were performed on days 28 and 35 using multiple Mann-Whitney tests. Error bars represent means with SEM. This expansion correlated with the levels of IFN-γ detected in the circulation (Figures 17A-17C). Furthermore, treatment promoted the generation of memory CAR T cells and demonstrated long-lasting effects (Figure 10E and Figures 17A-17C). *P<0.05, **P<0.01. Error bars represent the mean with SEM.

[0250] Mice treated with CAR engagers showed no signs of toxicity based on clinical observation and weight measurements (Figure 10H). Subsequent analysis performed 2 months after CAR T cell injection demonstrated substantial presence of CAR T cells, including memory CAR T cells, in CAR engager-treated mice (Figures 10F-10J, Figures 16A-16C, and Figures 17A-17C). In the CAR+PBS group, CAR T cells were detected in the spleen, but these mice died due to tumor growth approximately 20 days after CAR T cell injection. BCMA-muIL2 treatment also increased the presence of CAR T cells in the bone marrow compared to the PBS or VHH-muIL2 cohorts, although the difference was not significant compared to the spleen. Data were analyzed by two-way ANOVA with Tukey's multiple comparison test. *P<0.05, ***P<0.001, ****P<0.0001. Individual flow data are shown in Figures 16A-16C. Error bars represent the mean with SEM. Data in Figure 10I demonstrate that persistent CAR T cells in the spleens of BCMA-muIL2-treated mice were CCR7 T cells that were not present in the CAR+VHH-muIL2 or CAR+PBS cohorts. + CD45RA + CD62L + Showing that they exhibited a stem cell memory phenotype.

[0251] tSNE analysis revealed the presence of distinct memory T cell populations based on surface marker expression of CD8α, CD4, CD45, CD45RA, CD45RO, CD62L, CD69, PD-1, HLA-DR, CCR7, and BCMA-CAR. CAR T cells were detected in the bone marrow but not in the spleen of the VHH-muIL2-treated group. In the BCMA-muIL2 group, the remaining CAR T cells were primarily CD8 T cells, whereas the majority of bone marrow CAR T cells in the VHH-muIL2 group were CD4 T cells. Further analysis is shown in Figures 17A-17C. The Flt-SNE mapping shown in Figure 17C is of CAR T cells derived from mice treated with PBS, BCMA-muIL2, and VHH-muIL2 as shown in Figure 10A. The expression of 10 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 three PBS mice, three BCMA-muIL2 mice, and four VHH-muIL2 mice was analyzed by flow cytometry. + α-BCMA-CAR +Immune cells were concatenated to form a total of approximately 9,800 (PBS spleen), 8,100 (BCMA-muIL2 spleen), 7,600 (PBS bone marrow), 14,200 (BCMA-muIL2 bone marrow), and 1,420 (VHH-muIL2 bone marrow). The entire high-dimensional dataset was merged to generate a single Flt-SNE map for each condition, with the signal intensities of various phenotypic markers defining a specific immune phenotype represented on a continuous color scale of blue-green-yellow-red. Flt-SNE was performed using the following parameters: maximum iterations: 1,000, theta: 0.5, learning rate: 200, perplexity: 20. In the spleens of the VHH-muIL2 cohort, there were insufficient numbers of CAR T cells to perform Flt-SNE. To improve visibility, the dots representing the VHH-muIL2 bone marrow samples were enlarged, as fewer cells were detectable in these mice. In BCMA-muIL2-treated mice, CAR T cells were significantly more abundant than in the spleens. + The majority of cells are CD8 + cells, whereas the VHH-muIL2 sample showed CD4 + The cells are CAR + In particular, the CAR+PBS cohort comprised the majority of CAR cells. + Although BCMA-muIL2-treated mice showed low or no expression of CD45RA, CD45RO, or CD62L, BCMA-muIL2-treated mice showed elevated levels of these memory markers. + Showed the group

[0252] Thus, treatment not only promoted robust growth and eradication of tumor cells using low doses of CAR T cells, but also promoted the development of long-lasting memory cells, demonstrating the efficacy of BCMA-muIL2 CAR engager treatment in enhancing tumor cell clearance and the generation of long-lasting memory cells by CAR T cells.

