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WO2025059162A1 - Car-engager containing il-2 variants to enhance the functionality of car t cells - Google Patents

Car-engager containing il-2 variants to enhance the functionality of car t cells Download PDF

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Publication number
WO2025059162A1
WO2025059162A1 PCT/US2024/046173 US2024046173W WO2025059162A1 WO 2025059162 A1 WO2025059162 A1 WO 2025059162A1 US 2024046173 W US2024046173 W US 2024046173W WO 2025059162 A1 WO2025059162 A1 WO 2025059162A1
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Prior art keywords
car
engager
cells
amino acid
bcma
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French (fr)
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Heydar MORAVEJ
Mohammad RASHIDIAN
Taha RAKHSHANDEHROO
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Dana Farber Cancer Institute Inc
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Dana Farber Cancer Institute Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A61K40/4215—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • IL-2R The cognate IL-2 receptor (IL-2R) includes three subunits, namely IL-2 subunit alpha (IL-2R ⁇ ; CD25), IL-2 subunit beta (IL-2R ⁇ ; CD122), and the IL-2 subunit gamma (IL-2R ⁇ ; CD132).
  • IL-2 may also induce an alternative differentiation pathway of T cells, resulting in the generation of distinct “better” effector CD8 + T cells (Hashimoto et al., Nature 610(7930):173-181 (2022)). This process may rely, at least in part, on IL-2 binding to IL-2R ⁇ . Additionally, IL-2R ⁇ - biased agonists may drive T cells towards a terminally differentiated state (Codarri et al., Nature 610(7930):161-172 (2022)). [0005] Known IL-2 variants may have different affinities for different IL-2R subunits and may offer advantages in connection with therapies.
  • a weak affinity IL-2 (muIL2) that contains the amino acid substitutions of H16A and F42A, relative to wild-type IL-2, has increased selectivity for T cells and less toxicity through reduced global IL-2 binding and activation of all IL- 2R expressing cells.
  • the binding affinity of muIL2 for human IL2R ⁇ and IL2R ⁇ is decreased 110- and 3-fold, respectively, compared with wild-type IL-2 (Quayle et al., Clin. Cancer Res.26(8):1953- 1964 (2020)).
  • Challenges persist with IL-2-based therapies in the clinic, with many clinical trials failing to meet their primary endpoints (Raeber et al., Ebiomedicine 90:104539 pp. 1-25 (2023)).
  • CD19 is a B-cell co-receptor expressed on B cells and a wide variety of blood-borne malignancies.
  • CD19 CAR T cells were initially approved for the treatment of acute lymphoblastic leukemia (ALL) and have subsequently been approved for Burkitt’s Lymphoma and Mantle Cell Lymphoma.
  • BCMA is a receptor expressed on the surface of B-cell lineage cells and a major marker of multiple myeloma (MM).
  • MM is associated with an uncontrollable expansion of plasma cells in the bone marrow, which can progress to extra-medullary lesions forming elsewhere in the body.
  • BCMA CAR T cell therapy has shown great promise against MM with studies showing an overall response rate of 80% even in patients with extra-medullary lesions (Gagelmann et al., Eur. J. Haematol.104(4):318-327 (2020)).
  • challenges persist with CAR T cell therapy.
  • a recent meta-analysis of 22 CAR T cell clinical studies highlighted its ineffectiveness in solid tumors with a poor average overall response rate of 9% (Hou et al., Dis. Markers 2019:3425291 pp. 1-11 (2019)).
  • IL-2 variants chimeric antigen receptor (CAR)-engagers containing an IL-2 variant, and methods of enhancing activity of chimeric CAR immune cells are 2 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 expected to address the above needs.
  • the IL-2 variants per se or connected with another active moiety, such as an antibody or binding fragment thereof, augment anti-cancer therapy.
  • the CAR- engagers containing an IL-2 variant enhance CAR immune cells, driving them toward generation of memory cells, and preventing exhaustion.
  • a first aspect of the present disclosure is directed to an IL-2 variant, which differs from wild type IL-2 (SEQ ID NO: 102) in terms of from three to eight amino acid substitutions selected from amino acid residues H16, D20, R38, F42, Y45, E62, L72, and V91 of SEQ ID NO: 1.
  • the three to eight amino acid substitutions are selected from H16A, H16R, H16S, D20A, D20Q, R38D, F42A, Y45A, E62N, L72G, and V91H.
  • Related aspects include nucleic acids encoding the IL-2 variants, vectors containing the nucleic acid, cells transformed with the vector, methods of making the IL-2 variants, pharmaceutical compositions containing the IL-2 variants, and uses thereof to treat cancer.
  • One such use involves enhancement of adoptive cell therapy such as CAR T therapy.
  • another aspect of the present disclosure is directed to a chimeric antigen receptor (CAR)- engager designed for use with CAR-immune cell therapy such as CAR-T therapy, wherein the CAR- engager contains a first moiety comprising that binds an epitope on an extracellular domain (ED) of the CAR connected to a second moiety comprising an immune effector domain comprising the IL- 2 variant.
  • the ED includes one or more extracellular binding domains (EBDs) of the CAR and any other extracellular portions of the CAR, e.g., a linker that connects antibody fragments, or EBDs, etc.
  • EBDs extracellular binding domains
  • connection between the first moiety and the second moiety may be peptidic or non-peptidic (covalent), such that the CAR-engager may be a continuous protein or polypeptide, or a moiety that contains two proteinaceous entities connected by a covalent bond.
  • Another aspect of the present disclosure is directed to a heterodimeric CAR-engager, containing a first moiety that binds an epitope on an EB of the CAR connected to first dimerization domain, and a second moiety containing an immune effector domain which comprises the IL-2 variant connected to a second dimerization domain, wherein the first and the second dimerization domains bind to form a heterodimeric CAR-engager.
  • nucleic acids that encode the CAR-engager in the embodiments wherein the CAR-engager is a continuous protein
  • nucleic acids that encode the second moiety of the heterodimeric CAR-engager protein vectors containing the nucleic acids encoding the CAR- engager protein, vectors containing the first and/or second proteins of the heterodimeric CAR- engager, cells transformed with the vector(s), pharmaceutical compositions that contain the CAR- engager and a pharmaceutically acceptable carrier, and methods of making the CAR-engager protein.
  • a further aspect of the present disclosure is directed to a method of treating cancer.
  • the method entails administering to a subject a first course of an effective amount of CAR-engager therapy.
  • the subject will have received a prior administration of immune cells that express a CAR that contains an extracellular domain that binds an antigen present on a cancer cell that contains the ectodomain, and the ectodomain of the CAR-engager, a transmembrane domain, and an intracellular domain comprising a stimulatory domain.
  • Working examples e.g., Example 11, disclosed herein demonstrate that CAR-engagers with different IL-2 variants enhance CAR immune cells that target BCMA and CD19, drives them toward generation of memory cells, and prevents exhaustion of the CAR-immune cells.
  • the working examples also demonstrate that the timing and dosage amounts of the CAR-engager therapy may optimize its effects on the prior CAR immune cell therapy in terms enhancing CAR immune cells that target a cancer antigen by driving them toward generation of memory immune cells, and exhaustion-preventative proliferation of the CAR immune cells. And thus, the working examples present a hypothesis of a fundamental mechanism of action as between the CAR-engager and the CAR immune cells.
  • the working examples demonstrate that the two binding events, namely the binding between the immune effector domain of the CAR-engager and the cognate receptor on the immune cells, and the binding between the moiety of the CAR-engager that binds the EB of the CAR, produce a synergistic, molecular “cross-talk” between the intracellular domain (endodomain) of the cognate receptor and the endodomain stimulatory regions of the CAR, respectively, that results in production of IFN- ⁇ and TNF- ⁇ , and ultimately the generation of memory immune cells, and exhaustion-preventative proliferation of the CAR immune cells.
  • FIG. 1 schematically illustrates the domains of a CAR-engager according to some embodiments that contains a first moiety containing an antigen that is used as a target for a CAR, 4 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 such as the ectodomain of CD19 or BCMA on the surface of cancer cells, a dimerization domain, such as CH3, and a second moiety containing an immune cell effector domain (ICE).
  • the CAR- engager may be a monomer, a dimer, or a multimer.
  • FIGs.2A – 2I are a set of illustrations and line plots that show three CAR-engagers.
  • FIG.2A – 2I are a set of illustrations and line plots that show three CAR-engagers.
  • FIG.2A schematically illustrates a CAR-engager that contains a BCMA ectodomain and a Neo2/15 synthetic cytokine immune cell effector domain.
  • FIG.2B schematically illustrates a CAR-engager that contains a BCMA ectodomain and two weak affinity mutated IL-2 (mIL2) synthetic cytokine immune cell effector domains.
  • FIG. 2C schematically illustrates a CAR-engager that contains a BCMA ectodomain and a 4-1BBL immune cell effector domain.
  • FIG.2D is a line plot that shows dose-dependent staining of CAR T cells that bind CD19 or non-transduced T cells (NT T cells) with CAR-engager or control proteins.
  • FIG.2E is a line plot that shows dose-dependent staining of CAR T cells that bind BCMA or non-transduced T cells (NT T cells) with CAR-engager or control proteins.
  • FIGs.2F and 2G are a line and bar plot, respectively, that together show dose-dependent activation of CAR T cells after CAR-engager treatment.
  • FIG. 2H is a line plot showing that the BCMA-muIL2 CAR-engager does not block the killing efficacy of the CAR T cells.
  • FIG. 2I is a line plot that shows phosphorylation of signal transducer and activator of transcription (STAT5) in the BCMA CAR T cells.
  • FIGs.3A – 3B are a set of illustrations and line plots showing the effects of CAR-engagers on non-transduced T cells.
  • FIG.3A schematically illustrates the experimental design.
  • FIG.3B is a set of line plots that show T cell count and carboxyfluorescein succinimidyl ester (CFSE) staining of non-transduced, activated T cells treated with teceleukin, a CAR-engager containing an BCMA ectodomain and two mutated weak affinity IL-2 (muIL2), or CAR-engager containing an BCMA ectodomain and a Neoleukin domain.
  • CFSE carboxyfluorescein succinimidyl ester
  • FIG. 4A – 4C are a set of illustrations and line plots showing that CAR-engagers specifically activate CAR T cells.
  • FIG.4A schematically illustrates the experimental design.
  • FIG. 4B is a bar plot that shows the percentage of CD69 + anti-BCMA CAR-transduced, activated T cells after treatment with BCMA CAR-engager, BCMA CAR-engager without an immune cell effector domain control, or a non-antigen-specific CAR-engager control.
  • FIG. 4C is a bar plot that shows the percentage of CD69 + anti-CD19 CAR-transduced, activated T cells after treatment with CD19 CAR-engager or a non-antigen-specific CAR-engager control.
  • FIG.5 is a line plot showing that CAR-engagers do not inhibit BCMA CAR T cell killing and that the percentage of OPM2 target cell survival after incubation with CAR T cells and CAR- engagers (red) or non-transduced T cells (blue).
  • FIGs. 6A – 6C are a set of illustrations and photographs showing that CAR-engagers reduce tumor burden in vivo.
  • FIG.6A schematically illustrates the experimental design.
  • FIGs.6B – 6C are a set of photographs that show tumor burden in mice before and after CAR T cell infusion and CAR-engager treatment.
  • FIGs.7A – 7C are a set of flow cytometry plots showing the tumor burden in mice after CAR T cell infusion and CAR-engager treatment.
  • FIG. 7A is a set of flow cytometry plots that shows OPM2 tumor burden in blood, spleen, and lymph nodes.
  • FIG.7B is a set of flow cytometry plots that shows OPM2 tumor burden in bone marrow and lung.
  • FIG.7C is a set of flow cytometry plots that shows OPM2 tumor burden in liver, kidney, and the eye tumor site.
  • eGFP OPM2 cells
  • PerCP signal control is shown on the x-axis.
  • FIGs.8A – 8C are a set of flow cytometry plots showing human CD45 + and CAR- T cells in mice after CAR T cell infusion and CAR-engager treatment.
  • FIG.8A is a set of flow cytometry plots that shows CAR T cells in blood, spleen, and lymph nodes.
  • FIG.8B is a set of flow cytometry plots that shows CAR T cells in bone marrow and lung.
  • FIG.8C is a set of flow cytometry plots that shows CAR T cells in the liver, kidney, and the eye tumor site.
  • CD45 staining is shown on the y- axis and CAR-engager labeled with AF647 staining is shown on the x-axis.
  • FIGs.9A –9E are a set of schematics, line plots, and box plots showing that CAR-engager treatment results in enhanced activity and persistence of CAR T cells in vivo.
  • FIG.9A is a line plot that shows circulating half-life of the BCMA CAR-engagers.
  • FIG.9B schematically illustrates the experimental design.
  • FIGs. 9C and 9D are a set of flow cytometry plots and box plots that show selective expansion and persistence of BCMA CAR T cells.
  • FIG. 9E is a box plot that shows the percentage of CD8 + CAR T cells after CAR-engager treatment.
  • FIGSNE stochastic neighbor embedding
  • FIG.10E is a set 6 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 of bar plots that show generation of memory CAR T cells.
  • FIGs. 10F and 10G are a set of flow cytometric plots and bar plots showing that a substantial number of CAR T cells two months post- CAR T cell injection.
  • FIG.10H is a line plot showing that mice maintained consistent body weight throughout the experiment.
  • FIG.10I is a set of flow cytometric plots that show CAR T cells from CAR-engager treated mice have a stem-cell memory phenotype.
  • FIG. 10E is a set 6 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 of bar plots that show generation of memory CAR T cells.
  • FIGs. 10F and 10G are a set of flow cytometric plots and bar plots showing that a substantial number of C
  • FIG. 10J is a set of tSNE plots displaying FlowSOM defined clusters among persisting BCMA CAR T cells.
  • FIGs. 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.
  • FIG. 11A schematically illustrates the experimental design.
  • FIG.11B is a set of photographs that show tumor burden in mice before and after CAR T cell infusion and CAR-engager treatment.
  • FIG.11C is a set of flow cytometric plots showing persistence of CAR T cells.
  • FIG.11D is a set of bar plots showing in vitro killing assays of persistent T cells.
  • FIG.11E is a set of t-SNE plots showing immune cell markers from CD8 + T cells.
  • FIGs. 12A – 12B is a schematic and a set of bar plots showing that CAR-E treatment expands CAR T cells in vivo in the absence of tumor cells.
  • FIG.12A schematically illustrates the experimental design.
  • FIG. 12B is a set of bar plots that show counts of CAR T cells 30 days-post injection.
  • FIGs.13A – 13B are a set of flow cytometry plots showing that neither the BCMA-muIL2 nor the VHH-muIL2 treatment exhibited binding to any specific population within human PBMCs.
  • FIG. 13A is a set of flow cytometry plots showing that BCMA-muIL2 does not bind to human PBMCs.
  • FIG.13B is a set of flow cytometry plots showing that VHH-muIL2 does not bind to human PBMCs.
  • FIGs. 14A – 14C are a set of photomicrographs and dot plots showing specific binding and gradual internalization of the BCMA-muIL2 in CAR T cells.
  • FIG. 14A – 14C are a set of photomicrographs and dot plots showing specific binding and gradual internalization of the BCMA-muIL2 in CAR T cells.
  • FIG. 14A is a set of photomicrographs that show cells stained with CellTracker Blue CMAC, incubated with the indicated treatment, each treatment labeled with Alexa647 (BCMA-muIL2) or dsRed (VHH- muIL2) for 1 to 5 hours and imaged. Photomicrographs are representative of >100 cell images.
  • FIG. 14B is a dot plot that shows quantitative analysis of the imaged cells.
  • FIG. 14C is a dot plot that 7 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 shows the correlation between Alexa647 mean intensity (BCMA-muIL2) and dsRed mean intensity (VHH-muIL2). [00031] FIGs.
  • FIGS. 15A – 15C are a set of flow cytometry plots showing individual flow cytometric data corresponding to the pooled data presented in FIG. 9D.
  • FIG. 15A is a set of flow cytometry results from mice treated solely with CAR T cells.
  • FIG.15B is a set of flow cytometry results from mice treated with CAR T cells and VHH-muIL2.
  • FIG.15C is a set of flow cytometry results from mice treated with CAR T cells and BCMA-muIL2.
  • FIGs. 16A – 16C are a set of flow cytometry plots showing individual flow cytometric data of the mice shown in FIGs.10A –10J.
  • FIG.16A is a set of flow cytometric results from mice treated solely with CAR T cells.
  • FIG.16B is a set of flow cytometry results from mice treated with CAR T cells and VHH-muIL2.
  • FIG.16C is a set of flow cytometry results from mice treated with CAR T cells and BCMA-muIL2.
  • FIGs. 17A – 17C are a set of bar, line, and tSNE plots showing human T cell-derived cytokines in the serum of mice that received OPM2 cancer cells followed by a low dose of CAR T cells.
  • FIG.17A is a bar plot that shows levels of IFN ⁇ , GM-CSF, and TNF ⁇ . Serum samples were diluted at a ratio of 1:40.
  • FIG.17B is a set of line plots that shows IFN ⁇ levels between the BCMA-muIL2 group and the VHH-muIL2 group (error bars represent mean with standard deviation).
  • FIG. 17C is a set of Flt-SNE mapping of CAR T cells derived from the PBS, BCMA-muIL2 and VHH-muIL2 treated mice showing the expression of ten immune cell markers.
  • FIG.18 is a set of t-SNE mapping of CD4 + CAR + T cells derived from the five BCMA- muIL2 CAR-E treated mice showing the expression of nine immune cell markers.
  • FIGs. 19A – 19G are a set of flow cytometry, tSNE, bar, violin, and pie plots and heatmaps showing single-cell RNA sequencing analyses elucidate BCMA-muIL2 effect on CAR T cells.
  • FIG.19A is a set of flow cytometry plots showing CAR + cells analyzed 89 days after CAR-T administration.