[0253] Example 9: Persistent CAR T cells treated with CAR engager treatment remain functional for 3 months after infusion Mice received 1 million OPM2 cells followed by 0.5 million BCMA CAR T cells (Figure 11A). One group of mice received CAR engager treatment, administered twice weekly for 2 weeks, followed by once weekly for another 2 weeks (6 doses, 200 μg per dose on days 4, 10, 14, 17, 21, and 28; n = 5). A control group received VHH-muIL2 treatment at the same dose and schedule (n = 5), while an additional control cohort received tumor cells alone (n = 3). All mice receiving CAR T cells demonstrated an early response compared with control mice without CAR T cells (Figure 11B). All mice treated with CAR engager (5 of 5) and 3 of 5 mice in the VHH-muIL2 group survived for more than 3 months, including the duration of the experiment. One VHH-muIL2 mouse died within approximately one month, and the second mouse died of cancer cell recurrence with liver metastasis (Figure 11B, day 77). Surviving mice were euthanized 3 months after CAR T cell injection, and splenocytes and bone marrow cells were analyzed to assess the presence of CAR T cells. Notably, CAR engager-treated mice showed a significant abundance of CAR T cells that homed to and persisted in the bone marrow and spleen compared with mice that received CAR T cells with VHH-muIL2 treatment (Figure 11C). Considering the two-month treatment-free period before mice were sacrificed, these results further suggest that the treatment promoted the generation of memory cells among CAR T cells.

[0254] To demonstrate sustained CAR T cell functionality in CAR engager-treated mice, in vitro killing assays were performed using BCMA CAR T cells harvested from bone marrow and spleen. Bone marrow and splenocytes were analyzed by flow cytometry to detect and quantify CAR-expressing T cells. Bone marrow or splenocytes were co-incubated with OPM2 target cells at various E:T ratios (1:1 and 2:1) based on the CAR-expressing cells. Survival was determined after 24, 48, and 72 hours using flow cytometry analysis. Three-month-old CAR T cells demonstrated efficient tumor cell killing and long-term functionality (Figure 11D). Only one of the VHH-muIL2-treated mice exhibited sufficient CAR T cells to perform a similar killing assay and demonstrated tumor cell killing, although the efficiency was lower than that observed with CAR engager-treated CAR T cells (Figure 11D) (error bars represent the mean with standard deviation). Thus, CAR engager treatment robustly expands and promotes the persistence of CAR T cells while maintaining their killing capacity.

[0255] To further characterize the phenotype of these persistent CAR T cells, flow cytometry analysis was performed to assess the expression of a panel of T cell markers (CD45, BCMA CAR, CD4, CD8, CD62L, CD45RO, CD45RA, CD69, and PD-1). To facilitate interpretation, t-SNE mapping of splenocytes and bone marrow cells was performed (Figure 11E). Splenocytes and bone marrow from five BCMA-muIL2-treated mice were analyzed by flow cytometry using anti-CD45-Pacific Blue, anti-CD8-FITC, anti-CD4-PE Dazzle594, BCMA (antigen)-AlexaFluor647, anti-CD69-BV421, anti-PD-1-BV605, anti-CD45RA-APC-Cy7, anti-CD45RO-PerCP-Cy5.5, anti-CD62L-PE, and CCR7-AlexaFluor700). CD45 from five mice was analyzed by flow cytometry. + , CD8 + α-BCMA-CAR +Cells were concatenated to form a total of approximately 17,600 (spleen) cells and approximately 10,800 (bone marrow) cells. The entire high-dimensional dataset (excluding CD45, CD8, and CD4 parameters) was merged to create a single tSNE map with signal intensities of six phenotypic markers defining specific immune phenotypes represented on a continuous blue-green-yellow-red color scale. tSNE analysis was performed using 1,000 iterations, a perplexity of 30, and learning rates of 1,237 and 756 for spleen and bone marrow, respectively. The population labeled 1 appears to exhibit a memory-like phenotype and expresses higher levels of CD45RO, CD62L, and CD45RA. The population labeled 2 appears to exhibit an effector-like phenotype, expressing lower levels of CD45RO, CD62L, and CD45RA.

[0256] Flow cytometry analysis demonstrated that persistent CAR T cells express CD4 + Populations and CD8 + It was revealed that the CD8 + CAR cells consist of two distinct populations: effector cells and CD45RA + CD62L + These appeared to represent memory cells. The memory population showed higher expression levels of BCMA CAR and CD45 (Figure 11E). Similarly, CD4 + CAR T cells exhibited two populations: effector and memory cells (Figure 18). Insufficient numbers of CAR T cells were detected in VHH-muIL2-treated mice to perform similar flow cytometry analysis. Thus, CAR engager treatment results in the generation of long-lasting memory CAR T cells.

[0257] As shown in Figure 18, Figures 11A-11E, CD4 + CAR +t-SNE mapping of T cells. Expression of nine immune cell markers (aCD45-Pacific Blue, aCD8-FITC, aCD4-PE Dazzle594, BCMA (antigen)-AlexaFluor647, aCD69-BV421, aPD-1-BV605, aCD45RA-APC-Cy7, aCD45RO-PerCP-Cy5.5, aCD62L-PE) on splenocytes and bone marrow from five BCMA-muIL2-treated mice was analyzed by flow cytometry. + , CD4 + α-BCMA-CAR + Immune cells were concatenated to form a total of approximately 9,000 (spleen) cells and approximately 6,600 (bone marrow) cells. The entire high-dimensional dataset (excluding CD45, CD8, and CD4 parameters) was merged to generate a single t-SNE map with signal intensities of six phenotypic markers defining specific immune phenotypes represented on a continuous blue-green-yellow-red color scale. tSNE analysis was performed using 1,000 iterations, a perplexity of 30, and learning rates of 630 and 466 for spleen and bone marrow, respectively. The population labeled "1" appears to exhibit a memory-like phenotype and expresses higher levels of CD45RO, CD62L, and CD45RA. The population labeled "2" appears to exhibit an effector-like phenotype and expresses lower levels of CD45RO, CD62L, and CD45RA.

[0258] CARs isolated from mice treated with either BCMA-muIL2 or VHH-muIL2 control + Single-cell RNA sequencing (scRNAseq) analysis was performed on T cells. Although the presence of CAR T cells in VHH-muIL2-treated mice was limited, sufficient numbers of cells were obtained from one of the VHH-treated mice for experiments (Figure 19A). +Cells were sorted after staining with BCMA-AlexaFluor647 and TotalSeq-C hashing antibodies from BCMA-muIL2 or VHH-muIL2 treated mice as shown in red and green boxes, respectively. Figure 19A. 5000 CARs from BCMA-muIL2 mouse bone marrow and spleen. + cells, and 2500 CARs derived from VHH-muIL2 mouse bone marrow and spleen + Cells were loaded onto the 10X channel. scRNAseq analysis demonstrated that the predominant population of persistent CAR T cells in BCMA-muIL2-treated mice was CD8 + The CD8 and CD4 T cells consisted of BCMA-muIL2-treated mice (Figures 19B-19C), which revealed enrichment for genes associated with an activated T cell state (Figures 19D-19E). The heatmap in Figure 19D shows genes that were significantly differentially expressed between CAR engager-treated and VHH conditions in CD8 and CD4 CAR T cells, separated by the different associated conditions. Genes marked with an * are genes that were significantly differentially expressed between BCMA-muIL2-treated and VHH-muIL2-treated mice in the subset of interest. This was evidenced by elevated expression levels of granzyme family genes, other cytotoxicity-related genes, and MHC class II genes. Because the mice had already cleared their tumors more than 60 days prior, no significant differences in activation markers were observed between CAR T cells obtained from BCMA-muIL2- or VHH-muIL2-treated mice. BCMA-muIL2 treatment did not induce upregulation of exhaustion markers, indicating that the treatment did not induce exhaustion in persistent CAR T cells.

[0259] Next, T cell receptor (TCR) clonotype diversity was assessed in BCMA-muIL2- and VHH-muIL2-treated mice (Figures 19F-19G). Both groups showed similar diversity in the clonotypes present, indicating that BCMA-muIL2 CAR engager treatment can effectively promote the generation of a diverse TCR repertoire in persistent CAR T cells, as opposed to promoting the dominance of a limited set of TCR clones. The pie plot in Figure 19F shows TCR clonotype diversity, with each slice of the pie chart representing the proportion of different TCR clonotypes present. Colors were randomly assigned to different clonotypes. Clonotype diversity within the total cell count for each sample was visualized in a stacked bar graph (Figure 19G), where similar clonotypes with counts less than 50 were combined. To assess diversity within each sample, the Simpson index was calculated; higher values ​​indicate greater diversity. Overall, the results showed that BCMA CAR engagers could not only help CAR T cells completely eliminate tumor cells, but also robustly induce the generation of long-lasting and functional memory CAR T cells.