  • FIG. 19B is a tSNE plot that shows data after Harmony algorithm, showing proportion of CD4, CD8, and proliferating (CD4 and CD8) cells.
  • FIG.19C is a tSNE plot that shows split between the groups treated with BCMA-muIL2 or VHH-muIL2 treatments.
  • FIG.19D is a set of heatmaps of significantly differentially expressed genes in CD4 + CAR T cells and CD8 + CAT T cells after the indicated treatment.
  • FIG.19E is a set of violin plots of the gene scores between CD8 8 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 and CD4 cells, the scores being constructed using the normalized expression of the different genes for each phenotype in FIG. 19D.
  • FIG. 19F is a set of pie plots that shows the diversity of T-cell receptor (TCR) clonotypes.
  • FIG.19G is a bar plot that shows clonotype diversity within a sample’s total cell count. [00036] FIGs.
  • FIGS. 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.
  • FIG. 20A schematically illustrates the experimental design.
  • FIG. 20B is a set of flow cytometry plots that show selective trafficking, expansion, and persistence of BCMA CAR T cells.
  • FIGs.21A – 21C are a set of line and bar plots showing the effects of CAR-engagers on non-transduced T cells and CAR T cells.
  • FIGs.21A and 21B are a line and bar plot, respectively, that together show dose-dependent activation of CAR T cells after CAR-engager treatment (FIG.21A) and that the CAR-engager does not activate non-transduced T cells (FIG. 21B).
  • FIG.21C is a line plot showing that the CD19-muIL2 CAR-engager does not block the killing efficacy of the CD19 CAR T cells or non-transduced T cells (NT T cells).
  • FIGs. 22A – 22B are a set a of line plots showing activation of CAR T cells by CAR- engager variants.
  • FIG. 22A is a line plot showing that CAR-engager variant treatment results in STAT5 phosphorylation.
  • FIG.22B is a line plot showing that CAR-engager variant treatment results in CD69 expression on CAR T cells.
  • FIGs.23A – 23B are a set of line plots showing activation of rested CAR T cells by CAR- engager variants.
  • FIG. 23A is a line plot showing that CAR-engager variant treatment results in STAT5 phosphorylation in rested CAR T cells.
  • FIG.23B is a line plot showing that CAR-engager variant treatment results in CD69 expression on rested CAR T cells.
  • FIGs.24A – 24B are a set of line plots showing the percentage of phosphorylated STAT5 (pSTAT5) positive BCMA CAR T Cells containing FDA-approved CAR constructs induced by BCMA CAR-engagers.
  • FIG. 24A is a line plot that shows pSTAT5 positive BCMA CAR T cells containing the FDA-approved idecabtagene vicleucel (Ide-cel) construct.
  • FIG. 24B is a line plot showing pSTAT5 positive BCMA CAR T cells containing the FDA-approved Ciltacabtagene autoleucel (Cilta-cel) construct.
  • FIGs.25A – 25F are a set of line plots showing the percentage of phosphorylated STAT5 (pSTAT5) positive BCMA CAR T Cells containing FDA-approved CAR constructs induced by BCMA-muIL2 CAR-engagers.
  • FIGs.25A – 25C are a set of line plots that show pSTAT5 positive BCMA CAR T cells containing the FDA-approved Ide-cel construct.
  • FIGs.25D – 25F are a set of line plots that show pSTAT5 positive BCMA CAR T cells containing the FDA-approved Cilta-cel construct. For incubation of 24 hours, CAR T cells were washed at 2 hours to remove unbound any CAR-engager.
  • FIGs. 26A – 26B are a set of line plots showing BCMA CAR-engager dose-dependent activation of rested BCMA CAR T cells through CD69 expression.
  • FIG. 26A is a line plot that shows pSTAT5 positive BCMA CAR T cells containing the FDA-approved Ide-cel construct.
  • FIG. 26B is a line plot showing pSTAT5 positive BCMA CAR T cells containing the FDA-approved cilta-cel construct.
  • CD69 serves as an activation marker for human T cells.
  • FIGs. 27A – 27B are a set of line plots showing BCMA CAR-engager dose-dependent activation of rested BCMA CAR T cells through CD69 expression.
  • FIG. 27A is a line plot that shows pSTAT5 positive BCMA CAR T cells containing the FDA-approved idecabtagene vicleucel construct.
  • FIG. 27B is a line plot showing pSTAT5 positive BCMA CAR T cells containing the FDA-approved ciltacabtagene autoleucel (Cilta-cel) construct.
  • FIGs. 28A – 28B are a set of line plots showing BCMA CAR-engager dose-dependent activation of rested BCMA CAR T cells through CD69 expression.
  • FIG. 28A is a line plot that shows pSTAT5 positive BCMA CAR T cells containing the FDA-approved idecabtagene vicleucel construct.
  • FIG. 28B is a line plot showing pSTAT5 positive BCMA CAR T cells containing the FDA-approved ciltacabtagene autoleucel construct.
  • FIGs.29A – 29C are a set of line plots showing induction of CAR T cell proliferation in vitro from CAR-engager treatment with reduced impact on non-transduced T cells as compared to wild-type IL-2.
  • FIGs. 29A – 29B are a set of line plots that show incubation of activated BCMA CAR T cells with the ciltacabtagene autoleucel CAR construct, with varying concentrations of BCMA CAR-engager treatment, followed by assessment of CAR T cell proliferation using flow cytometry after 3 days.
  • the dashed line represents the number of CAR T cells without any treatment.
  • FIGs.30A – 30C are a set of line plots showing bio-layer interferometry (BLI) associate and dissociation between CAR-engagers containing the triple mutant IL-2 V7 or CAR-engagers containing the double-mutant IL-2 U4 exhibits lower affinity for IL-2Ra as compared to wild-type IL-2.
  • BBI bio-layer interferometry
  • FIG.30A is a line plot that shows BLI between wild-type IL-2 (Teceleukin) and IL-2Ra.
  • FIG. 30B is a line plot that shows BLI between the V7 CAR-engager and IL-2Ra.
  • FIG.30C is a line plot that shows BLI between the CAR-engager containing the U4 CAR-engager and IL-2Ra.
  • FIGs.31A – 31B are a set of schematics and line plots showing that the CAR-engagers containing IL-2 variants expand CAR-T cells in vivo, and that the expansion levels correlate with in vitro pSTAT5 signaling.
  • FIG.31A schematically illustrates the experimental design.
  • FIG.31B is a set of line plots that show CD4 and CD8 CAR T cells circulating in the blood. Mice were bled for 5 weeks on days 11, 18, 28, 35, 42 post injection, and CD4 and CD8 CAR T-cells were counted. All the three tested CAR-engagers expanded CAR T cells in vivo as compared to the control, PBS treatment group. V7 and Y2 CAR-engagers expanded CAR T cells more than X12 CAR-engagers.
  • FIGs.32A – 32C are a set of schematics, photographs, and line plots showing BCMA-V7 CAR-engagers enhances CAR-T cell activity when administered at day 3 or even day 14 post- CAR T-cell injection.
  • FIG. 32A schematically illustrates the experimental design.
  • Another cohort of mice received 6 doses of V7 CAR-engager treatment starting from day 3 post-injection of CAR-T cells, administered on days 3, 6, 10, 14, 21, 28.
  • the third cohort of mice received 6 doses of treatment starting from day 14, which is the post- CRS window in patients, and on days 14, 18, 21, 28, 35, 42.
  • FIG.32B is a set of photographs that shows BLI imaging on the indicated days to assess tumor growth in the different cohorts.
  • FIG.32C is a line plot that shows CAR T cells circulating in the blood. Mice were bled for 6 weeks, once per week starting from day 14, and CAR T cells were counted. V7 CAR-engager treatment expanded CAR T cells in both cohorts that received treatment, compared to the PBS treatment group.
  • FIGs. 33A – 33M are a set of schematics, line, bar, and dot plots showing substantial activation and transcriptomic changes in CAR T cells after CAR-E treatment.
  • FIG. 33A is a line plot that shows CAR-E induction of pSTAT5 activity in CAR T cells with either the full CAR 11 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 construct or the CAR-ICD- ⁇ construct.
  • FIGs.33B – 33D are a set of line plots that show CAR-E CD69 (FIG. 33B), IFN- ⁇ (FIG.33C), and TNF- ⁇ (FIG. 33D) staining in BCMA CAR T cells and BCMA CAR-ICD- ⁇ T cells.
  • FIGs.33E – 33G are a set of line plots that show CD69 (FIG.33E), IFN- ⁇ (FIG.
  • FIG. 33F schematically illustrates the experimental design for in vivo assessment of the efficacy of CAR-E on BCMA CAR T cells and BCMA CAR-ICD- ⁇ T cells.
  • FIG. 33I is a set of bar plots that show expansion and persistence of BCMA CAR T cells and BCMA CAR-ICD- ⁇ T cells in mouse organs 1 month after CAR T cell injection; **** P ⁇ 0.0001.
  • FIG.33J is a volcano Plot that shows the highest upregulated genes in CD8 + CAR T cells 4 hours after CAR- E treatment.
  • FIG. 33K is a volcano plot that shows the highest upregulated genes in CD8 + CAR- ICD- ⁇ T cells 4 hours after CAR-E treatment.
  • FIG. 33L is a heatmap that shows gene expression changes in CD8 + and CD4 + T cells after 4 hours of CAR-E treatment.
  • FIG.33M is a heatmap that shows gene expression changes in CD8 + and CD4 + T cells after 2 and 24 hours of CAR-E treatment.
  • FIG.34 is a set of bar plots showing transcriptome changes in CAR T cells after CAR-E treatment.
  • FIGs.35A – 35G are a set of schematics, photographs, line, and pie graphs showing that lower doses of CAR-engagers enhance CAR T cell activity and promote functional memory.
  • FIG.35A – 35G are a set of schematics, photographs, line, and pie graphs showing that lower doses of CAR-engagers enhance CAR T cell activity and promote functional memory.
  • FIG.35B is a set of photographs that show bioluminescence imaging (BLI) of monitored tumor burdens.
  • FIG. 35C is a survival analyses showing that all CAR-engager treated mice survived for the duration of the experiment.
  • FIG.35D is a line graph that shows the quantification of BLI analyses from FIG.35B.
  • FIG.35E is a line graph that shows flow cytometric analyses of CAR T-cell presence in blood.
  • FIG.35F is a line graph that shows IFN- ⁇ levels.
  • FIG.35G is a set of pie graphs that show the CAR T cells in the bone marrow and spleen of mice treated with CAR-engager.
  • FIGs.36A – 36G are a set of schematics, bar, and line graphs showing treatment of CAR- engager expands CAR T cells in in vivo in the absence of tumor cells in a dose-dependent manner.
  • FIG. 36A schematically illustrates the experimental design.
  • FIGs. 36B – 36C are a set of bar and line graphs showing flow cytometric analysis of spleen and bone marrow tissues harvested 30 days post-injection of CAR T cells.
  • FIG. 36D – 36E are a set of bar graphs that show analyses of persisting CAR T cells and different subsets of memory CAR T cells in the spleen and bone marrow 12 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 of the CAR-E-treated mice.
  • FIG. 36F is a bar graph showing that both the antigen and the low- affinity IL-2 components of CAR-engager are essential for its impact.
  • FIG. 37 a set of flow cytometry plots showing anti-human-CD45 and BCMA-CAR staining in blood samples of mice that received human CAR T cells with and without CAR-E treatment.
  • FIGS. 39A – 39D are a set of heatmaps and tSNE plots showing CAR-E promotion of phyotypic diversity of bone marrow and splenocyte-derived CAR T cells.
  • FIG. 39A – 39D are a set of heatmaps and tSNE plots showing CAR-E promotion of phyotypic diversity of bone marrow and splenocyte-derived CAR T cells.
  • FIG. 39A is a heatmap that shows eight FLOWSOM-derived metaclusters in bone marrow samples.
  • FIG.39B is a heatmap that shows eight FLOWSOM-derived metaclusters in spleen samples.
  • FIG.39C is a tSNE plot that shows CAR T populations in bone marrow samples.
  • FIG. 39D is a tSNE plot that shows CAR T populations in spleen samples.
  • FIGs. 40A – 40D are a set of flow cytometry plots showing anti-human-CD45 and BCMA-CAR staining in individual mice that received human CAR T cells and CAR-E treatment.
  • FIG.40A is a set of flow cytometry plots of mice that received CAR T cells and PBS control.
  • FIG. 40B is a set of flow cytometry plots of mice that received CAR T cells and 2 mg/kg BCMA-muIL2.
  • FIG.40C is a set of flow cytometry plots of mice that received CAR T cells and 4 mg/kg BCMA- muIL2.
  • FIG.40D is a set of flow cytometry plots of mice that received CAR T cells and 8 mg/kg BCMA-muIL2.
  • FIGs. 41A – 41E are a set of flow cytometry plots showing anti-human-CD45 and BCMA-CAR staining in individual mice that received human CAR T cells and CAR-E treatment.
  • FIG. 41A is a set of flow cytometry plots of mice that received CAR T cells and BCMA-CH3 control.
  • FIG.41B is a set of flow cytometry plots of mice that received CAR T cells and low dose IL-2.
  • FIG.41C is a set of flow cytometry plots of mice that received CAR T cells and VHH-muIL2.
  • FIG. 41D is a set of flow cytometry plots of mice that received CAR T cells and BCMA-muIL2.
  • FIG.41E is a set of flow cytometry plots of mice that received CAR T cells only.
  • FIG.42 is a set of line plots showing CAR-engagers each containing an immune cell effector domain containing an U4, V6, V7, or Y2 IL-2 variant and their selective and potent induction of proliferation in CAR T cells but not non-transduced T cells.
  • FIG.43 is a set of line plots showing phosphorylation of STAT5 in CAR T cells and non- transduced T cells following CAR-E treatment.
  • FIG. 44 is a set of line plots showing CD69 expression on CAR T cells and non- transduced T cells following CAR-E treatment.
  • FIG.45 is a set of line plots showing TNF- ⁇ and IFN- ⁇ secretion from CAR T cells and non-transduced T cells following CAR-E treatment.
  • FIG.46A – 46B are a set of line plots showing CAR-E binding to IL-2R ⁇ and IL-2R ⁇ by Biolayer Interferometry. DETAILED DESCRIPTION OF THE DISCLOSURE [00063] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated to facilitate the understanding of the present disclosure.
  • transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.
  • the transitional phrase “consisting of” excludes any element or method step not specified in the claim (or the specific element or method step with which the phrase “consisting of” is associated).
  • the transitional phrase “consisting essentially of” limits the scope of a claim to the specified elements and method or steps and “unrecited elements and method steps that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure.
  • the disclosure provides an IL-2 variant, which differs from wild type IL-2 (SEQ ID NO: 102) in terms of from three to eight amino acid substitutions selected from amino acid residues H16, D20, R38, F42, Y45, E61, E62, L72, and V91 of SEQ ID NO: 102.
  • the three to eight amino acid substitutions are selected from H16A, H16R, H16S, D20A, D20Q, R38D, F42A, Y45A, E61A, E62N, L72G, and V91H.
  • IL-2 variant refers to non-naturally occurring variant of IL-2 capable of binding to the cognate IL-2 receptor(s) on an immune cell and initiating signal transduction through that receptor to achieve substantially the same effect as the naturally occurring cytokine.
  • the amino acid sequence of wild-type IL-2 is set forth below (SEQ ID NO: 102): 1 aptssstkkt qlqlehllld lqmilnginn yknpkltrml tfkfympkka telkhlqcle 61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [00070] All descriptions of the IL-2 variants of the present disclosure are made in reference to SEQ ID NO:102.
  • the IL-2 variant includes a first amino acid substitution (of the three to eight amino acid substitutions), from N-terminus to C-terminus, is selected from H16A, H16R, H16S, D20A, or D20Q, a second amino acid substitution is selected from R38D, F42A, and V91H, and a third amino acid substitution is selected from Y45A and E62N. [00071] In some embodiments, the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S. In other embodiments, the IL-2 variant includes a first amino acid substitution of D20A or D20Q.
  • the IL-2 variant includes a second amino acid substitution of F42A.
  • the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S and a second amino acid substitution of F42A.
  • the IL-2 variant includes a first amino acid substitution of D20A or D20Q and a second amino acid substitution of F42A.
  • the IL-2 variant includes a third amino acid substitution of Y45A. In other embodiments, the third amino acid substitution is E62N.
  • the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, and a third amino acid substitution of Y45A.
  • the IL-2 variant includes a first amino acid substitution of D20A or D20Q, a second amino acid substitution of F42A, and a third amino acid substitution of Y45A.
  • the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, and a third amino acid substitution of Y45A.
  • the IL-2 variant includes a first amino acid substitution of D20A or D20Q, a second amino acid substitution of F42A, and a third amino acid substitution of Y45A. [00076] In some embodiments, the IL-2 variant includes a fourth amino acid substitution selected from R38D, E61A, L72G, and V91H. [00077] In some embodiments, the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, a third amino acid substitution of Y45A or E62N, and a fourth amino acid substitution selected from D20A, D20Q, R38D, Y45A, E61A, L72G, and V91H.
  • the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, a third amino acid substitution of Y45A or E62N, a fourth amino acid substitution selected from D20A, D20Q, E61A, L72G, and V91H, and a fifth amino acid substitution of R38D.
  • the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, a third amino acid substitution of Y45A or E62N, a fourth amino acid substitution selected from D20A, D20Q, E61A, and V91H, a fifth amino acid substitution of R38D, and a sixth amino acid substitution of L72G.
  • the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, a third amino acid substitution of Y45A, 16 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 a fourth amino acid substitution selected from D20A, D20Q, E61A, and V91H, a fifth amino acid substitution of R38D, a sixth amino acid substitution of L72G, and a seventh amino acid substitution of E62N.
  • the IL-2 variant has amino acid substitutions D20Q, F42A, and Y45A, and has the amino acid sequence set forth below (SEQ ID NO: 112). Amino acid substitutions relative to wild-type IL-2 for SEQ ID NOs: 112-123 are boxed in the sequences below.
  • the IL-2 variant has amino acid substitutions H16A, F42A, and E62N, and has the amino acid sequence set forth below (SEQ ID NO: 113): 1 aptssstkkt qlqleallld lqmilnginn yknpkltrml takfympkka telkhlqcle 61 enlkpleevl nlaqsknfhl rprdlisnin vivlelkgse
  • CAR-engagers may also reduce the cellular dose needed for CAR immune cell therapy, which may result in reduced adverse side effects (e.g., cytokine release syndrome) caused by the larger doses typically used in the clinic and therefore, the CAR-engagers are also referred herein as CAR-enhancers or CAR-E.
  • CAR-enhancers CAR-E.
  • CAR-engager binding to CAR is reversible, the CAR engager does not induce immune synapse formation of CAR on the CAR immune cell surface. Therefore, CAR- engagers do not block CAR-mediated killing of cancer cells.
  • CAR immune cells often do not persist in the body during minimal residual disease (MRD), which, as known in the art, is associated with limited cancer antigens.
  • MRD minimal residual disease
  • the disclosure provides a CAR-E which contains a first proteinaceous moiety and a second proteinaceous moiety.
  • the first proteinaceous moiety binds an epitope on the extracellular domain (ED) of a CAR.
  • the first proteinaceous moiety binds an epitope present on the extracellular binding domain (EBD) of the CAR that binds a cancer antigen on the surface of a cancer cell.
  • the second proteinaceous moiety which is an immune effector domain, comprises an IL-2 variant of the present disclosure.
  • CAR-engager is a contiguous protein, where the first proteinaceous entity and the proteinaceous entity are connected by a peptide bond. In some embodiments, the first proteinaceous entity and the second proteinaceous entity are connected by click chemistry. [00096] In some embodiments, the CAR-engager is formulated and administered as a monomeric protein or proteinaceous entity. In other embodiments, the CAR-engager is formulated and administered in the form of a dimer, either as a homodimer or a heterodimer protein or proteinaceous entity.
  • the first proteinaceous moiety of the CAR-engager is designed to bind an epitope present on the extracellular binding domain of the CAR that targets an antigen on the surface of a cancer cell.
  • the first moiety of the CAR-engager is an ectodomain that binds an EBD of a CAR presented on an immune cell.
  • the ectodomain of a cancer antigen is the portion of the antigen on the surface of a cancer cell that binds a T cell receptor or a CAR on 19 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 an immune cell.
  • the CAR-engager may include the entire extracellular domain of a cancer antigen.
  • Ectodomains may be derived from (e.g., identified in) a cancer antigen in accordance with standard techniques. See, e.g., and Gershoni et al., Biodrugs 21(3):145-156 (2007) and Francino-Urdaniz and Whitehead, RSC Chem. Biol. 2(6):1580-1589 (2021).
  • the term “derived from” as used herein when referring to a protein and nucleic acid refers to a sequence that originates and is identified from the sequence of a parent (e.g., wild-type or endogenous) protein and nucleic acid, respectively.
  • a sequence derived from a parent sequence may be a portion (fragment) of the parent sequence and/or may vary by at least one position from the parent amino acid or nucleotide sequence.
  • Protein variants may include amino acid substitutions, insertions, and/or deletions.
  • an amino acid sequence derived from a parent sequence may constitute a fragment of the parent sequence and be identical for a specific range of amino acids of the parent but does not include amino acids outside that specific region.
  • Nucleic acid variants may include substitutions or in-frame insertions or deletions (i.e., insertions or deletions that do not result in downstream frame shift of the nucleic acid codons).
  • Unique cancer antigens may be determined by known methods. For example, cancer genomes can be compared with normal cell genomes to identify neoantigens. In some embodiments, caner transcriptomes are compared to normal cell transcriptomes. Computational methods may then be utilized to identify suitable binding sites for a CAR. Most often the CAR binds a portion of the extracellular domain of an antigen. In some embodiments, the CAR and the corresponding cancer antigen are known in the art. [000100] In some embodiments, the ectodomain of the CAR-engager contains the entire extracellular domain of a cancer antigen.
  • the CAR-engager contains a portion of the extracellular domain of a cancer antigen which is targeted by a CAR.
  • the ectodomain of the CAR- engager contains the extracellular domain of BCMA.
  • the amino acid sequence of a representative CAR-engager that contains a BCMA extracellular domain is MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA (SEQ ID NO: 1).
  • the ectodomain of the CAR-engager contains two repetitions of the extracellular domain of BCMA.
  • the amino acid sequence of a representative CAR-engager that contains two repetitions of the BCMA extracellular domain is set forth below (SEQ ID NO: 2): 1 mlqmagqcsq neyfdsllha cipcqlrcss ntppltcqry cnasvtnsvk gtnagggsgg 61 gsprgsgggs mlqmagqcsq neyfdsllha cipcqlrcss ntppltcqry cnasvtnsvk 121 gtn [000103]
  • the ectodomain of the CAR- engager contains a variant of the extracellular domain of CD19.
  • amino acid sequence of a representative CAR-engager that contains a variant of CD19 extracellular domain is set forth below (SEQ ID NO: 3): 1 peeplvvkve egdeawlpcl kgtsdgptqq ltwsresplk pflkvsfgvp glgvhvrpna 61 vslvisnvsq qmggfylcqp gppsekawqp gwtvnvegsg elfrwnvsdl gglgcglknr 121 ssegpsspsg klmspklyvw akdrpeiweg eppclpprds lnqslsrdmt vapgstlwls 181 cgvppdsvsr gplswthvhp kgpksllsle lkddrpardm wvt
  • the ectodomain of the CAR-engager is KDRPEIWEGEPP (SEQ ID NO: 106), which corresponds to amino acid residues 142-153 of SEQ ID NO: 5.
  • the ectodomain of the CAR- engager contains at least a portion of the extracellular domain of CD20.
  • the amino acid sequence of a representative CAR-engager that contains a CD20 extracellular domain is KISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQS (SEQ ID NO: 6).
  • the ectodomain of the CAR- engager contains at least a portion of the extracellular domain of CD22.
  • the amino acid sequence of a representative CAR-engager that contains a CD22 extracellular domain is set forth below (SEQ ID NO: 7): 1 dsskwvfehp etlyawegac vwipctyral dgdlesfilf hnpeynknts kfdgtrlyes 61 tkdgkvpseq krvqflgdkn knctlsihpv hlndsgqlgl rmesktekwm erihlnvser 121 pfpphiqlpp eiqesqevtl tcllnfscyg ypiqlqwlle gvpmrqaavt stsltik
  • the ectodomain of the CAR- engager contains the Ig domains 2 and 3 (2-3) of CD22.
  • the amino acid sequence of a representative CAR-engager that contains Ig domains 2-3 of CD22 is set forth below (SEQ ID NO: 103): 1 phiqlppeiq esqevtltcl lnfscygypi qlqwllegvp mrqaavtsts ltiksvftrs 61 elkfspqwsh hgkivtcqlq dadgkflsnd tvqpkleikv tpsdaivreg dsvtmtcevs 121 ssnpeyttvs wlkdgtslkk qntftlnlre vtkdqsgkyc cqvsndvgpg rseevflq [000111] In some embodiments,
  • the amino acid sequence of a representative CAR-engager that contains Ig domain 3 of CD22 is set forth below (SEQ ID NO: 104): 1 pkleikvtps daivregdsv tmtcevsssn peyttvswlk dgtslkkqnt ftlnlrevtk 61 dqsgkyccqv sndvgpgrse evflq [000112]
  • the ectodomain of the CAR-engager contains the Ig domains 5 through 7 (5-7) of CD22.
  • amino acid sequence of a representative CAR-engager that contains Ig domains 5-7 of CD22 is set forth below (SEQ ID NO: 105): 1 pkkvttviqn pmpiregdtv tlscnynssn psvtryewkp hgaweepslg vlkiqnvgwd 61 nttiacaacn swcswaspva lnprdvrvrk ikplseihsg nsvslqcdfs sshpkevqff 121 wekngrllgk esqlnfdsis pedagsyscw vnnsigqtas prrlrvsmsp gdqvmegksa 181 tltcesdanp pvshytwfdw nnqslpyhsq klrlepvkvq
  • the amino acid sequence of a representative CAR-engager that contains a Claudin 18.2 first extracellular domain is set forth below (SEQ ID NO: 8): 1 dqwstqdlyn npvtavfnyq glwrscvres sgftecrgyf tllglpamlq avr [000114]
  • the amino acid sequence of a representative CAR-engager that contains a Claudin 18.2 second extracellular domain is set forth below (SEQ ID NO: 9): 1 vtnfwmstan mytgmggmvq tvqtrytfga a [000115]
  • the ectodomain of the CAR- engager contains at least a portion of the extracellular domain of SLAMF7.
  • the amino acid sequence of a representative CAR-engager that contains a SLAMF7 extracellular domain is set forth below (SEQ ID NO: 10): 1 sgpvkelvgs vggavtfplk skvkqvdsiv wtfnttplvt iqpeggtiiv tqnrnrervd 61 fpdggyslkl sklkkndsgi yyvgiysssl qqpstqeyvl hvyehlskpk vtmglqsnkn 121 gtcvtnltcc mehgeedviy twkalgqaan eshngsilpi swrwgesdmt ficvarnpvs 181 rnfsspilar klcegaaddp dssm [000116]
  • the CAR targets PD-1 the CAR targets
  • a representative CAR-engager that contains a PD-1 extracellular domain is set forth below (SEQ ID NO: 11): 1 fldspdrpwn pptfspallv vtegdnatft csfsntsesf vlnwyrmsps nqtdklaafp 61 edrsqpgqdc rfrvtqlpng rdfhmsvvra rrndsgtylc gaislapkaq ikeslraelr 121 vterraevpt ahpspsprpa gqfqtlv [000117]
  • the ectodomain of the CAR-engager contains a variant of the extracellular domain of PD-1.
  • the ectodomain of the CAR-engager contains the N-loop of PD-1.
  • the amino acid sequence of a representative CAR-engager that contains the N- loop of the PD-1 extracellular domain is LDSPDRPWNP (SEQ ID NO: 107), which corresponds to amino acid residues 2-11 of SEQ ID NO: 11.
  • the ectodomain of the CAR-engager contains the CD-loop of PD- 1.
  • the amino acid sequence of a representative CAR-engager that contains the CD-loop of the PD- 1 extracellular domain is NQTDKLAAFPEDRSQPGQDCRFRVTQ (SEQ ID NO: 108), which corresponds to amino acid residues 51-76 of SEQ ID NO: 11.
  • the ectodomain of the CAR-engager contains at least a portion of the extracellular domain of KIT.
  • amino acid sequence of a representative CAR-engager that contains a KIT extracellular domain is set forth below (SEQ ID NO: 12): 1 qpsvspgeps ppsihpgksdassivgdeir llctdpgfvk wtfeildetn enkqnewite 61 kaeatntgky tctnkhglsn siyvfvrdpa klflvdrsly gkedndtlvr cpltdpevtn 121 yslkgcqgkp lpkdlrfipd pkagimiksv krayhrlclh csvdqegksv lsekfilkvr 181 pafkavpvvs vskasyllre geeftvtcti kdvssvyst wkrensqtkl qekyn
  • amino acid sequence of a representative CAR-engager that contains a TROP2 extracellular domain is set forth below (SEQ ID NO: 13): 1 htaaqdnctc ptnkmtvcsp dgpggrcqcr algsgmavdc stltskclll karmsapkna 61 rtlvrpseha lvdndglydp dcdpegrfka rqcnqtsvcw cvnsvgvrrt dkgdlslrcd 121 elvrthhili dlrhrptaga fnhsdldael rrlfreryrl hpkfvaavhy eqptiqielr 181 qntsqkaagd vdigdaayyf erdikgeslf qgrggldlr
  • the amino acid sequence 31 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 of a representative CAR-engager that contains a CD38 extracellular domain is set forth below (SEQ ID NO: 14): 1 vprwrqqwsg pgttkrfpet vlarcvkyte ihpemrhvdc qsvwdafkga fiskhpcnit 61 eedyqplmkl gtqtvpcnki llwsrikdla hqftqvqrdm ftledtllgy laddltwcge 121 fntskinyqs cpdwrkdcsn npvsvfwktv srrfaeaacd vvhvmlngsr skifdknstf 181 gsvevhnlqp
  • the amino acid sequence of a representative CAR-engager that contains MSLN is set forth below (SEQ ID NO: 15): 1 malptarpll gscgtpalgs llfllfslgw vqpsrtlage tgqeaapldg vlanppniss 61 lsprqllgfp caevsglste rvrelavala qknvklsteq lrclahrlse ppedldalpl 121 dlllflnpda fsgpqactrf fsritkanvd llprgaperq rllpaalacw gvrgsllsea 181 dvralgglac dlpgrfvaes aevllprlvs cpgpldqdqq eaaraalqgg gppygppstw 241 svstmdalrg ll
  • the ectodomain of the CAR-engager contains a portion of the MSLN protein.
  • the ectodomain of the CAR-engager is IPXGYLVLDLSMQEALS (SEQ ID NO: 17), where X is any amino acid.
  • the ectodomain of the CAR-engager is YXVXDLSMQEL (SEQ ID NO: 18), where X is any amino acid.
  • CAR-E first moiety CAR-binding antibodies and derivatives thereof [000124]
  • the first moiety of the CAR-engager may be an antibody that binds an epitope on the ED of the CAR, or an ED-binding derivative thereof.
  • Antibody derivatives include antibody fragments (e.g., a scFv and nanobody fragments).
  • the first moiety of the CAR- engager is an anti-anti-CD19 antibody binding moiety that binds an epitope on a CAR the EBD of which binds CD19.
  • a representative anti-anti-CD19 binding moiety is set forth below (SEQ ID NO: 124): 32 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 1 qvqlqqpgae lvrpgasvkl scktsgysft rywmnwvkqr pgqglewigm ihpsdsetrl 61 nqkfkdkatl tvdnssstay mqlssptsed savyycasiy yeeawgqgtl vtvsaggggs 121 ggggsggggs diqmtqspas lsasvgetvt itcrasgnih nylawyqqkq gkspqllvyn aktladsvps rfsgsgsgtq yslkinslqp edfgsyy
  • the first moiety of the CAR-engager may be an anti-(G4S) binding moiety that binds an epitope on a CAR the linker of which has at least two repeats of GGGGS (SEQ ID NO: 71).
  • a representative heavy chain variable region (VH) of an anti-(G4S) binding moiety is set forth below (SEQ ID NO: 125): 1 qsvkesggrl vtpgtpltlt ctvsgfslss naidwvrqap gkglewigil grsgstyyas 61 wakgrftisr tssttvdlki tspttedtat yfcarhfylw gpgtlvtvss [000127]
  • a representative light chain variable region (VL) of an anti-(G4S) binding moiety is set forth below (SEQ ID NO: 126): 1 aqvltqtasp vsaavggtvt incqasqsvy snylswyqqk pgqppkllma ttstlepgvp 61 srfkgsgsgt
  • a representative anti- ⁇ light chain binding moiety is set forth below (SEQ ID NO: 110).
  • Anti-anti-mouse binding moieties are known in the art, 33 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 see, Kochenderfer et al., J. Immunother. 32(7):689-702 (2009) and Cheng et al., Cytometry A. 103(1):16-26 (2023).
  • Additional antibodies and derivatives thereof that bind CAR EDs that may be useful are known in the art, see, e.g., U.S. Patent 9,701,758 and U.S. Patent Application Publication 2005/0287148, both of which are incorporated herein by reference in their entireties.
  • the second moiety is an immune cell effector domain that comprises an IL-2 variant as described herein.
  • the IL-2 variant binds the one or more of the cognate receptor subunits of IL-2R ⁇ , IL-2R ⁇ , and IL-2R ⁇ on the immune cell (containing the CAR). This binding event modulates the activity of the CAR-immune cell.
  • modulate(s),” and “modulation” as used herein embrace both activation and inhibition of the CAR immune cell.
  • the CAR-engager contains a plurality (i.e., two, three, or more) of immune cell effector domains, wherein at least one of which is an IL-2 variant described herein, and wherein any two or more of immune effector domains may be the same as or different from each other.
  • the CAR-engager contains two immune effector domains containing two IL-2 variants as disclosed herein. In some embodiments, the CAR-engager contains three immune effector domains containing three IL-2 variants as disclosed herein.
  • the second immune cell effector domain is a cytokine, an immune cell-activating moiety, or an immune cell-inhibiting moiety, and variants and fragments thereof, that bind to their cognate targets on the immune cells.
  • cytokine includes low molecular weight extracellular polypeptides/glycoproteins that promote, modulate, and regulate the immune response (i.e., increase or decrease activity, differentiation, or proliferation).
  • Representative examples of cytokines include chemokines, interferons (IFNs), interleukins (ILs), lymphokines and tumor necrosis factors (TNFs).
  • immune cell activating variant of a cytokine as used herein refers to non-naturally occurring variant of a cytokine capable of binding to a cytokine receptor on an immune cell and initiating signal transduction through that receptor to achieve substantially the same effect as the naturally occurring cytokine.
  • the second immune cell effector domain is an immune cell activating moiety, e.g., immune cell activating cytokines and immune cell-activating variants and 34 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 fragments thereof.
  • Immune cell activating moieties activate, promote, or maintain the activity of immune cells.
  • the second immune cell effector domain is derived from CD40, CD48, CD58, CD70, CD80, CD86, CD112, glucocorticoid-induced TNFR-related protein ligand (GITRL; TNFSF18), herpesvirus entry mediator (HVEM; TNFSF14), Semaphorin 3B (SEMAA; SEMA3B), Signaling lymphocytic activation molecule family member 1 (SLAM; SLAMF1; CD150), T cell immunoglobulin and mucin domain containing 4 (TIM4), TNF superfamily member 4 (TNFSF4; OX40L), TNF superfamily member 8 (TNFSF8; CD30L), interleukin-2 (IL-2), IL-7, IL-9, IL-10, IL-12, IL-15, IL-18, IL-21, IL-27, CCL21, 4-1BBL (also known as TNF superfamily member 9; TNFSF9), or an immune cell-activating variant
  • GITRL TNFSF18
  • the amino acid sequences of representative immune cell activating moieties are from which the second immune cell effector domain may be derived are provided at the NCBI Accession numbers set forth in Table 2, and are incorporated herein by reference.