[0260] Example 10: CAR engagers expand CAR T cells in the absence of tumor antigens CAR T cell expansion typically occurs in patients post-infusion, with peak expansion observed approximately 10-14 days post-infusion (Rodriguez-Otero et al., N. Engl. J. Med. 388(11):1002-1014 (2023)).

[0261] Eradication of minimal residual disease (MRD) promotes long-lasting, complete responses. However, the limited presence of corresponding antigens associated with MRD may not adequately support the proliferation and efficacy of conventional CAR T cells. To demonstrate efficacy in the absence of tumor antigens, NSG mice were injected with 250,000 BCMA CAR T cells alone in the absence of tumor cells. These mice received two 25 μg doses of BCMA-muIL2 on days 1 and 8 after CAR T cell injection. The control group received VHH-muIL2 treatment (n=4 per group). On day 30, the mice were euthanized, and their spleens and bone marrow were evaluated for the presence of CAR T cells. BCMA-muIL2-treated mice had higher numbers of CAR T cells in the spleen. + The BCMA CAR engagers expanded CAR T cells in vivo, even in the absence of tumor cells, showing higher numbers of CAR+ T cells in the bone marrow (approximately 6.8-fold higher) and higher numbers of CAR+ T cells in the bone marrow (approximately 5.5-fold higher) (Figures 12A-12B); error bars represent the mean with standard deviation. Overall, these findings demonstrate that CAR engagers can expand CAR T cells even in the absence of tumor antigens. Furthermore, the efficacy of this treatment was evident even at lower doses and frequencies.

[0262] Example 11: CD19 CAR-E does not inhibit the killing effect of CD19 CAR T cells We observed that BCMA-binding CAR engagers bind to BCMA CAR T cells but do not inhibit their killing efficacy (Figures 2D-2H). To examine the effects of CAR-E containing other cancer antigens on antigen-specific CAR T cells, we performed immunohistochemistry using CD19 CAR T cells and patient-derived CD19 CAR T cells in the presence of various concentrations of CD19 CAR engagers. +A killing assay was performed using leukemia cells. Notably, the results showed no inhibition of killing even at the highest tested concentration (1000 nM of CAR engager) (Figure 21C). Nalm6 cells were co-incubated with CD19 CAR T cells (solid) or non-transduced T cells (open) in the presence of various concentrations of CD19-muIL2 CAR-E treatment (E:T ratio 1:1; 30,000 cells each). After 48 hours, viable (PI) cells were detected. - ) Nalm6 cells were counted, and for each experiment the N was 3. The experimental findings disclosed herein with CD19 are consistent with findings in the BCMA cancer models and BCMA CAR-E described above.

[0263] All patent and non-patent publications—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 was specifically and individually indicated to be incorporated by reference.

[0264] 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 can be made to the exemplary embodiments and other arrangements can be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.

Claims

1. 1. A chimeric antigen receptor (CAR) engager protein comprising: a first portion comprising the ectodomain of an antigen present on a cancer cell; a second portion linked to the first portion, the second portion comprising a first immune cell effector domain; CAR engager.

2. The ectodomain is selected from the group consisting of 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 1B (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 2. The CAR engager of claim 1, wherein the CAR engager is derived from E7, IL1RAP, IL3RA, IL13Rα2, KDR, KIT, KLRK1, L1CAM, MAGEA1, MAGEA4, MET, MME, MSLN, MUC1, MUC16, MS4A1, NCAM1, PD-1, PMEL, PROM1, PSCA, ROR1, ROR2, SDC1, SLAM7, TEM1, TROP2, TNF receptor superfamily member (TNFRSF) 8, TNFRSF10B, TNFRSF13C, TNFRSF17, ULBP1, or ULBP2.

3. 3. The CAR engager of claim 2, wherein the ectodomain is derived from BCMA.

4. 4. The CAR engager of claim 3, wherein the ectodomain comprises the amino acid sequence MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA (SEQ ID NO: 1).

5. 5. The CAR engager of claim 4, wherein the ectodomain comprises the amino acid sequence MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAGGGSGGGSPRGSGGGSMLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTN (SEQ ID NO: 2).

6. 3. The CAR engager of claim 2, wherein the ectodomain is derived from CD19.