  • Table 2 Gene Name, Symbols, and NCBI Accession Numbers of Representative Immune Cell- Activating Proteins Gene Name Gene Symbols Protein Accession No(s).
  • the immune cell effector domain contains the weak affinity variant of IL-2 (muIL2), having the amino acid sequence set forth below (SEQ ID NO: 19): 1 aptssstkkt qlqleallld lqmilnginn yknpkltrml takfympkka telkhlqcle 61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [000138]
  • the second immune cell effector domain is an IL-2 variant that has an H16A substitution (i.e., an alanine (A) at amino acid residue 16 in place of the histidine (H)) and
  • the second immune cell effector domain contains at least a portion of the weak affinity IL-2 variant, having together, the amino acid sequence set forth below (SEQ ID NO: 20): 1 aptssstkkt qlqleallld lqmilnginn yknpkltrml takfympkka telkhlqcle 61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tltggggsgg ggsggggggsgg ggsaptsst kktqlqleal lldlqmilng 181 innyknpklt rmltakfymp kkatelkhlq cleeelkple evlnlaqskn fhlrpr
  • the second immune cell effector domain contains at least a portion of the wild-type IL-2, having the amino acid sequence set forth below (SEQ ID NO: 102): 1 aptssstkkt qlqlehllld lqmilnginn yknpkltrml tfkfympkka telkhlqcle 61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [000141]
  • the muIL2 (SEQ ID NO: 19) second immune cell effector domain has a dissociation constant (KD) of about 1200 nM for IL-2R ⁇ (CD25), representing a 110-fold decrease as compared to wild type
  • the second immune cell effector contains at least a portion of IL- 7.
  • the amino acid sequence of a representative IL-7 is set forth below (SEQ ID NO: 21): 1 mfhvsfryif glpplilvll pvassdcdie gkdgkqyesv lmvsidqlld smkeigsncl 61 nnefnffkrh icdankegmf lfraarklrq flkmnstgdf dlhllkvseg ttillnctgq 121 vkgrkpaalg eaqptkslee nkslkeqkkl ndlcflkrll qeiktcwnki lmgtkeh [000143]
  • the second immune cell effector domain contains at least a portion of IL-18.
  • the second immune cell effector domain contains at least a portion of IL-21.
  • the second immune cell effector domain contains at least a portion of IL-27.
  • amino acid sequence of a representative IL-27 is set forth below (SEQ ID NO: 25): 1 mgqtagdlgw rlsllllpll lvqagvwgfp rppgrpqlsl qelrreftvs lhlarkllse 61 vrgqahrfae shlpgvnlyl lplgeqlpdv sltfqawrrl sdperlcfis ttlqpfhall 121 gglgtqgrwt nmermqlwam rldlrdlqrh lrfqvlaagf nlpeeeeeee eeeeerkgl 181 lpgalgsalq gpaqvswpql lstyrllhsl elvlsravre llllskaghs vwplgfptls
  • the second immune cell effector domain contains two repeats of Neo-2/15, having the amino acid sequence of SEQ ID NO: 26. [000149] In some embodiments, the second immune cell effector domain contains at least a portion of 4-1BBL. 4-1BBL is also known as TNF ligand superfamily member 9 (TNFSF9).
  • the amino acid sequence of a representative 4-1BBL is provided at NCBI Accession No. NP_003802, incorporated herein by reference.
  • the second immune cell effector domain 38 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 contains at least a portion of the extracellular domain of 4-1BBL.
  • the second immune cell effector domain contains a portion of the extracellular domain of 4-1BBL, having the amino acid sequence set forth below (SEQ ID NO: 27): 1 dpaglldlrq gmfaqlvaqn vllidgplsw ysdpglagvs ltgglsyked tkelvvakag 61 vyyvffqlel rrvvagegsg svslalhlqp lrsaagaaal altvdlppas searnsafgf 121 qgrllhlsag qrlgvhlhte ararhawqlt qgatvlglfr vtpeipa [000150]
  • the CAR-engager contains three immune cell effector domains, e.g., wherein the first immune cell effector domain is an IL-2 variant and the second and the third immune cell effector domains are the extra
  • the second immune cell effector domain may be a fragment, e.g., a single-chain variable antibody fragment (scFv), that binds and activates the CAR immune cell.
  • the second immune cell effector domain is an scFv that binds an epitope on 4- 1BB, CD2, CD27, CD28, CD30 (TNFRSF8), CD40L, CD226, CTLA4, GITR, IL-2R, LIGHT, OX40, PD-1, TIM2, SLAM, or TIM1.
  • the second immune cell effector domain is derived from a commercially available anti-CTLA4 antibody, antibody fragment, or derivative thereof, e.g., bavunalimab (formerly pavunalimab/XmAb 22841), botensilimab, cadonilimab, ipilimumab (Yervoy®), quavonlimab, tremelimumab (Imjudo®), volrustomig, vudalimab, or zalifrelimab.
  • the second immune cell effector domain is a scFv that binds CTLA4.
  • the amino acid sequences of representative heavy and light chains of antibodies that bind CTLA4 are set forth in Table 3.
  • Table 3 Amino acid Sequences of Representative anti-CTLA Antibody Heavy and Light Chains botensilimab heavy chain (SEQ ID NO: 28) 1 evqlvesggg lvkpggslrl scaasgftfs sysmnwvrqa pgkglewvss isssssyiyy s l d t t q s p t Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 1 qvqlvesggg vvqpgrslrl scaasgftfs sytmhwvrqa pgkglewvtf isydgnnkyy 61 adsvkgrfti srdnskntly lqmnslraed taiyycartg wlgpfdywgq gt
  • the second immune cell effector domain is derived from a commercially available anti-OX40 antibody, antibody fragment (e.g., scFv), or derivative thereof, e.g., tavolimab, or vonlerolizumab (Pogalizumab; MOXR 0916).
  • scFv antibody fragment
  • tavolimab tavolimab
  • vonlerolizumab Pogalizumab; MOXR 0916.
  • Table 4 Amino acid Sequences of Representative anti-OX40 Antibody Heavy and Light Chains tavolimab heavy chain (SEQ ID NO: 38) 1 qvqlqesgpg lvkpsqtlsl tcavyggsfs sgywnwirkh pgkgleyigy isyngityhn s s g y e r s p t y t y v y k g s p t [ 000 56] n some embod ments, t e mmune ce e ector doma n conta ns t e V av ng t e amino acid sequence set forth below (SEQ ID NO 42): 1 diqmtqspss lsasvgdrvt itcrasqdis nylnwyqqkp gkapklliyy tsrlrsgvps 61 r
  • the second immune cell effector domain is derived from a commercially available anti-PD-1 antibody, antibody fragment (e.g., scFv), or derivative thereof, e.g., atezolizumab, avelumab, bintrafusp alfa, cosibelimab, danburstotug, durvalumab (Imfinzi®), inbakicept, lodapolimab, pimivalimab, or socazolimab.
  • Table 5 Amino Acid Sequences of Representative anti-PD-1 Antibody Heavy and Light Chains atezolizumab heavy chain (SEQ ID NO: 44) 1 evqllesggg lvqpggslrl scaasgftfs syimmwvrqa pgkglewvss iypsggitfy v t s 42 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 121 psdeqlksgt asvvcllnnf ypreakvqwk vdnalqsgns qesvteqdsk dstyslsstl 181 tlskadyekh kvyacevthq glsspvtksf nrgec pimivalimab heavy chain (SEQ ID NO: 50) [ ving the amino acid sequence set forth below
  • Immune cell inhibiting moieties repress or block immune cell activity and function.
  • the immune cell-inhibiting moiety may be derived from CD80, CD86, CD112, CD155, CD276 (B7-H3), Ceacam-1, FGL1, galectin- 3, HLA-E, HVEM, PD-L1, PD-L2, VISTA, or VTCN1 (B7-H4).
  • the amino acid sequences of representative immune cell-inhibiting proteins from which the second immune cell effector domain may be derived are provided at the NCBI Accession numbers set forth in Table 6, and are incorporated herein by reference.
  • the second immune cell effector domain contains at least a portion of the extracellular domain of CD86.
  • amino acid sequence of a representative CD155 extracellular domain is set forth below (SEQ ID NO: 56): 1 wpppgtgdvv vqaptqvpgf lgdsvtlpcy lqvpnmevth vsqltwarhg esgsmavfhq 61 tqgpsysesk rlefvaarlg aelrnaslrm fglrvedegn ytclfvtfpq gsrsvdiwlr 121 vlakpqntae vqkvqltgep vpmarcvstg grppaqitwh sdlggmpnts qvpgflsgtv 181 tvtslwilvp ssqvdgknvt ckvehesfek pqlltvnltv yyppevsisg ydnnw
  • the amino acid sequence of a representative galecin-3 extracellular domain is set forth below (SEQ ID NO: 60): 1 madnfslhda lsgsgnpnpq gwpgawgnqp agaggypgas ypgaypgqap pgaypgqapp 61 gaypgapgay pgapapgvyp gppsgpgayp ssgqpsatga ypatgpygap agplivpynl 121 plpggvvprm litilgtvkp nanrialdfq rgndvafhfn prfnennrrv ivcntkldnn 181 wgreerqsvf pfesgkpfki qvlvepdhfk vavndahllq ynhrvkklne isklgisgdi 241 dl
  • the second immune cell effector domain contains at least a portion of the extracellular domain of nectin-2 (CD112, HVEB).
  • amino acid sequence of a representative nectin-2 extracellular domain is set forth below (SEQ ID NO: 63): 1 qdvrvqvlpe vrgqlggtve lpchllppvp glyislvtwq rpdapanhqn vaafhpkmgp 61 sfpspkpgse rlsfvsakqs tgqdteaelq datlalhglt fürgnytce fatfpkgsvr 121 gmtwlrviak pknqaeaqkv tfsqdpttva lciskegrpp ariswlssld weaketqvsg 181 tlagtvtvts rftlvpsgra dgvtvtckve hesfeepali pvtlsvrypp evsisgyddn 241 wyl
  • the amino acid sequence of a representative PD- L1extracellular domain is set forth below (SEQ ID NO: 64): 1 ftvtvpkdly vveygsnmti eckfpvekql dlaalivywe medkniiqfv hgeedlkvqh 61 ssyrqrarll kdqlslgnaa lqitdvklqd agvyrcmisy ggadykritv kvnapynkin 121 qrilvvdpvt seheltcqae gypkaeviwt ssdhqvlsgk tttnskree klfnvtstlr 181 intttneify ctfrrldpee nhtaelvipe lplahppner [000173]
  • the second immune cell effector domain contains at least
  • the second immune cell effector domain contains at least a portion of the extracellular domain of VTCN1 (B7-H4).
  • VTCN1 The amino acid sequence of a representative VTCN1 is set forth below (SEQ ID NO: 66): 1 liigfgisgr hsitvttvas agnigedgil sctfepdikl sdiviqwlke gvlglvhefk 61 egkdelseqd emfrgrtavf adqvivgnas lrlknvqltd agtykcyiit skgkgnanle 121 yktgafsmpe vnvdynasse tlrceaprwf pqptvvwasq vdqganfsev sntsfelnse 181 nvtmkvvsvl ynvtinntys cmiendiaka tgdikvtese ikrrshlqll nskas Dimerization domain 47 Include Draft Include
  • the CAR-engager forms and is administered in the form of a homodimer or a homo-multimer.
  • the homodimer thus contains two CAR-engager entities.
  • the order of the first moiety, the second moiety and the dimerization domain are not critical.
  • the dimerization domain is disposed between the first moiety and the second moiety.
  • the CAR-engager is in the form of a heterodimer, which contains a first moiety connected to a first dimerization domain and a second moiety connected to a second dimerization domain.
  • the first and second dimerization domains dimerize the first and second moieties to form a heterodimer.
  • the first and second dimerization domains contain a knob-in-hole configuration.
  • One of the dimerization domains contains a protuberance (knob) and the other dimerization domain contains a cavity (hole) that is sterically compensatory to the protuberance, where the tertiary structure of the protuberance is positionable within the tertiary structure of the cavity.
  • Dimerization domains with knob-in-hole configurations may have directed amino acid mutations where the protuberance is an amino acid that has a larger side chain volume than present on a dimerization domain derived from a natural source (e.g., IgA, IgD, IgG, IgM, or IgE) and the cavity is an amino acid that has a smaller side chain volume than present on a dimerization domain derived from a natural source.
  • the protuberance is an amino acid change from a threonine (T) to a lysine (K) and the corresponding cavity is an amino acid change from a leucine (L) to an aspartic acid (D) or a lysine (K).
  • the first dimerization domain contains two amino acid substitutions, for example, a threonine (T) to a lysine (K) and a leucine (L) to a lysine (K), while the second dimerization domain contains a leucine (L) to an aspartic acid (D) or a glutamic acid (E) and a tyrosine (Y) to a glutamic acid (E) or aspartic acid (D).
  • the knob-in-hole dimerization domains are based on opposed charges.
  • the first dimerization domain contains a positively charged amino acid and the second dimerization domain contains a negatively charged amino acid sterically opposable to the positively charged amino acid on the first dimerization domain.
  • Additional protuberance and cavity arrangements are known in the art. See, e.g., U.S. Patents 5,821,333, 7,183,076, 8,642,745, 9,248,182, 9,309,311, 9,527,927, 9,562,109, 9,890,204, 48 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 10,138,303, and 11,168,344 and U.S.
  • the dimerization domains may be derived from IgA, IgD, IgG, IgM, or IgE.
  • the first and the second dimerization domains may contain the same or different amino acid sequences, provided that they bind each other.
  • the first and the second dimerization domains are the IgG1 constant heavy (CH) 3 domain.
  • the amino acid sequence of a representative IgG1 CH3 domain is set forth below (SEQ ID NO: 67): 1 epkspksadk thtapqprep qvytlppsrd eltknqvslt clvkgfypsd iavewesngq 61 pennykttpp vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg 121 k [000182]
  • the first and the second dimerization domains are the IgG1 constant heavy CH2 domain.
  • the amino acid sequence of a representative IgG1 CH2 domain is set forth below (SEQ ID NO: 68): 1 pcpapellgg psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna 61 ktkpreeqyn styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kak [000183]
  • the first and the second dimerization domains are the IgG1 CH2 and CH3 domains.
  • the CH2 and CH3 domains may be interconnected by a linker.
  • the first, the second, or both the first and the second dimerization domains contain a fragment crystallizable region (Fc).
  • the Fc contains L234A and L235A substitutions relative to wild-type Fc that abolishes binding of the Fc to (1) the Fc- ⁇ receptor and (2) the complement component 1q (C1q), referred herein as a “silent Fc”.
  • the silent Fc maintains binding to the neonatal Fc receptor (FcRn) (and therefore extending circulatory half-life of CAR-E which contains the silent Fc to several days).
  • Silent Fc also provides a stabilizing effect to the CAR- E (comparable to the stabilizing effect of wild-type Fc).
  • the silent Fc also has a P329G substation; these three L234A, L235A, and P329G substitutions are also commonly referred to as “PG-LALA”.
  • Linkers [000184] In some embodiments, the CAR-engager contains one or more linkers.
  • a linker is “flexible” when the peptide bonds allow rotation of the amino acid residues within the linker and allow the first and the second moieties to move and bind to their respective cognate receptors on the 49 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 CAR-expressing immune cell or steric spacing (i.e., a spacer) between the first and the second moieties.
  • a linker may be disposed between any two CAR-engager components (also referred to herein as domains, entities, moieties, portions).
  • a linker may be disposed between the dimerization domain and the adjacent domain.
  • a linker may be disposed between the dimerization domain and the second moiety.
  • the CAR-engager contains two linkers, where a first linker is disposed between the first moiety and the dimerization domain, and a second linker is disposed between the dimerization domain and the second moiety.
  • the linker comprises an amino acid having the sequence GGGX, GGGGX (SEQ ID NO: 69), or GSSGSX (SEQ ID NO: 70), where X is any nucleotide, typically either cysteine (C) or serine (S), or repeating sequence thereof.
  • the linker has the amino acid sequence GGGGS (SEQ ID NO: 71), GSPRG (SEQ ID NO: 72), GGGGSGGGGS (SEQ ID NO: 73), GGGGSGGGGSGGGGS (SEQ ID NO: 74), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 75), GSPRGGGGSGGGGSGGGGS (SEQ ID NO: 76), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 77), KESGSVSSEQLAQFRSLD (SEQ ID NO: 78), EGKSSGSGSESKST (SEQ ID NO: 79), or GSAGSAAGSGEF (SEQ ID NO: 80).
  • the linker may be derived from IgA, IgD, IgE, IgG, or IgM. In some embodiments, the linker may be derived from the hinge region of CD3 ⁇ , CD4, CD8 ⁇ , CD28, IgG1, IgG2, or IgG4. Amino acid sequences of representative linkers are listed in Table 7. Table 7: Amino acid Sequences of Representative Linkers Linker Sequence A 50 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000189] In some embodiments, the CAR-engager is in the form of a fusion protein, where the components are linked by peptide bonds.
  • the CAR-engager contains proteinaceous entities that may be covalently connected interconnected by click chemistry, which is a type of chemical connection formed by a method of controlled chemical ligation.
  • the connection may be an azide-alkyne connection, an oxime or hydrazine connection, a tetrazine-transcyclooctene connection, an azide-nitrone connection, a thiol-alkene connection, an alkene-tetrazole connection, an alkene-tetrazine connection, an alkene-azide connection, a conjugated diene-alkene connection, or an isonitrile-tetrazine connection.
  • CAR-E or the IL-2 variant may be encoded in a nucleic acid which is expressed in a cell to produce the CAR-E.
  • nucleic acid refers to a polymer of nucleotides, each of which are organic molecules consisting of a nucleoside (a nucleobase and a five-carbon sugar) and a phosphate.
  • nucleotide unless specifically stated or obvious from context, includes nucleosides that have a ribose sugar (i.e., a ribonucleotide that forms ribonucleic acid, RNA) or a 2’-deoxyribose sugar (i.e., a deoxyribonucleotide that forms deoxyribonucleic acid, DNA).
  • Nucleotides serve as the monomeric units of nucleic acid polymers or polynucleotides.
  • the four nucleobases in DNA are guanine (G), adenine (A), cytosine (C) and thymine (T).
  • the four nucleobases in RNA are guanine (G), adenine (A), cytosine (C) and uracil (U).
  • Nucleic acids are linear chains of nucleotides (e.g., at least 3 nucleotides) chemically bonded by a series of ester linkages between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar (i.e., ribose or 2’-deoxyribose) in the adjacent nucleotide.
  • nucleotides e.g., at least 3 nucleotides
  • nucleic acid sequence of a representative wild-type IL-2 is set forth below (NCBI Accession No. NM_000586.4; SEQ ID NO: 111;): 51 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 1 ctatcaccta agtgtgggct aatgtaacaa agagggattt cacctacatc cattcagtca 61 gtctttgggg gtttaaagaa attccaaaga gtcatcagaa gaggaaaaat gaaggtaatg 121 ttttttcaga caggtaaagt ctttgaaaat atgtgtaata tgtaaacat tttgacaccc 181 ccataatatt ttccagaat tacagtata aattgcatc
  • nucleic acid encoding the CAR-engager includes a signal peptide- encoding nucleic acid disposed 5’ to the nucleic acid encoding the first moiety.
  • signal peptide refers to a short (e.g., 5-30 or 10-100 amino acids long) stretch of amino acids that directs the transport of the protein during translation.
  • CAR-engagers containing a signal peptide will be secreted from the cell. Typically, the signal peptide is cleaved from the CAR-engager before secretion.
  • the signal peptide may be connected to the nucleic acid encoding the first moiety or the nucleic acid encoding the second moiety.
  • the signal peptide may be derived from Ig- ⁇ -3 heavy chain (IGHG3), albumin, CD8 ⁇ , CD33, erythropoietin (EPO), IL-2, human or mouse Ig-kappa chain V- III (IgK VIII), tissue plasminogen activator (tPA), or secreted alkaline phosphatase (SEAP).
  • IGHG3 Ig- ⁇ -3 heavy chain
  • EPO erythropoietin
  • IL-2 human or mouse Ig-kappa chain V- III
  • IgK VIII tissue plasminogen activator
  • SEAP secreted alkaline phosphatase
  • Signal peptides may also be synthetic (i.e., non-naturally occurring). Amino acid sequences of representative signal peptides are listed in Table 8.
  • Table 8 Amino acid Sequences of Representative Signal Peptides Signal peptide Sequence 52 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 CD33 (SEQ ID NO: 92) MPLLLLLPLLWAGALA EPO (SEQ ID NO: 93) MGVHECPAWLWLLLSLLSLPLGLPVLG [000196] IL-2 variant-encoding nucleic acids and CAR-engager-encoding nucleic acids may be introduced into a cell by a suitable vector.
  • the CAR-encoding nucleic acids may be introduced into one or more cells by separate vectors.
  • a vector is configured so as to contain the elements necessary to effect transport into the immune cell and effect expression of the nucleic acid(s) after transformation.
  • Such elements include an origin of replication, a poly-A tail sequence, a selectable marker, and one or more suitable sites for the insertion of the nucleic acid sequences, such as a multiple cloning site (MCS), one or more suitable promoters, each promoter operatively linked to the insertion sites of the nucleic acid sequences and the selectable marker, and additional optional regulatory elements.
  • MCS multiple cloning site
  • promoter refers to a nucleic acid sequence that regulates, directly or indirectly, the transcription of a corresponding nucleic acid coding sequence to which it is operably linked, which in the context of the present disclosure, is an IL-2 variant, and in other aspects, a CAR-engager protein that contains the variant.
  • a promoter may function alone to regulate transcription, or it may act in concert with one or more other regulatory sequences (e.g., enhancers or silencers, or regulatory elements that may be present in the nucleic acid sequences or the vector). Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (toward the 5’ region of the sense strand).
  • Promoters typically range from about 100-1000 base pairs in length. 53 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000198]
  • the term “operatively linked” as used herein is to be understood that a nucleic acid sequence is spatially situated or disposed in the vector relative to another nucleic acid sequence, e.g., a promoter is operatively linked to drive the expression of a nucleic acid coding sequence (e.g., the CAR-engager-encoding nucleic acid sequence).
  • a single vector contains a single promoter operatively linked to the CAR-engager-encoding nucleic acid of the enhancer.
  • a single vector contains a single promoter operatively linked to the first moiety-encoding nucleic acid and the second moiety-encoding nucleic acid.
  • the nucleic acids are separated by a nucleic acid encoding a self-cleaving peptide or an internal ribosome entry site (IRES).
  • the single vector contains a first promoter operatively liked to the first moiety- encoding nucleic acid and a second promoter operatively liked to the first moiety -encoding nucleic acid.
  • two vectors are constructed.
  • a first vector contains a promoter operatively linked to the first moiety-encoding nucleic acid and a second vector contains a promoter operatively linked to the second moiety-encoding nucleic acid.
  • the vector contains a strong mammalian promoter, for example a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, synthetic promoters (e.g., RPBSA (synthetic, from Sleeping Beauty), or CAG (synthetic, CMV early enhancer element, chicken ⁇ -Actin, and splice acceptor of rabbit ⁇ -Globin)) or promoters derived from the ⁇ -actin, phosphoglycerate kinase (PGK), or factor EF1 ⁇ genes.
  • CMV cytomegalovirus
  • SV40 simian virus 40
  • synthetic promoters e.g., RPBSA (synthetic, from Sleeping Beauty), or CAG (sy
  • the promoter may contain a core region located close to the nucleic acid coding sequence.
  • the promoter is modified to remove methylation sensitive motifs (e.g., a cytosine nucleotide is followed by a guanine nucleotide, or “CpG”), or by the addition of a regulatory sequence that binds transcriptional factors that repress DNA methylation.
  • the vector includes A/T-rich, nuclear matrix interacting sequences, known as scaffold matrix attachment regions (S/MAR), which enhance transformation efficiency and improve the stability of transgene expression.
  • the vector is a viral vector, for example, a retroviral vector, a lentiviral vector, an adenoviral vector, a herpesvirus vector, an adenovirus, or an adeno-associated virus (AAV) vector.
  • lentiviral vectors have been described, for example, in U.S. Patents 5,665,577, 5,981,276, 6,013,516, 7,090,837, 8,119,119 and 10,954,530.
  • 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.
  • the vector may include both viral and non- viral elements.
  • the vector is a plasmid.
  • the plasmid may also contain other elements e.g., that facilitate transport and expression of the nucleic acid in an immune cell.
  • the plasmid may be linearized with restriction enzymes, in vitro transcribed to produce mRNA, and then modified with a 5’ cap and a 3’ poly-A tail.
  • the vector multiple plasmids, a first plasmid encoding a first proteinaceous entity (e.g., the ectodomain of the CAR-engager) and a second plasmid encoding a second proteinaceous entity (e.g., the immune effector domain of the CAR-engager).
  • the IL-2 variants and CAR-Es may be expressed in a genetically modified (or transformed) cell containing a vector that contains a nucleic acid encoding an IL-2 variant, the CAR- E, or components of the CAR-E for the purpose of making and purifying protein.
  • Cells useful for the cloning and other manipulations of these vectors are conventional. Cells from various strains of E. coli may be used for replication of the vectors and other steps in the construction of the CAR-engagers of this disclosure.
  • Suitable host cells or cell lines for the expression of the nucleic acids encoding the IL-2 variants and the nucleic acid-encoding CAR-engagers include eukaryotic cells.
  • the cells are a mammalian cell line.
  • the cells are mammalian cells such as CHO (e.g., DG44, CHO-S), fibroblast cells (e.g., 3T3, COS), embryonic cells (e.g., PER.C6, HEK (e.g., HEK.293)), somatic cell hybrids (e.g., Sp2/0), and cancer cells, for example, myeloma cells (e.g., NS0 (NS zero)).
  • the nucleic acids encoding the CAR- engager is expressed in a CHO or a myeloma cell.
  • Human cells may be used, thus enabling the expressed CAR-engager to be modified with human glycosylation patterns.
  • suitable mammalian cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Green et al., eds., Molecular Cloning: A Laboratory Manual, 5 th ed., Cold Spring Harbor Laboratory Press, New York, 2012. 55 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000208]
  • the cells are prokaryotic.
  • Prokaryotic (i.e., bacterial) cells may prove useful as host cells suitable for the expression of the nucleic acids encoding CAR-engagers (see, e.g., Pluckthun, Immunol. Rev.130:151-188 (1992)).
  • any CAR-engagers produced in a bacterial cell would be screened for retention of function (e.g., CAR binding ability of an expressed CAR-E).
  • the CAR-engager expressed by the bacterial cell was produced in a properly folded form, that bacterial cell would be a desirable host, or in alternative embodiments the CAR-engager may express in the bacterial host and then be subsequently re-folded.
  • various strains of E. Coli used for expression are well-known as host cells in the field of biotechnology.
  • Various strains of B. Subtilis, Streptomyces, other bacilli and the like may also be employed.
  • the IL-2 variant or the CAR-engager protein is isolated from the cell (e.g., cell lysates) or from the medium in which the cell is cultured. Protein isolation techniques are known in the art.
  • Representative isolation techniques include chromatography, affinity chromatography, nickel- nitrilotriacetic acid (Ni-NTA) affinity chromatography, high performance liquid chromatography (HPLC), hydroxylapatite chromatography, protein A- Sepharose, gel electrophoresis, and dialysis.
  • the affinity chromatography resin is a Protein A affinity chromatography resin or a Protein G affinity chromatography resin. Additional protein isolation systems and methods are known in the art. See, e.g., U.S. Patents 516,9936, 6,267,958, 8,357,778, 9,630,165, 9,708,399, 10,023,608, 10,207,229, 11,369,703, and 11,390,668, U.S.
  • compositions containing an IL-2 variant or CAR-E may be formulated in a pharmaceutically acceptable carrier.
  • an effective amount refers to a sufficient amount of an IL-2 variant or a CAR-engager to provide the desired effect, e.g., which may include any one or more of killing at least cancer cell, reversing, alleviating, ameliorating, inhibiting, diminishing, slowing down, arresting, stabilizing, achieving remission, or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a cancer.
  • IL-2 variant and the CAR-engager administered to a subject will vary between wide limits, depending 56 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 upon the location, type, and severity of the cancer, the age, body weight, and condition of the individual to be treated, etc. A physician will ultimately determine appropriate doses to be used.
  • the IL-2 variant and/or the CAR-engager in the pharmaceutical composition may be in the form of a monomer (in embodiments lacking a dimerization domain), homodimer, or heterodimer, as described herein. [000211] IL-2 variants of the present disclosure may be used alone or in combination with another active agents to treat cancer.
  • High-dose IL-2 treatment can overcome regulatory T cell (Treg)-associated IL-2 trapping and allow extra IL-2 to activate tumor infiltrating lymphocytes (TILs) for treating cancer, for example, metastatic renal cell carcinoma and melanoma.
  • TILs tumor infiltrating lymphocytes
  • patients who respond to high-dose IL-2 treatment frequently suffer from intolerable toxicities (See, e.g., Li et al., Nat. Commun.8(1):1762 (2017)), which has limited its clinical use.
  • IL-2 variants of the present disclosure may constitute a more attractive alternative in terms of binding to IL-2R ⁇ on Tregs (See, e.g., Mott et al., J. Mol. Biol.247(5):979-94 (1995)) or increasing the binding to IL-2R ⁇ on effector cells (See, e.g., Levin et al., Nature 484(7395):529-33 (2012) and Sun et al., Nat. Commun. 10(1):3874 pp. 1-12 (2019)).
  • IL-2 variants of the present disclosure may be conjugated to other active moieties, such as antibodies. See, Ren, et al., supra.
  • the IL-2 variants of the present disclosure may be fused to an anti–PD-1 antibody. These fusion proteins may show better intratumoral T cell binding, more potent antitumor effects, and may also overcome PD-L1 therapy resistance.
  • the IL-2 variants may also be conjugated to anti-tumor antibodies. See, e.g., Sun et al., Nat. Commun.10(1):3874 pp.1-12 (2019).
  • Compositions may be provided as sterile solid or liquid preparations.
  • Solid preparations may be reconstituted and diluted into a liquid preparation before use, e.g., with carriers containing isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous solutions, which may be buffered to a selected pH.
  • Liquid carriers include aqueous and non-aqueous carriers alike. Representative examples of liquid carriers include sterile water for injection, saline, Lactated Ringer Injection solution, phosphate buffered saline, soluble sugars (e.g., dextrose), dimethyl sulfoxide (DMSO), ethanol, and suitable mixtures thereof.
  • the liquid carrier includes a protein dissolved or dispersed therein, representative examples include serum albumin (e.g., human serum albumin, recombinant human albumin), gelatin, and casein.
  • the liquid carrier includes a water-miscible polyol (e.g., glycerol, propylene glycol, liquid 57 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 polyethylene glycol, and the like).
  • the compositions are typically isotonic, i.e., they have the same osmotic pressure as blood.
  • Citric acid, sodium chloride, sugars, polyalcohols, and isotonic electrolyte solutions may be used to achieve the desired isotonicity.
  • other excipients may be added, e.g., wetting, dispersing, or emulsifying agents, gelling and viscosity enhancing agents, preservatives and the like as known in the art.
  • the compositions include citric acid, ethylenediaminetetraacetic acid (EDTA), and polysorbate 20 with a pH range between about 6.8 to about 7.2.
  • the methods may entail administration of the IL-2 variant, per se, or in the form of a fusion with another active moiety, such as an antibody or binding fragment thereof, that binds a receptor on an immune cell, e.g., a TIL, or antibody that binds an epitope on an antigen present on a tumor cell.
  • the administration of the IL-2 variant is used to treat kidney cancer (e.g., metastatic renal cell carcinoma), melanoma, colon cancer, lung cancer, or ovarian cancer.
  • the method entails administering to a subject in need thereof a pharmaceutical composition containing a CAR-engager described herein.
  • Cancers treatable in accordance with the disclosed methods broadly include hematopoietic cancers and cancers characterized by the presence of a solid tumor.
  • subject or “patient” as used herein includes all members of the animal kingdom prone (or disposed) to or suffering from the indicated cancer. In some embodiments, the subject is a human.
  • a subject “having cancer” or “in need of” treatment broadly embraces subjects who have been positively diagnosed, including subjects having active disease who may have been previously treated with one or more rounds of therapy, and subjects who are not currently being treated (e.g., in remission) but who might still be at risk of relapse, and subjects who have not been positively diagnosed but who are predisposed to cancer (e.g., on account of the basis of prior medical history and/or family medical history, or who otherwise present with a one or more risk factors such that a medical professional might reasonably suspect that the subject was predisposed to cancer).
  • treat refers to the aft-recognized indicia of therapeutic efficacy, intervention, process performed on, or the administration of an active agent to the subject in need thereof with the therapeutic objective (“therapeutic effect”) of reversing, alleviating, ameliorating, inhibiting, diminishing, slowing down, arresting, stabilizing, or preventing the onset, progression, development, metastases, severity or recurrence of a symptom, improvement in survival time, total/complete or partial remission, complication or condition, or biochemical indicia associated with cancer.
  • therapeutic objective therapeutic effect
  • the cancer is a hematopoietic cancer.
  • Representative hematological cancers include plasma cell neoplasm (e.g., myeloma, multiple myeloma, relapsed or refractory multiple myeloma, plasma cell myeloma, extramedullary multiple myeloma, monoclonal gammopathy of unknown significance (MUGS), asymptomatic smoldering multiple myeloma, or solitary plasmacytoma), lymphoma (e.g., Hodgkin’s lymphoma (HL), non-Hodgkin’s lymphoma, Burkitt lymphoma, Waldenstrom macroglobulinemia, plasmablastic lymphoma, plasmacytoid lymphoma, B-cell lymphoma, high-grade B-cell lymphoma
  • plasma cell neoplasm e.g., myeloma, multiple myeloma, relapsed
  • the cancer is characterized by the presence of a solid tumor.
  • the cancer is a bladder cancer (e.g.,transitional cell carcinoma, also called urothelial carcinoma), kidney cancer (e.g., renal cell carcinoma (RCC), kidney renal clear cell carcinoma (KIRC), transitional cell cancer, or Wilms tumor), skin cancer (e.g., melanoma, skin cutaneous melanoma (SKCM), basal cell carcinoma, and squamous cell carcinoma of the skin), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC)), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN) also called head and neck squamous cell carcinoma (HNSC), laryngeal and hypopharyngeal cancer, nasal cavity and paranasal sinus cancer, na
  • a bladder cancer e.g.,
  • the disclosed methods treat malignant mesothelioma, ovarian cancer, breast cancer (e.g., TNBC), pancreatic cancer, lung cancer, liver cancer, glioblastoma, gastric cancer, endometrial cancer, cervical cancer, biliary cancer, uterine serous carcinoma, cholangiocarcinoma, neuroblastoma, sarcoma, or melanoma.
  • the disclosed methods further include an anti-CD19 immune cell therapy, a CAR-E that binds CD19, and are used to treat HL, non-Hodgkin’s lymphoma, ALL, CLL, chronic lymphocytic leukemia, Burkitt lymphoma, DLBCL, PMBL, high-grade B-cell lymphoma, FL, MCL, or multiple myeloma (MM).