7. The ectodomain has the amino acid sequence PEEPLVVKVEEGDEAWLPCLKGTSDGPTQQLTWSRESPLKPFLKVSFGVPGLGVHVRPNAVSLVISNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPC 7. The CAR engager of claim 6, comprising amino acids having at least about 85% sequence identity to LPPRDSLNQSLSRDMTVAPGSTLWLSCGVPPDDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVTGTRLFLPRATAQDAGKYYCHRGNLTMSFHLEVKARPVSAHTKLRTGGWK ​​(SEQ ID NO: 3).

8. 8. The CAR engager of claim 7, wherein the ectodomain comprises the amino acid sequence of SEQ ID NO:

3.

9. The ectodomain has the amino acid sequence PEEPLVVKVEEGDEAWLPCLKGTSDGPTQQLTWSRESPLKPFLKVSFGVPGLGVHVRPNAVSLVISQVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWQVSDLGGLGCGLKQRSSEGPSSPSGKL MSPKLYVWAKDRPEIWEGEPPCLPPRDSLQQSLSRDMTVAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVTGTRLFLPRATAQDAGKYYCHRGQLTMSFHLEVKARPVSAHTKLRTGGWK ​​(SEQ ID NO: 4) or PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGF YLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGE 9. The CAR engager of claim 8, comprising: PPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWK ​​(SEQ ID NO: 5).

10. 3. The CAR engager of claim 2, wherein the ectodomain is derived from CD20.

11. 11. The CAR engager of claim 10, wherein the ectodomain comprises the amino acid sequence KISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQS (SEQ ID NO: 6).

12. 3. The CAR engager of claim 2, wherein the ectodomain is derived from SLAMF7.

13. 13. The CAR engager of claim 12, wherein the ectodomain comprises the amino acid sequence SGPVKELVGSVGGAVTFPLKSKVKQVDSIVWTFNTTPLVTIQPEGGTIIVTQNRNRERVDFPDGGYSLKLSKLKKNDSGIYYVGIYSSSLQQPSTQEYVLHVYEHLSKPKVTMGLQSNKNGTCVTNLTCCMEHGEEDVIYTWKALGQAANESHNGSILPISWRWGESDMTFICVARNPVSRNFSSPILARKLCEGAADDPDSSM (SEQ ID NO: 10).

14. The CAR engager of claim 2, wherein the ectodomain is derived from PD-1.

15. 15. The CAR engager of claim 14, wherein the ectodomain comprises the amino acid sequence FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV (SEQ ID NO: 11).

16. 3. The CAR engager of claim 2, wherein the ectodomain is derived from KIT.

17. The ectodomain has the amino acid sequence QPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTN YSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPV VSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARV NDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIY MNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYD 17. The CAR engager of claim 16, comprising: RLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTP (SEQ ID NO: 12).

18. 3. The CAR engager of claim 2, wherein the ectodomain is derived from CD38.

19. The ectodomain has the amino acid sequence: VPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRK 19. The CAR engager of claim 18, comprising DCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSSCTSEI (SEQ ID NO: 14).

20. 3. The CAR engager of claim 2, wherein the ectodomain is derived from CD22.

21. The ectodomain has the amino acid sequence DSSKWVFEHPETLYAWEGACCVWIPCTYRALDGDLESFILFHNPEYNKNTSKFDGTRLYESTKDGKVPSEQKRVQFLGDKNKNCTLSIHPVHLNDSGQLGLRMESKTEKWMERIHLNVSERPFPPPHIQLPPEIQESQEVTLTCLLNFSCYGYPIQLQWLLEGV PMRQAAVTSTSLTIKSVFTRSELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKHTPKLEIKVTPSDAIVREGDSVTMTCEVSSS NPEYTTVSWLKDGTSLKKQNTFTLNLREVTKDQSGKYCCQVSNDVGPGRSEEVFLQVQYAPEPSTVQILHSPAVEGSQVEFLCMSLANPL PTNYTWYHNGKEMQGRTEEKVHIPKILPWHAGTYSCVAENILGTGQRGPGAELDVQYPPKKVTTVIQNPMPIREGDTVTLSCNYNSSNP SVTRYEWKPHGAWEEPSLGVLKIQNVGWDNTTIACAACNSWCSWASPVALNVQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEV 21. The CAR engager of claim 20, comprising: QFFWEKNGRLLGKESQLNFDSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSMSPGDQVMEGKSATLTCESDANPPVSHYTWFDWNNQSLPYHSQKLRLEPVKVQHSGAYWCQGTNSVGKGRSPLSTLTVYYSPETIGRR (SEQ ID NO: 7).