  • HL non-Hodgkin’s lymphoma
  • ALL CLL
  • chronic lymphocytic leukemia Burkitt lymphoma
  • DLBCL DLBCL
  • PMBL high-grade B-cell lymphoma
  • FL FL
  • MCL multiple myeloma
  • the disclosed methods further include anti-BCMA immune cell therapy and a CAR-E that binds BCMA, and are used to treat MM, HL, non-Hodgkin’s lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), plasma cell leukemia, SLE, acute AMR, chronic AMR, or AL-amyloidosis.
  • the cancer is characterized as being in a state of minimal residual disease (MRD).
  • MRD is a state at which a cancer patient has a small number of cancer cells that remain in the body after treatment.
  • the number of remaining cells may be so small that they do not cause any physical signs or symptoms of the cancer, and often may not be detectable through traditional methods, such as viewing cells under a microscope and/or by tracking abnormal serum proteins in the blood.
  • the amount of cancer antigens present in a subject in a state of MRD are limited. And this limited presence of the cancer antigen may not adequately support the proliferation and efficacy of CAR immune cells.
  • the additional presence of a CAR-engager presents the CAR immune cells with not only additional cancer antigen, but also a supportive IL-2 variant, and in some embodiments, a second immune cell effector domain, that may modulate the activity of the CAR immune cell to promote proliferation, efficacy, and/or persistence.
  • the subject receiving an administration of CAR-engager is in a state of MRD.
  • the method of treating cancer involves treatment of a state of minimal residual disease (MRD) in the subject.
  • the method of treating cancer involves elimination of MRD in the subject.
  • MRD minimal residual disease
  • samples from either a blood draw or a bone marrow aspiration may be used. The most widely used tests to measure MRD are flow cytometry, polymerase chain reaction (PCR) and next-generation sequencing.
  • the CAR extracellular domain is derived from a commercially available anti-BCMA antibody, BCMA-binding fragment, or derivative thereof, e.g., belantamab (Blenrep®), linvoseltamab (REGN5458), pacanalotamab (AMG 420), pavurutamab (AMG 701), and teclistamab (Tecvayli®).
  • the extracellular domain of the CAR binds the BCMA ectodomain of the CAR-engager that has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the CAR extracellular domain contains a single variable heavy (VHH) or variant thereof.
  • the CAR extracellular domain is derived from a commercially available anti-Claudin 18.2 antibody, anti-Claudin 18.2-binding fragment, or derivatives thereof, e.g., osemitamab and zolbetuximab (Vyloy®).
  • the extracellular domain of the CAR binds a Claudin 18.2 ectodomain of the CAR-engager.
  • the CAR-engager contains a Claudin 18.2 ectodomain that has any one of the amino acid sequences SEQ ID NOs: 8-9. [000238]
  • the CAR binds SLAMF7.
  • the extracellular domain of the CAR binds the SLAMF7 63 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 ectodomain of the CAR-engager. In some embodiments, the extracellular domain of the CAR binds the SLAMF7 ectodomain of the CAR-engager having the amino acid sequence SEQ ID NO: 10. [000239] In some embodiments, the CAR binds PD-1. CAR extracellular domains that bind to PD- 1 are known in the art. See, e.g., U.S. Patents 10,124,023 and 11,136,392, and U.S.
  • the CAR extracellular domain is derived from a commercially available anti-PD-1 antibody, anti- PD-1-binding fragment, or derivative thereof, e.g., balstilimab, budigalimab, cadonilimab, cemiplimab (Libtayo®), cetrelimab, dostarlimab (Jemperli®), izuralimab, nivolumab (Opdivo®), pacmilimab, pembrolizumab (Keytruda®), penpulimab, peresolimab, pidilizumab, retifanlimab, rosnilimab, sintilimab, spartalizumab, tislelizumab, toripalimab, volrustomig, vudalimab, zeluvalimab, and zi
  • balstilimab e.g., balstilimab, budigalimab, cadoni
  • the extracellular domain of the CAR binds a PD-1 ectodomain of the CAR-engager. In some embodiments, the extracellular domain of the CAR binds the PD-1 ectodomain of the CAR-engager that has any one of the amino acid sequences SEQ ID NO: 11 and 107-108. [000240] In some embodiments, the CAR binds Mast/stem cell growth factor receptor Kit (KIT; also known as Receptor tyrosine kinase KIT proto-oncogene). CAR extracellular domains that bind KIT are known in the art. See, e.g., U.S.
  • KIT Mast/stem cell growth factor receptor Kit
  • the CAR extracellular domain is derived from a commercially available anti-KIT antibody, anti-KIT-binding fragment, or derivative thereof, e.g., barzolvolimab.
  • the extracellular domain of the CAR binds the KIT ectodomain of the CAR-engager.
  • the extracellular domain of the CAR binds the KIT ectodomain of the CAR-engager having the amino acid sequence SEQ ID NO: 12. [000241]
  • the CAR binds TROP2.
  • CAR extracellular domains that bind TROP2 are known in the art. See, e.g., U.S. Patents 11,602,525, and 11,768,203, and U.S. Patent Application Publications 2018/0296689, 2021/0169852, and 2022/0204582.
  • the CAR extracellular domain is derived from a commercially available anti-TROP2 antibody, anti- TROP2-binding fragment, or derivatives thereof, e.g., datopotamab and sacituzumab.
  • the extracellular domain of the CAR binds a TROP2 ectodomain of the CAR-engager.
  • the CAR-engager contains a TROP2 ectodomain that has the amino acid sequence SEQ ID NO: 13. 64 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000242]
  • the CAR binds CD38.
  • CAR extracellular domains that bind CD38 are known in the art. See, e.g., U.S. Patents 10,709,775, 10,799,536, 10,836,998, and 11,365,394 and U.S.
  • the CAR extracellular domain is derived from a commercially available anti-CD38 antibody, anti-CD38-binding fragment, or derivative thereof, e.g., daratumumab (Darzalex®), isatuximab (Sarclisa®), and mezagitamab.
  • the extracellular domain of the CAR binds the CD38 ectodomain of the CAR- engager.
  • the extracellular domain of the CAR binds the CD38 ectodomain of the CAR-engager having the amino acid sequence SEQ ID NO: 14. [000243] In some embodiments, the CAR binds MSLN. CAR extracellular domains that bind MSLN are known in the art. See, e.g., U.S. Patents 10,550,179, 10,640,569, 10,730,954, 11,648,268, and 11,702,472, and U.S.
  • the CAR extracellular domain is derived from a commercially available anti-MSLN antibody, anti-MSLN-binding fragment, or derivative thereof, e.g., amatuximab.
  • the extracellular domain of the CAR binds a MSLN ectodomain of the CAR-engager.
  • the extracellular domain of the CAR binds the MSLN ectodomain of the CAR-engager that has any one of the amino acid sequences SEQ ID NO: 15 and 17-18.
  • the intracellular domain of the CAR contains a signaling domain that enables intracellular signaling and immune cell function.
  • the signaling domain may include a primary signaling domain and/or a co-stimulatory signaling domain.
  • the intracellular domain is capable of delivering a signal approximating that of natural ligation of an ITAM- containing molecule or receptor complex such as a TCR receptor complex.
  • the signaling domain includes a plurality, e.g., 2 or 3, costimulatory signaling domains, e.g., selected from 4-1BB, CD3 ⁇ , CD28, CD27, ICOS, and OX40.
  • the signaling domain may include a CD3 ⁇ domain as a primary signaling domain, and any of the following pairs of co-stimulatory signaling domains from the extracellular to the intracellular direction: 4-1BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28-4-1BB; OX40- CD28; CD28-OX40; 4-1BB-CD3 ⁇ ; CD3 ⁇ -4-1BB; CD28-CD3 ⁇ ; CD3 ⁇ -CD28; CD28-4-1BB and 4- 1BB-CD28.
  • the primary signaling domain is derived from CD3 ⁇ , CD27, CD28, CD40, KIR2DS2, MyD88, or OX40.
  • the co-stimulatory signaling 65 include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 domain is derived from one or more of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8 ⁇ , 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 signal transducer ((HCST)), DAP12 (TYROBP), Dectin-1, Fc ⁇ RI, Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII, IL-2RB, ICOS, KIR2DS2, MyD88, OX40, and ZAP70.
  • HCST hematopoietic cell signal transducer
  • DAP12 TYROBP
  • a representative CAR with a CD3 ⁇ stimulatory signaling domain is the FDA-approved CAR-expressing immune cells tisagenlecleucel (Kymriah®).
  • Representative CARs with CD3 ⁇ and 4-1BB co-stimulatory signaling domains are the FDA-approved CAR-expressing immune cells idecabtagene vicleucel (Abecma®), lisocabtagene maraleucel (Breyanzi®), and ciltacabtagene autoleucel (Carvykti®).
  • Representative CARs with CD28 and CD3 ⁇ co-stimulatory signaling domains are the FDA-approved CAR-expressing immune cells brexucabtagene autoleucel (Tecartus®) and axicabtagene ciloleucel (Yescarta®).
  • the CAR immune cell is a T cell.
  • the CAR immune cell is a NK cell. Additional CAR immune cells are known in the art, e.g., U.S.
  • the CAR immune cells may be autologous or allogeneic.
  • immune cells or progenitors thereof can be isolated from a tissue of body fluid from one subject prior to administration to the same subject (autologous) or a different, compatible subject (allogeneic). Most typically, the CAR immune cells are administered once.
  • the number of CAR immune cells administered to a subject will vary between wide limits, depending upon the location, type, and severity of the cancer, the age, body weight, and condition of the individual to be treated, etc. A physician will ultimately determine appropriate number of cells and doses to be used. Typically, the CAR immune cells will be given in a single, one-time dose.
  • Dosage amounts (e.g., numbers) of CAR immune cells effective to treat cancer are known in the ar. In some embodiments, the effective number of the CAR immune cells is between about 1 ⁇ 10 4 to about 1 ⁇ 10 10 cells per subject. In some embodiments, the effective number of the CAR immune cells is between about 1 ⁇ 10 5 to about 1 ⁇ 10 10 cells per subject.
  • the effective number of the CAR immune cells is about the number of cells given in FDA-approved 66 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 CAR T cell therapies, which is between about 1 ⁇ 10 6 to about 1 ⁇ 10 10 cells per kg of subject body weight. In some embodiments, the effective number of the CAR immune cells is between about 1 ⁇ 10 5 to about 6 ⁇ 10 8 cells per kg of subject body weight. [000251] Since the CAR-engager promotes functionality and persistence of CAR immune cells, CAR therapy that contemplates coordinate administration of the CAR-engager may entail use of fewer CAR immune cells compared to FDA-approved CAR T cell therapies.
  • the CAR immune cells may be administered to a subject for the treatment of a cancer by any medically acceptable route.
  • the CAR immune cells are typically delivered intravenously, although they may also be introduced into other convenient sites (e.g., to an affected organ or tissue) or modes, as determined by an attending physician.
  • Administration of the CAR-E [000253] Broadly, the order in which the CAR-engager and CAR immune cells are administered during the same course of treatment may be varied, provided that they are able to interact in vivo and cause the desired effect.
  • the CAR-engager is co-administered substantially simultaneously to the subject with the CAR immune cells. In some embodiments, the CAR-engager is contacted with the CAR immune cells in vitro before co-administration to the subject. In some embodiments, the CAR-engager is administered to the subject subsequent to the administration of the CAR immune cells. In some embodiments, the CAR-engager is administered to the subject prior to the administration of the CAR immune cells.
  • the methods entail administration of an effective amount of the CAR-engager to a cancer patient who had received, is receiving, or will receive an administration of immune cells containing a CAR that contains an extracellular domain to which the CAR-engager binds, a transmembrane domain, and an intracellular domain comprising a stimulatory domain.
  • the order in which the CAR-engager and CAR immune cells are administered during the same course of treatment may not be critical, provided that they are able to interact in vivo and cause the desired effect.
  • the CAR-engager is co-administered substantially simultaneously to the subject with the CAR immune cells.
  • the CAR-engager is contacted with 67 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 the CAR immune cells in vitro before co-administration to the subject.
  • the CAR-engager is administered to the subject prior to the administration of the CAR immune cells.
  • the CAR-engager is administered to the subject subsequent to the administration of the CAR immune cells, e.g., upon a determination that the CAR-immune cells have lost vitality or persistence in the subject. This determination may be made in accordance with known techniques.
  • a sample is obtained from the subject after the administration of the immune cells.
  • the concentration of immune cells present within the sample may be used to calculate the difference between the concentration of the immune cells administered to the subject and the concentration of the immune cell measured in the sample.
  • the CAR-engager may be administered once the measured immune cell concentration is less than the administered immune cell concentration. In some embodiments, the CAR-engager is administered once the measured immune cell concentration 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 administered concentration of immune cells.
  • administration of the CAR-engager is conducted at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 2 months, at least, at least about 3 months, at least about 6 months, at least about 9 months, or at least about a year after administering the CAR-immune cells.
  • the CAR-engager is administered for at least one cycle, for example, administered once a week, once every two weeks, once every three weeks. The cycle may be repeated, for example, for 2 cycles, 3 cycles, 5 cycles, or8 cycles.
  • the CAR-engager is administered for a period of consecutive days before cyclic administration, for example, administered once a day for five days and once every three weeks thereafter. In some embodiments, the CAR-engager is administered once every 3 weeks (a 21-day cycle) as an infusion over about 30 to about 90 minutes. In some embodiments, the CAR-engager is administered for 5 consecutive days every 21 days and repeated for 8 cycles. [000258] In some embodiments, the CAR-engager is administered as an intravenous infusion over a period of time. Representative infusion times are 30 minutes, 60 minutes, and 90 minutes. In some embodiments, the infusion time is between 30 and 60 minutes.
  • the first administration is infused into a patient for 90 minutes and subsequent administrations are infused into a patient for 30 minutes.
  • the treatment method may entail administration of a first course of CAR-engager therapy that is initiated up to about 6 months after administration of the CAR immune cell therapy.
  • the term “effective amount” as used herein refers to a sufficient amount of CAR-engager to provide the desired effect, e.g., the amount of a CAR-engager to bind to a CAR-expressing immune cell.
  • the first course of CAR-engager therapy is initiated at any time up to about 4 years after the CAR immune cell therapy. In some embodiments, the first course of CAR- engager therapy is initiated at any time up to about 3 years after the CAR immune cell therapy. In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 2 years after the CAR immune cell therapy. In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 1 year after the CAR immune cell therapy. In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 9 months after the CAR immune cell therapy.
  • the first course of CAR-engager therapy is initiated at any time up to about 6 months after the CAR immune cell therapy.
  • the first course of CAR-engager therapy is initiated at any time up to about 5 months after the CAR immune cell therapy.
  • the first course of CAR-engager therapy is initiated at any time up to about 4 months after the CAR immune cell therapy.
  • the first course of CAR-engager therapy is initiated at any time up to about 3 months after the CAR immune cell therapy.
  • the first course of CAR-engager therapy is initiated at any time up to about 2 months after the CAR immune cell therapy.
  • the first course of CAR-engager therapy is initiated at any time up to about 1 month after the CAR immune cell therapy.
  • the first course of CAR-engager therapy is initiated at any time up to about 4 weeks after the CAR immune cell therapy.
  • the first course of CAR-engager therapy is initiated at any time up to about 3 weeks after the CAR immune cell therapy.
  • the first course of CAR-engager therapy is initiated about 2 weeks after the CAR immune cell therapy.
  • the first course of CAR-engager therapy is initiated 2 weeks after the CAR immune cell therapy.
  • the first course of CAR-engager therapy entails administering a total of about 1 to about 6 doses (e.g., 2 doses, 3 doses, 4 doses, 5 doses, or 6 doses) of the CAR- engager.
  • the first course of the CAR-engager therapy entails administration of about 1 dose per week, about 2 doses per week, about 3 doses per week, or about 4 doses per week.
  • the first course of CAR-engager therapy is conducted over a period of time of about 1 to about 3 weeks with administration of about 1 to about 3 doses of CAR-engager per week.
  • the dosage amounts of the CAR-engager administered during the first course of CAR- engager therapy may range from about 1 to about 8 mg/kg of patient body weight.
  • the dosage (effective amount) of the CAR-engager is about 1 mg/kg, 2 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, or about 8 mg/kg.
  • the present methods further include administration of a second, subsequent course of CAR-engager therapy to the subject.
  • the subject may have relapsed, or is at risk of relapse.
  • the CAR-engager administered in the second course of CAR- engager therapy may be the same as or different from the CAR-engager administered in the first course of CAR-engager therapy.
  • the CAR-engager administered in the second course of CAR- engager therapy may be administered within the same period of time after the CAR immune cell therapy as described above or in the same amounts of time described above, but after the first course of CAR-engager therapy.
  • Combination Therapy [000274]
  • the present methods may include co-administration of another anti-cancer therapy.
  • co-administered includes substantially contemporaneous administration, by the same or separate dosage forms, or sequentially, e.g., as part of the same treatment regimen or by way of successive treatment regimens.
  • the first of the two therapies may still be detectable at effective concentrations at the site of treatment.
  • the sequence and time interval may be determined such that they can act together (e.g., synergistically to provide an increased benefit than if they were administered otherwise).
  • the therapeutics may be administered at the same 70 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 time or sequentially in any order at different points in time; however, if not administered at the same time, they may be administered sufficiently close in time so as to provide the desired therapeutic effect, which may be in a synergistic fashion.
  • the terms are not limited to the administration of the active agents at exactly the same time.
  • the subject may also have had an additional anti-cancer therapy.
  • the additional therapy may be administered (1) prior to CAR immune cell therapy, (2) after the CAR immune cell therapy but before the first course of CAR-E therapy, (3) after the first course of CAR-E therapy but before the second course of CAR-E therapy, or (4) after the second course of CAR-E therapy.
  • the additional anti-cancer therapy is chemotherapy, radiotherapy, immunotherapy, targeted therapy, pro-apoptotic therapy, or cell cycle regulation therapy, therapy with thalidomide, lenalidomide, bortezomib, and/or melphalan.