22. 21. The CAR engager of claim 20, wherein the ectodomain comprises the amino acid sequence APRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEVQFFWEKNGRLLGKESQLNFDSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSMSPGDQVMEGKSATLTCESDANPPVSHYTWFDWNNQSLPYHSQKLRLEPVKVQHSGAYWCQGTNSVGKGRSPLSTLTVYYSPETIGRR (SEQ ID NO: 107).

23. 23. The CAR engager of any one of claims 1 to 22, further comprising a first linker connecting the first portion and the second portion.

24. 24. The CAR engager of claim 23, further comprising a dimerization domain disposed between said first linker and said second portion comprising said first immune cell effector domain.

25. 25. The CAR engager of claim 24, further comprising a second linker connecting said dimerization domain and said second moiety, wherein said first linker and said second linker may be the same or different.

26. 26. The CAR engager of claim 25, wherein the first linker and the second linker are flexible.

27. 27. The CAR engager of claim 26, wherein the first linker and / or the second linker is derived from the hinge region of CD3zeta, CD4, CD8α, CD28, IgG1, IgG2, or IgG4.

28. 27. The CAR engager of claim 26, wherein the first linker and / or the second linker comprise 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.

29. 27. The CAR engager of claim 26, 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 GSPRGGGGSGGGGGSGGGGS (SEQ ID NO: 76).

30. 30. The CAR engager of any one of claims 24-29, wherein the dimerization domain is derived from IgA, IgD, IgG, IgM, or IgE.

31. 31. The CAR engager of claim 30, wherein said dimerization domain comprises an IgG1 constant heavy (CH)3 domain.

32. 31. The CAR engager of claim 30, wherein the dimerization domain further comprises an IgG CH2 domain and an IgG CH3 domain.

33. 33. The CAR engager of any one of claims 1-32, wherein the first immune cell effector domain comprises a cytokine or immune cell activating moiety.

34. 34. The CAR engager of claim 33, 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.

35. 34. The CAR engager of any one of claims 1 to 33, wherein the second portion further comprises a plurality of immune cell effector domains.

36. 36. The CAR engager of claim 35, wherein the second portion further comprises a second immune cell effector domain, wherein the first and second immune cell effector domains can be the same or different.

37. 37. The CAR engager of claim 36, wherein said second immune effector domain comprises a weak affinity variant of IL-2 having the amino acid sequence APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 19).

38. the first immune cell effector domain and the second immune cell effector domain each have the amino acid sequence APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL 38. The CAR engager of claim 37, having the sequence: TGGGGSGGGGSGGGGGSGGGGGSAPTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 20).

39. 37. The CAR engager of claim 36, wherein the first immune cell effector domain and the second immune cell effector domain each have the amino acid sequence of SEQ ID NO: 102 with an H16A substitution.

40. 37. The CAR engager of claim 36, wherein the first immune cell effector domain and the second immune cell effector domain each have the amino acid sequence of SEQ ID NO: 102 with an F42A substitution.

41. 37. The CAR engager of claim 36, wherein the first or second immune cell effector domain comprises the amino acid sequence GSHMPKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS (SEQ ID NO: 26).

42. 42. The CAR engager of claim 41 , wherein said first and second immune cell effector domains each comprise the amino acid sequence of SEQ ID NO:

26.

43. 36. The CAR engager of claim 35, wherein the first immune cell effector domain comprises 4-1BBL or an immune cell-activating variant thereof.

44. 44. The CAR engager of claim 43, wherein said first immune cell effector domain comprises the amino acid sequence DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPA (SEQ ID NO: 27).

45. 36. The CAR engager of claim 35, wherein said second portion further comprises a third immune cell effector domain, and any two or more of said first, second, and third immune cell effector domains can be the same or different.

46. 45. The CAR engager of claim 44, wherein each of the first, second, and third immune cell effector domains comprises the amino acid sequence of SEQ ID NO:

27.

47. 34. The CAR engager of claim 33, wherein said first cellular effector domain is a single chain variable antibody fragment (scFv) that binds to and activates an immune cell.

48. 48. The CAR engager of claim 47, wherein said first cell effector domain is 4-1BB, CD2, CD27, CD28, CD30, CD40L, CD226, CTLA4, GITR, IL-2R, LIGHT, OX40, PD-1, TIM2, SLAM, or TIM1.