  • Expansion and differentiation agents may also be provided prior to, during, or after administration of the CAR immune cells to increase differentiation, expansion, and/or persistence of the CAR immune cells (e.g., T cells and NK cells).
  • CAR immune cells e.g., T cells and NK cells.
  • Anti-cancer agents that may be used in combination with the IL-2 variants and/or chimeric antigen receptor (CAR)-engagers are known in the art. See, e.g., U.S. Patent No.9,101,622 (Section 5.2 thereof).
  • an "anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by 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 a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of cancerous cells. This process may involve contacting the cancer cells with recipient cells and the agent(s) or multiple factor(s) at the same time.
  • the IL-2 variants and/or CAR-engagers of the present disclosure are used in conjunction with or following prior therapies such as chemotherapeutic, 71 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 radiotherapeutic, immunotherapeutic intervention, targeted therapy, pro-apoptotic therapy, or cell cycle regulation therapy.
  • the IL-2 variants and/or CAR-engagers of the present disclosure are used in conjunction with high-dose chemotherapy prior to the administering of the genetically modified immune cells.
  • bone marrow cells or peripheral blood stem cells are administered subsequent to the high-dose chemotherapy.
  • the IL-2 variants and/or CAR-engagers of the present disclosure are used in conjunction with an effective amount of thalidomide, lenalidomide, bortezomib, or a combination thereof.
  • Additional potentiating treatments that may be used in conjunction with the IL-2 variants and/or CAR-engagers of the present disclosure include melphalan.
  • Melphalan (Alkeran®, Evomela®), an alkylating antineoplastic agent, is used for high-dose conditioning prior to hematopoietic stem cell transplant 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 regiments before bone marrow transplant, and in some cases, malignant melanoma.
  • Melphalan may be administered in pill form by mouth.
  • Immunotherapy including immune checkpoint inhibitors may be employed to treat a diagnosed cancer.
  • Immune checkpoint molecules include, for example, 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®).
  • 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, e.g., U.S. Patent Application Publications 2012/0321637, 2014/0194442, and 2020/0155520. 72 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 Chemotherapy [000283] Anti-cancer therapies also include a variety of combination therapies with both chemical and radiation-based treatments.
  • Combination chemotherapies include, for example, Abraxane®, altretamine, docetaxel, Herceptin®, methotrexate, Novantrone®, Zoladex®, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, Taxol®, gemcitabien, Navelbine®, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, or any analog or derivative variant of the foregoing and also combinations
  • Radiotherapy also include radiation-based, DNA-damaging treatments.
  • Combination radiotherapies include what are commonly known as gamma-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells which cause a broad range of damage on DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells and will be determined by the attending physician.
  • Radiotherapy may include external or internal radiation therapy.
  • External radiation therapy involves a radiation source outside the subject’s body and sending the radiation toward the area of the cancer within the body.
  • Internal radiation therapy uses a radioactive substance sealed in needles, seeds, wires, or catheters that are placed directly into or near the cancer.
  • the lentivirus was harvested at 48, 72, and 96 hours (h) post transfection, sedimented at 20,000 x g for 2h, and resuspended in optiMEM media. A new batch of HEK293 cells was then subjected to three rounds of transduction with the virus. Cells were allowed to recover in DMEM complete media and were subjected to puromycin selection to retain only cells that integrated the lentivirus plasmid. Cells were then expanded in four 15 cm culture dishes until they reached confluency, washed carefully with PBS, and incubated in serum-free DMEM for 24 to 48h. The supernatant was harvested, and protein expression was confirmed via SDS-PAGE and immunoblotting.
  • Proteins were purified by adsorption onto a nickel nitriloacetic acid (Ni-NTA) metal affinity column. Non- specifically bound proteins were removed by washing with 40 mM imidazole. The imidazole concentration was increased to 250 mM, allowing recovery of the protein of interest. The protein was further purified via size-exclusion chromatography and were stored in 50 mM HEPES buffer, pH 7.5 at -80 °C until use. [000289] Some CAR-engagers were isolated by passage through an affinity chromatography resin, typically in the presence of a neutral phosphate buffer.
  • the affinity chromatography resin was then subjected to an acidic buffer with a pH of about 3 to about 4, thereby washing CAR-engager off of the affinity chromatography resin.
  • a basic buffer may be used to neutralize the acidic buffer, then a tangential flow filtration of the neutralized buffer can be performed with a formulation buffer, to isolate a concentrated and purified solution containing the CAR-engager.
  • the CAR construct that binds human BCMA contains an scFv derived from the anti-human BCMA antibody clone MSK54, followed by human 39BB and CD3 ⁇ intracellular signaling domains.
  • the human signaling CAR constructs were transduced into HeLa cells that stably produce gamma-retrovirus pseudotyped with the envelope of the feline endogenous virus (RD114), which has been shown to transduce human hematopoietic cells (HSC) with high efficiency (Ward et al., Mol. Ther. 8(5):804-12 (2003)).
  • High viral titer clones were isolated by 74 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 limiting dilution.
  • the high expression clone was seeded and grown in DMEM complete media containing 10% FBS until 80% confluency. Media was exchanged with RPMI complete media containing 10% FBS. After 24 hours, the virus-containing media was harvested, sterile-filtered using a 0.45 ⁇ m PES filter, and utilized for producing CAR T cells. [000291]
  • the production of CAR T cells was adapted from previous studies. See, for example, Li et al., Methods Mol. Biol.1514:111-118 (2017).
  • whole blood was obtained from apheresis leukoreduction collars of platelet healthy donors, due to a high number of viable white blood cells.
  • the whole blood was subjected to centrifugation through a Ficoll gradient to isolate PBMCs.
  • Whole PBMCs were utilized without selecting for CD8 + T cells.
  • PBMCs were resuspended in RPMI media containing 10% Fetal Bovine Serum (FBS), 200 IU/mL IL-2, 60 ng/mL IL-7, 10 ng/mL IL-15, 2 ⁇ g/mL anti-human CD3 (OKT3 clone), and 0.5 ⁇ g/mL anti-human CD28 (CD28.1 clone) at a cell concentration of 4 ⁇ 10 6 cells/mL in 3 mL of media per well in a 6-well plate.
  • FBS Fetal Bovine Serum
  • mice were used to establish a multiple myeloma mouse model in NSG mice.
  • In vivo experiments were initiated by tail vein intravenous injection of 1 ⁇ 10 6 OPM2 cells expressing GFP and Firefly Luciferase followed by biweekly Bioluminescent Imaging (BLI).
  • BLI Bioluminescent Imaging
  • mice were intravenously injected through the tail vein with CAR T cells.
  • BLI was performed biweekly afterwards to assess tumor burden. Quantification was measured using photons/sec using Aura software.
  • 75 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000293] Organ analysis.
  • mice were sacrificed and spleen, bone marrow, blood, liver, kidney, and lungs were harvested and weighed.
  • the liver, kidney, and lungs were minced, digested with collagenase (final concentration of 1 ⁇ g/mL collagenase), and incubated at 37 °C for 1 h.
  • Spleens were crushed, and bone marrow was aspirated using a 30-gauge insulin needle. All processed cells were pushed through a 70 ⁇ m strainer to produce a single cell suspension of cells. Cells were resuspended in 1 mL of ammonium-chloride- potassium (ACK) lysis buffer to deplete the sample of red blood cells for 2 m at room temperature.
  • ACK ammonium-chloride- potassium
  • the resulting single cell suspensions were washed with fluorescence-activated single cell sorting (FACS) buffer of PBS and 0.5% BSA, stained, and analyzed using flow cytometry.
  • FACS fluorescence-activated single cell sorting
  • the OPM2 cell line which endogenously express BCMA, were engineered to express green fluorescent protein (GFP) and Firefly Luciferase.
  • GFP green fluorescent protein
  • Firefly Luciferase PBMC
  • Peripheral Blood Mononuclear Cells were obtained by Ficoll gradient of apheresis leukoreduction collars of platelet healthy donors.
  • Tumor burden was assessed using the IVIS® Lumina Series III (Perkin Elmer) after intraperitoneal injection of D-Luciferin (150 mg/kg, from a 15 mg/ml solution) at the indicated time. Each mouse was imaged in groups of up to five mice in the supine position at the same time points (5 min). BLI intensity was analyzed by Aura imaging analysis software (Spectral Instruments Imaging). Peripheral blood from mice was obtained 76 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 by submandibular bleeding in an EDTA-coated tube and analyzed for CAR T cell detection and expansion. In brief, volume of the blood was determined in order to calculate absolute values.
  • the samples were incubated with the indicated treatment, labeled with Alexa647, at the specified time and temperature.
  • the cells were imaged using the Leica THUNDER Imager.
  • the intensity cut-off for the AlexaFluor647 channel was set at 2000 for all images, except for the VHH-muIL2 samples shown on the right (condition 4), for which, the image sensitivity was enhanced by 25-fold (intensity cut- off 80) to visualize the Alexa647 signal.
  • ELISA ELISA analyses were performed to measure the levels of human T cell-derived cytokines in the serum of mice that received OPM2 cancer cells followed by a low dose of CAR T cells, as shown in FIG. 19A.
  • pSTAT5 assay Initially, CAR T were incubated in complete RPMI media without the presence of cytokines (rested) for 24 h. Cells were then stained with CellTraceTM Blue (Invitrogen) or CellTrackerTM Red (Invitrogen) for 30 minutes at 37 °C for the indicated conditions. Cells were washed once with complete RPMI + 10% FBS media. CAR T cells stained with CellTrackerTM Red were blocked using recombinant BCMA-CH3 (100 nM) for 20 minutes on ice while cells stained with CellTraceTM Blue were not blocked.
  • CellTraceTM Blue Invitrogen
  • CellTrackerTM Red Invitrogen
  • Cells were then washed once with complete RPMI + 10% FBS media followed by seeding of approximately 2 ⁇ 10 5 cells (either the two separately stained cells co-cultured or separately cultured) per well of a 96-well plate in the presence of serial dilutions of treatments or cytokine controls at 37 °C. After 5 minutes of incubation, cells were immediately fixed with 1.5% formaldehyde in PBS for 10 minutes at room temperature. Cells were then permeabilized with ice cold 100% methanol for 20 minutes on ice 4 °C.
  • Phenotype of the cells was then determined using projection of our sample to the Seurat pbmc_multimodal dataset using the FindTransferAnchors() and FindQuery() workflow.
  • the FindMarkers() function was used to find differentially expressed genes between chosen groups.
  • the heatmaps were generated with the DoHeatmap() function with a downsampling of 500 cells.
  • Gene Scores were obtained by creating a list of the genes of interest which was given as the feature for the function AddModuleScore().
  • a 96 well plate was seeded with approximately 2 ⁇ 10 5 cells/well in the presence of 10 nM treatment or controls at 37 °C for 2 hours. Cells were washed with FACS buffer and left at 37 °C for 2 or 22 hours (4 hour and 24-hour timepoints). Cells were stained with anti-CD8-FITC (1:50, Biolegend), anti-CD4-PE/Dazzle594TM (1:50, Biolegend) and Alexa647 labeled BCMA and sorted on the Sony Sorter MA900.10,000 CD4 and 10,000 CD8 cells were sorted per condition. SMART- Seq mRNA library preparation kit (Takara Bio) was utilized to generate mRNA libraries, with each replicate tagged with a unique index.
  • RNA Seq-Analysis Gene counts for the samples were obtained by trimming the fastQ files and transcript quantification using the RNAlysis software. Gene names were obtained from the homo sapiens ensembl database with biomaRt, and differential expression between different conditions was determined using the DESeq2 pipeline. Volcano plots were drawn using the EnhancedVolcano library, with a cutoffs at logFC >
  • GSEA was performed using the pipeline of the fgsea package with the ranking metric being -log 10 (p-value)*sign (fold change) and basing the computations on the hallmark pathways of the MSigDB collection.
  • Example 2 BCMA-containing CAR-engager in vitro characterization.
  • a Fusion protein consisting of the human BCMA ectodomain was fused with a two low- affinity mutated human IL-2 (muIL2) domains, and to improve pharmacokinetics and enhance stability, the CH3 domain (Feige et al., Trends Biochem.
  • IL-2 induces an alternative differentiation pathway of T cells, resulting in the generation of distinct “better effector” CD8 + T cells (Hashimoto et al., Nature 610(7930):173-181 (2022)). This process may rely, at least in part, on IL-2 binding to IL- 2R ⁇ . Additionally, IL-2R ⁇ -biased agonists may drive T cells towards a terminally differentiated state (Codarri et al., Nature 610(7930):161-172 (2022)).
  • CAR-engagers may be able to overcome the need for IL-2R ⁇ in the alternative differentiation pathway by anchoring the low-affinity IL-2 on the surface of the CAR T cells via the antigen-to-CAR binding, thereby promoting the generation of memory CAR T cells.
  • a potential synergistic effect between CAR signaling and IL-2 signaling may also exist.
  • BCMA CAR-engager Minimal binding of BCMA CAR-engager to non-transduced T cells was observed as well as minimal binding of VHH-muIL2, a control construct which replaces the BCMA ectodomain with an irrelevant nanobody (VHH) in the CAR-engager CH3-muIL2 construct.
  • VHH irrelevant nanobody
  • This observation further suggests that the binding of the CAR-engager to CAR T cells is primarily driven by the BCMA antigen, and that the muIL2 exhibits weak binding to both CAR T and non-transduced T cells.
  • the BCMA-muIL2 CAR-engager did not exhibit binding to any immune cell populations in human peripheral blood mononuclear cells (PBMCs) (FIGs.15A – 15B).
  • PBMCs peripheral blood mononuclear cells
  • BCMA CAR-engager the functional effects of BCMA CAR-engager on BCMA CAR T cells were evaluated.
  • BCMA CAR T cells were incubated with varying concentrations of the BCMA-muIL2 CAR-engager for 24 h, followed by assessment 80 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 of the expression of the CD69 activation marker (Cibrián and Sánchez-Madrid, Eur. J. Immunol. 47(6):946-953 (2017)) using flow cytometric analysis.
  • OPM2 cells were co-incubated with BCMA CAR T cells (shown in red) or non- transduced T cells (shown in gray) (E:T ratio 1:1; 30,000 cells of each) in the presence of varying concentrations of the BCMA-muIL2 CAR-E treatment.
  • Live (PI-) OPM2 cells were counted 48 hours later, with an N of 3 for each of the experiments. Error bars in FIG.2H represent mean with standard deviation. Without being bound by theory, this finding might be attributed to the reversibility of CAR-engager binding to CAR, while the killing process, which involves the clustering effect and synapse formation between CAR and cancer antigen, is an irreversible event.
  • the BCMA CAR-engager selectively induces STAT5 activity in CAR T cells through the cis-delivery of the low-affinity IL-2 to the same targeted CAR T cells.
  • IL-2 is known to exhibit strong activity on T cells, and the phosphorylation of STAT5 both serves as a reliable indicator of IL-2/IL-2R engagement and correlates with downstream effects such as phenotypic marker expression and cell proliferation (Jones et al., J. Immunol.205(7):1721-1730 (2020)).
  • BCMA CAR T cells were exposed to varying concentrations of the BCMA CAR-engager.
  • Wild-type IL-2 exhibited a lower EC50 (approximately 0.001 nM) suggesting a difference in signaling kinetics.
  • the two-step process involved in STAT5 activity mediated by the BCMA-muIL2 CAR-engager involves: (i) binding of the antigen-to-CAR on T cell surfaces and (ii) subsequent interaction of the low-affinity IL-2 with nearby IL-2R, which, without being bound by theory, may be the reason for the measured difference.
  • wild-type IL-2 requires only binding to IL-2R, enabling it to more rapidly induce STAT5 activity.
  • the VHH-muIL2 can activate STAT5 solely through the low- affinity IL-2, which may explain its requirement for higher concentrations to induce STAT5 activity in T cells.
  • BCMA CAR T cells pre-blocked with BCMA-CH3 showed a significant decrease in pSTAT5 levels to the same degree as control VHH-muIL2, validating that the potency of the CAR- engager is mediated by antigen-to-CAR binding (FIG.2I).
  • FOG.2I antigen-to-CAR binding
  • non- blocked and pre-blocked BCMA CAR T cells were co-cultured in the presence of varying concentrations of CAR-engager.
  • Pre-blocked CAR T cells had lower pSTAT5 levels compared to their co-cultured non-blocked CAR T cells, indicating that CAR-engager affects the targeted CAR T cells (cis-activation) but not adjacent cells.
  • BCMA CAR T cells were treated with the indicated treatments for 5 min at 37 °C followed by STAT5 phosphorylation assessment.
  • peripheral blood mononuclear cells PBMCs
  • PBMCs peripheral blood mononuclear cells
  • the activated T cells were treated with teceleukin (recombinant human IL-2 without glycosyl units), a CAR-engager that contains a N-terminal ectodomain that binds BCMA ( ⁇ 7 kDa), a CH3 domain ( ⁇ 14 kDa), and two repeats of the weak affinity variant of IL-2 immune cell effector domain, with the overall structure of BCMA-CH3-muIL2-muIL2 and referred herein as BCMA-muIL2, or a CAR-engager that contains an ectodomain that binds BCMA, a CH3 domain, and the Neo-2/15 immune cell effector domain, with the overall structure of BCMA-CH3-Neo-2/15, referred to herein as BCMA-Neo-2/15, for 4 days.
  • teceleukin recombinant human IL-2 without glycosyl units
  • a CAR-engager that contains a N-terminal ectodomain that binds BCMA (
  • T cells were counted and stained with carboxyfluorescein succinimidyl ester (CFSE) and analyzed for mean fluorescence intensity (MFI) of CFSE to determine T cell division.
  • CAR-engagers BCMA-muIL2 or BCMA-Neo-2/15 effected T cell counts and division (CFSE staining), similar to teceleukin, which is known to activate T cells.
  • the CAR-engager treatment resulted in less sensitivity as compared to the teceleukin treatment.