49. 49. The CAR engager of claim 48, wherein the first cellular effector domain comprises an scFv that binds to CTLA-4.

50. The scFv comprises a VL domain having the amino acid sequence of EIVLTQSPGTLSLSPGERATTLSCRAQSVSRYLGWYQQKPGQAPRLLIYGASTRATGIPDRFSGSGSGTDFTLTITRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 36), and a VL domain having the amino acid sequence of EVQLVESGGGLV 50. The CAR engager of claim 49, comprising a VH domain having the amino acid sequence of KPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLFGPFDIWGQGTLVTVSS (SEQ ID NO: 37).

51. 49. The CAR engager of claim 48, wherein said first cellular effector domain binds to OX40.

52. the scFv comprising a VL domain having the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIK (SEQ ID NO: 42) and a VL domain having the amino acid sequence of EVQLVQSGAEV 52. The CAR engager of claim 51 , comprising a VH domain having the amino acid sequence of KKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVSS (SEQ ID NO: 43).

53. 49. The CAR engager of claim 48, wherein said first cellular effector domain binds to PD-1.

54. The scFv comprises a VL domain having the amino acid sequence of: EIVMTQSPATTLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPRTFGQGTKVEIK (SEQ ID NO: 52) and a VL domain having the amino acid sequence of: QVQLVESGG 54. The CAR engager of claim 53, comprising a VH domain having the amino acid sequence of: GVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASNGDHWGQGTLVTVSS (SEQ ID NO: 53).

55. 55. The CAR engager of any one of claims 1-54, wherein said first immune cell effector domain comprises an immune cell inhibitory moiety.

56. 56. The CAR engager of claim 55, wherein said 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).

57. 57. The CAR engager of claim 56, wherein said first immune cell effector domain is derived from PD-L1.

58. 58. The CAR engager of claim 57, wherein said first immune cell effector domain comprises the amino acid sequence FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER (SEQ ID NO: 64).

59. 57. The CAR engager of claim 56, wherein said first immune cell effector domain is derived from CD80.

60. 60. The CAR engager of claim 59, wherein said first immune cell effector domain comprises the amino acid sequence VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDN (SEQ ID NO: 54).

61. 57. The CAR engager of claim 56, wherein said first immune cell effector domain is derived from CD276 (B7-H3).

62. the first immune cell effector domain has the amino acid sequence LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEV 62. The CAR engager of claim 61 , comprising QVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEA (SEQ ID NO: 57).

63. 57. The CAR engager of claim 56, wherein said first immune cell effector domain is derived from VTCN1 (B7-H4).

64. the first immune cell effector domain has the amino acid sequence LIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLE 64. The CAR engager of claim 63, comprising: YKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKAS (SEQ ID NO: 66).

65. 65. The CAR engager of any one of claims 1 to 64, wherein the CAR engager is in the form of a fusion protein, and the first and second portions are joined by a peptide bond.

66. 65. The CAR engager of any one of claims 1 to 64, wherein the first moiety comprising the ectodomain is linked to the second moiety comprising the first immune cell effector domain or the first linker via an azide-alkyne bond, an oxime or hydrazine bond, a tetrazine-transcyclooctene bond, an azide-nitrone bond, a thiol-alkene bond, an alkene-tetrazole bond, an alkene-tetrazine bond, an alkene-azide bond, a conjugated diene-alkene bond, or an isonitrile-tetrazine bond.

67. 67. The CAR engager of any one of claims 24 to 66, which is in the form of a homodimer comprising two CAR engagers.

68. A heterodimeric CAR engager comprising: a first portion comprising an ectodomain of an antigen present on a cancer cell linked to a first dimerization domain; and a second portion comprising a first immune cell effector domain linked to a second dimerization domain; wherein the first and second dimerization domains dimerize to form a heterodimer. CAR engager.

69. 69. The CAR engager of claim 68, wherein said first dimerization domain comprises a ridge and said second dimerization domain comprises a cavity sterically compensatory to said ridge, said ridge being positionable within said cavity.

70. 66. A nucleic acid encoding the CAR engager of claim 65.

71. A nucleic acid encoding an ectodomain of an antigen present on a cancer cell fused to a first dimerization domain.

72. A nucleic acid encoding an immune cell effector domain fused to a second dimerization domain.

73. 73. The nucleic acid of any one of claims 70 to 72, further comprising a sequence encoding a signal peptide.

74. A vector comprising the nucleic acid of any one of claims 70 to 73.

75. A cell comprising the vector of claim 74.

76. 76. The cell of claim 75, which is a mammalian cell.

77. 77. The cell of claim 76, which is a bacterial cell.

78. 70. A pharmaceutical composition comprising the CAR engager of any one of claims 1 to 69 and a pharmaceutically acceptable carrier.

79. 79. The pharmaceutical composition of claim 78, wherein the cell population comprises an effective number of immune cells comprising a CAR comprising an extracellular domain that binds to the ectodomain of a CAR engager, a transmembrane domain, and an intracellular domain comprising a stimulatory domain.

80. 1. A method of making a CAR engager, comprising: Culturing the cell of any one of claims 75 to 77 in a medium under conditions in which the nucleic acid encoding the CAR engager is expressed; and and isolating the CAR engager from the cell and / or medium.

81. 1. A method of treating cancer comprising: administering to a subject an effective amount of a CAR engager of any one of claims 1-69; wherein prior to, substantially simultaneously with, or subsequent to administration of the CAR engager, the subject is administered an effective number of immune cells comprising a chimeric antigen receptor (CAR) comprising an extracellular domain that binds to the ectodomain of the CAR engager, a transmembrane domain, and an intracellular domain that comprises a stimulatory domain (CAR immune cells); method.

82. 82. The method of claim 81, wherein the first immune cell effector domain of the CAR engager comprises a cytokine or immune cell activating moiety.

83. 82. The method of claim 81, wherein the first immune cell effector domain of the CAR engager comprises an immune cell inhibitory moiety.

84. 84. The method of any one of claims 81-83, wherein the subject is administered the CAR immune cells prior to administration of the CAR engager.

85. 85. The method of claim 84, wherein the CAR immune cells are administered at least about 6 months, at least about 9 months, or at least about 1 year prior to administering the CAR engager.

86. 84. The method of any one of claims 81-83, wherein the CAR engager and the CAR immune cells are administered simultaneously.

87. 87. The method of claim 86, wherein said co-administering comprises administering said CAR immune cells and said CAR engager substantially simultaneously.

88. 87. The method of claim 86, further comprising contacting the CAR engager with the CAR immune cells in vitro prior to administering the CAR engager and immune cells.

89. 87. The method of claim 86, comprising: measuring the concentration of immune cells present in a sample obtained from the subject after administering the immune cells; and calculating the difference between the concentration of the immune cells administered to the subject and the measured concentration of immune cells. method.

90. 90. The method of claim 89, wherein said co-administering comprises administering the CAR engager if the measured CAR immune cell concentration is lower than the administered immune cell concentration.

91. 91. The method of any one of claims 81-90, wherein the CAR immune cell is a T cell or an NK cell.

92. T cells are CD8 ＋ 92. The method of claim 91, wherein the cell is a T cell.

93. The method of any one of claims 81 to 92, wherein the cancer is a hematopoietic cancer.

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

95. 95. The method of claim 94, wherein the cancer is multiple myeloma.

96. 93. The method of any one of claims 81 to 92, wherein the cancer is characterized by a solid tumor.

97. 97. The method of claim 96, wherein the cancer is renal cell carcinoma, breast cancer, ovarian cancer, neuroblastoma, glioblastoma, gliosarcoma, head and neck cancer, hepatocellular carcinoma, cervical cancer, pancreatic cancer, lung cancer, fallopian tube cancer, prostate cancer, kidney cancer, bladder cancer, gastrointestinal cancer, melanoma, colorectal cancer, or esophageal cancer.

98. 98. The method of any one of claims 81-97, further comprising administering high dose chemotherapy to the subject prior to administering the CAR immune cells.

99. 99. The method of claim 98, further comprising administering bone marrow cells or peripheral blood stem cells to the subject.

100. 100. The method of any one of claims 81-99, further comprising administering to the subject an additional active agent comprising one or more of thalidomide, lenalidomide, and bortezomib.

101. The effective number of CAR immune cells is approximately 1 x 10 per kg of subject body weight. ４ ~approximately 6 x 10 ５ The method of any one of claims 81 to 100, wherein the cell is a cell.

102. 102. The method of any one of claims 81 to 101, wherein the subject is in a minimal residual disease state.

103. 1. A chimeric antigen receptor (CAR) engager comprising: a first portion comprising the ectodomain of an antigen linked to a second portion comprising a first immune cell effector domain.

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