  • Systemic administration of IL-2 is associated with severe side effects (Rosenberg, J. Immunol. 192(12):5451-5458 (2014); Dutcher et al., J.
  • Example 4 CAR-engagers activate CAR T cells specifically through the ectodomain [000311] To show that CAR-engager immune cell effector domains stimulate immune cells, the following experiment was performed. As illustrated in FIG.4A, PBMCs were stimulated with anti- CD3, anti-CD28, IL-2, IL-7, and IL-15 to produce activated T cells, which were then transduced with a vector containing a CAR. The activated CAR-expressing T cells (CAR T cells) were rested for 24 hours and then treated with CAR-engagers containing an immune cell effector domain or CAR-engagers lacking an immune cell effector domain as an ectodomain control.
  • CAR T cells activated CAR-expressing T cells
  • CAR-engagers containing BCMA ectodomain, a CH3 domain, and containing either 4- 1BBL (BCMA-41BBL), weak affinity IL-2 (BCMA-muIL2), or Neo-2/15 (BCMA-Neo-2/15) immune cell effector domains were tested for T cell activation.
  • BCMA-41BBL 4- 1BBL
  • BCMA-muIL2 weak affinity IL-2
  • Neo-2/15 BCMA-Neo-2/15
  • ectodomain specificity control containing a nanobody that binds FN1 (clone NJB2, abbreviated NJB2-VHH) fused to a CH3 domain, and the Neo-2/15 stimulatory (NJB2-VHH-Neo-2/15) was tested for T cell activation.
  • the ectodomain specificity controls have similar overall structure as the CAR-engagers used in this experiment (protein domain-CH3-muIL2-muIL2 or protein domain-CH3-Neo-2/15).
  • Ectodomain specificity controls and CAR-engagers were incubated with CAR T cells for 10 hours, and the cells were stained for CD69 as an activation marker and measured by flow cytometry.
  • CAR-engagers containing a cancer antigen ectodomain specifically activate CAR T cells that expresses a CAR that recognizes CAR-engager’s cancer antigen ectodomain.
  • Example 5 CAR-engagers stimulate CAR T cell killing target cells 84 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000314] To show that CAR-engagers do not inhibit CAR T l cell killing, the following experiment was performed. CAR T cells were produced as described above and co-incubated with CAR- engagers and BCMA + multiple myeloma cancer cells.
  • CAR T cells were incubated with OPM2 BCMA + cells at an E:T ratio of 1:1 for 1 day and analyzed for target cell survival as compared to target cells without T cell coincubation (FIG.5A).
  • BCMA ectodomain CAR-engagers with either a muIL2 (BCMA-CH3-muIL2) or a 4- 1BBL (BCMA-CH3-41BBL) immune cell effector domain did not inhibit killing of OPM2 cells (FIG.5B).
  • Example 6 CAR-engagers reduce tumor burden, extend survival, and extend CAR T cell persistence in vivo [000316] To show that CAR-engagers reduce tumor burden, extend CAR T cell in vivo persistence, and extend survival, the following experiment was performed. 1 ⁇ 10 6 OPM2 BCMA + multiple myeloma cancer cells were intravenously (i.v.) injected into NOD-scid IL2R ⁇ null (NSG) mice 10 days before infusing a suboptimal dose of 5 ⁇ 10 5 anti-BCMA CAR T cells by i.v. injection.
  • mice were treated twice weekly for two weeks, followed by once weekly with 200 ⁇ g/mouse of CAR-engager containing a BCMA ectodomain, a CH3 domain, and two weak affinity IL-2 immune cell effector domains (BCMA-CH3-muIL2-muIL2) by intraperitoneal (i.p.) injection (FIG.6A).
  • CAR-engager containing a BCMA ectodomain, a CH3 domain, and two weak affinity IL-2 immune cell effector domains (BCMA-CH3-muIL2-muIL2) by intraperitoneal (i.p.) injection.
  • FIG.6A two weak affinity IL-2 immune cell effector domains
  • CAR-engager treated group all mice completely cleared OPM2 tumor cells from the bone marrow, as no signal was detected by imaging.
  • FIGs. 7A-7C show flow cytometry with GFP on the y-axis.
  • GFP + OPM2 cells were detected at similar levels in the bone marrow, lung (FIG.7B), and liver (FIG.7C) of the no-CAR control mouse and the CAR-only mice.
  • FIG.7A One CAR-only mouse had significant levels of OPM2 cells in the blood and spleen (FIG.7A).
  • FIGs.7A The one mouse that received CAR T cell and CAR-engager treatment that developed an eye tumor had no to little OPM2 cells in the blood or spleen (FIG.7A), bone marrow or lung (FIG 7B), and liver (FIG. 7C).
  • FIGS. 8A – 8C show flow cytometry with anti-CD45 on the y-axis and BCMA + -CH3 tagged with Alexa Flour TM 647 (AF647) on the x-axis.
  • CD45 + CAR + T cells only persisted in CAR T cell and CAR-engager treated mice.
  • CAR-only treated mice had little to no CD45 + cells in all organs tested (FIGs.8A-8C).
  • CAR T cell + CAR-engager treated mice had CD45 + cells that also stained positive for the BCMA cancer antigen (which is also the CAR binding target) tagged with AF647, as shown on the x-axis.
  • CD45 + AF647 + double positive CAR T cells were detected in the blood, spleen, and lymph node (FIG. 8A), bone marrow and lung (FIG. 8B), and liver and kidney (FIG.8C). CD45 + single positive cells were only detected in large numbers in liver and kidney (FIG.8C). Little to no CD45 + AF647 + double positive CAR T cells were detected in the eye tumor site (FIG.8C). These results indicated that CAR-engagers reduce tumor burden, extend CAR T cell in vivo persistence, and extend survival.
  • Example 7 CAR-engager fate [000322] The CAR-engagers bind CAR T cells at the cell surface at 4 °C, and slowly internalize at 37 °C.
  • BCMA CAR T cells were exposed to AlexaFluor647-labeled BCMA-muIL2, BCMA-CH3, or VHH-muIL2 at a concentration of 2 nM. The cells were incubated at either 4 °C or 37 °C for various time intervals, followed by fixation and subsequent microscopy imaging.
  • the cytoplasm mean intensity was reported, as the dsRed is expressed inside the cell, whereas for the AlexaFluor 647 channel, the mean intensity for the entire cell was measured. [000323]
  • the CAR-engager rapidly clears from the circulation. Pulsing CAR T-cells with the CAR- engager treatment, where pulsing involves periods of stimulation followed by periods of resting, is superior to prolonged exposure to CAR-engagers as extended exposure can lead to exhaustion or the generation of terminally differentiated CAR T cells.
  • a CAR-engager with a short circulation half-life can be more effective at expanding CAR T cells, driving generation of memory CAR T cells, decrease potential competition with tumor antigen for CAR binding, and enhanced safety profile in patients. Therefore, the CH3 domain of IgG1 was used in the CAR-engager platform. Pharmacokinetic studies illustrated that the circulatory half-life of the CAR-engager was short (1- 1.5 hours) (FIG. 9A).
  • the sera were then obtained by centrifugation and used for the subsequent analysis.
  • An ELISA was performed to determine the concentration of the treatments in the sera.
  • the ELISA plates were coated with 5 ⁇ g/ml anti-His6 antibody overnight, followed by incubation with the sera for 2 h at room temperature.
  • An anti-FLAG HRP antibody was next used for the detection; the CAR-engager was engineered to have FLAG and His6 tags at the C-terminus. Based on the collected five time points, the initial concentration of the treatment in the sera was estimated to be 20% higher than the first (30 min) collected time point.
  • the BCMA CAR-E was >90% and >99% cleared from the circulation in 8 h and 24 h, respectively.
  • the BCMA CAR-engager enhances activity and persistence of CAR T-cells in a multiple myeloma (MM) model.
  • MM multiple myeloma
  • BCMA CAR T cells Two weeks after OPM2 cell injection, freshly prepared BCMA CAR T cells (0.5 million CAR + cells containing an 39BB-CD3 ⁇ CAR construct) were intravenously administered.
  • a cohort of mice received the BCMA-muIL2 CAR-engager treatment (FIG.9B).
  • BCMA-muIL2 CAR-E treatment 200 ⁇ g was administered twice per week for two weeks, followed by once per week until the endpoint. After one month or longer, mice were euthanized, and flow cytometric analyses were performed on the harvested organs.
  • BCMA CAR T cells were detected by co-staining with an anti-human CD45 antibody and Alexa647-labeled BCMA antigen. Similar results were obtained in repeated experiments. Additional control cohorts received VHH-muIL2 treatment with the same dose and schedule as BCMA-muIL2.
  • FIG.9D shows pooled data from these experiments.
  • Data were analyzed by group mean comparisons using one-way ANOVA and subsequent Tukey post-hoc analysis. Individual flow graphs for the pooled data are shown in FIGs.17A – 17C. Error bars represent mean with standard deviation.
  • Analysis of blood samples collected at various time points revealed a substantial expansion of CAR T cells in the circulation following CAR-engager treatment, with the peak expansion observed at week 4 (FIG. 10D).
  • Flow cytometric analyses of blood samples revealed robust expansion of CAR T cells in the treatment group compared to PBS or VHH-muIL2 cohorts.
  • mice treated with CAR-engager exhibited no signs of toxicity based on clinical observations and weight measurements (FIG. 10H).
  • Subsequent analysis conducted two months after CAR T cell injection demonstrated a substantial presence of CAR T cells, including memory CAR T cells, in the CAR-engager treated mice (FIGs.10F-10J, FIGs.18A – 18C, and FIGs.19A – 19C).
  • CAR T cells were detected in the spleen; however, these mice succumbed to tumor growth at around 20 days post-CAR T cell injection.
  • the BCMA-muIL2 89 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 treatment had also increased the presence of CAR T cells in bone marrow compared to PBS or VHH-muIL2 cohorts, but the difference was less significant than spleen. Data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test. *P ⁇ 0.05, ***P ⁇ 0.001, ****P ⁇ 0.0001. Individual flow data are shown in FIGs.18A – 18C. Error bars represent mean with S.E.M.
  • FIG. 19A – 19C The Flt-SNE mapping shown in FIG. 19C is of CAR T cells derived from the PBS, BCMA-muIL2 and VHH-muIL2 treated mice as shown in FIG. 10A.
  • CD45 + , ⁇ -BCMA-CAR + immune cells from the mice were concatenated to form a total of ⁇ 9800 (PBS spleen), ⁇ 8100 (BCMA-muIL2 spleen), ⁇ 7600 (PBS bone marrow), ⁇ 14200 (BCMA-muIL2 bone marrow), ⁇ 1420 (VHH-muIL2 Bone Marrow).
  • the entire high dimensional dataset was merged to create a single Flt-SNE map for each condition with the signal strength of various phenotypic markers defining specific immune phenotypes expressed with a blue-green-yellow-red continuous color scale.
  • FltSNE was conducted with the following parameters: max iterations: 1000, theta: 0.5, learning rate: 200, perplexity: 20. There were inadequate numbers of CAR T cells in VHH-muIL2 cohort spleen to conduct Flt-SNE. To enhance visibility, the dots representing the VHH-muIL2 bone marrow samples were enlarged, as fewer cells were detectable in these mice. In the BCMA-muIL2 treated mice, the majority of CAR + cells were CD8 + cells, while in the VHH-muIL2 samples, CD4 + cells constituted the majority of CAR + cells.
  • CAR + cells in the CAR+PBS cohort exhibited low or no expression of CD45RA, CD45RO, 90 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 or CD62L, whereas the BCMA-muIL2 treated mice showed a CAR + population with elevated expression levels of these memory markers.
  • the treatment not only facilitates robust proliferation and eradication of tumor cells using low doses of CAR T cells but also promotes the development of long-lasting memory cells, demonstrating the efficacy of BCMA-muIL2 CAR-engager treatment in enhancing the clearance of tumor cells by CAR T cells, and generation of long-lasting memory cells.
  • Bone marrow and splenocytes were analyzed via flow cytometry to detect and quantify CAR-expressing T cells and the bone marrow cells or splenocytes were co-incubated with OPM2 target cells at various E:T ratios (1:1 and 2:1) based on CAR-expressing cells. Survival was determined 24, 48, and 72 hours later using flow cytometric analysis. The three-month-old CAR T cells demonstrated efficient killing of tumor cells and long-term functionality (FIG.11D).
  • CD45 + , CD8 + , ⁇ -BCMA- CAR + cells from the five mice were concatenated to form a total of ⁇ 17600 (spleen) and ⁇ 10800 (bone marrow) cells.
  • the entire high dimensional dataset (excluding the CD45, CD8, and CD4 parameters) was merged to create a single tSNE map with the signal strength of six phenotypic markers defining specific immune phenotypes expressed with a blue-green-yellow-red continuous color scale.
  • tSNE analysis was performed using 1000 iterations, a perplexity of 30 and a learning rate of 1237 and 756 for spleen and bone marrow respectively.
  • CD45 + , CD4 + , ⁇ -BCMA-CAR + immune cells from the five mice were concatenated to form a total of ⁇ 9000 (spleen) and ⁇ 6600 (bone marrow) cells.
  • the entire high dimensional dataset (excluding the CD45, CD8, and CD4 parameters) was merged to create a single t-SNE map with the signal strength of six phenotypic markers defining specific immune phenotypes expressed with a blue-green-yellow-red continuous color scale.
  • tSNE analysis was performed using 1000 iterations, a perplexity of 30 and a learning rate of 630 and 466 for spleen and bone marrow, respectively.
  • CAR + cells were sorted after staining with BCMA-AlexaFluor647 and TotalSeq-C hashing antibodies from BCMA-muIL2 or VHH-muIL2 treated mice as shown in the red and green boxes, respectively in FIG.21A.5000 CAR + cells from BCMA-muIL2 mice bone marrow and spleen and 2500 CAR + cells from VHH- muIL2 mice bone marrow and spleen were loaded onto the 10X channel.
  • the scRNAseq analysis revealed that the predominant population of persistent CAR T cells in the BCMA-muIL2-treated mouse consisted of CD8 + T cells (FIGs.
  • FIG. 21D shows significantly differentially expressed genes between CAR-engager treatment and VHH conditions in CD8 and CD4 CAR T cells, split among different relevant conditions.
  • Genes marked with an * are the significantly differentially expressed genes between BCMA-muIL2 and VHH-muIL2 treated mice in the subset of interest. This was evidenced by elevated expression levels of granzyme family genes, other cytotoxicity-associated genes, and MHC class II genes.
  • CAR-engager expands CAR T cells in the absence of tumor antigens
  • CAR T cell expansion typically occurs following infusion in patients, with peak expansion observed around 10-14 days post-infusion (Rodriguez-Otero et al., N. Engl. J. Med. 388(11):1002-1014 (2023)).
  • Eradication of minimal residual disease (MRD) facilitates long-lasting and complete responses.
  • Example 11 CAR-engager containing immune cell effector domain IL-2 variants
  • the binding strength of the IL-2 variant to the CAR immune cell may have significant impacts on the efficacy of CAR-engager therapy.
  • CAR-engagers IL-2 variant containing different IL-2 variants were generated and integrated into CAR-engagers and are summarized in Table 9. Amino acid substitutions are relative to the natural (wild-type) sequence of human IL-2 (NCBI Accession No. NP_000577; SEQ ID NO: 102).
  • some of the CAR-engagers contain different dimerization domains, including CH3, and silent fragment crystallizable region (Fc).
  • Silent Fc contains at least two mutations relative to wild-type Fc that abolish Fc- ⁇ receptor and complement component 1q (C1q) binding, while maintaining the stabilizing effect and neonatal Fc receptor (FcRn) binding of wild-type Fc.
  • the at least two mutations include L234A and L235A, and are commonly referred to as (“LALA”).
  • CAR-engager variant E4 has a dimerization domain containing IgG1 with the silent Fc mutations of L234A and L235A (“LALA”) and further lacks an IL-2 variant.
  • the CAR-engager variant E4 was developed and used as a control molecule.
  • CAR-engagers containing a single IL-2 variant are listed in Table 9 as “One-IL-2” while CAR-engagers containing two repeats of IL-2 variants are listed in the below table as “IL-2.”
  • variants Q3, A8, A10, and A16 have two repeats of the indicated IL-2 variant on each molecule of CAR-engager.
  • the CAR-engager may be present as a dimer due to the presence of a dimerization domain.
  • CAR-engagers containing a wild-type CD19 SEQ ID NO: 5
  • CD19 wt wild-type CD19
  • CAR-engagers containing the variant of CD19 that has the amino acid sequence of SEQ ID NO: 3, are listed in Table 9 as “CD19.”
  • Table 1 CAR-engager ICE Variants
  • Variant CAR-engager structure IL-2 amino acid substitutions relative Activity 95 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024
  • FIG. 22A shows the percentage of BCMA CAR T cells stained positive for phosphorylated STAT5 (pSTAT5) after treatment by select CAR-engager ICE variants described in Table 9, as determined by flow cytometry.
  • FIG. 22B shows dose-dependent activation of rested BCMA CAR T cells after CAR- engager treatment after 24 hours.
  • CD69 serves as an activation marker for human T cells.
  • the dashed line at 0 nM represents the background expression of CD69 in the absence of any treatment or cytokines.
  • the dashed line “+Cytokine” indicates the CD69 expression 96 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 level of non-rested CAR T cells incubated in the presence of IL-2, IL-7, and IL-15 cytokines. The dashed lines are included as additional negative and positive controls.
  • FIG.23A shows STAT5 phosphorylation in BCMA CAR T cells after CAR-engager ICE variant treatment.
  • Several of the IL-2 variants exhibited the ability to induce STAT5 activity in BCMA CAR T cells.
  • FIGs. 24A –24B show STAT5 phosphorylation in BCMA CAR T cells induced by treatment with the BCMA CAR-engagers detailed in Table 9.
  • the CAR T cells contain the FDA- approved BCMA CAR constructs idecabtagene vicleucel (Ide-cel; ABECMA®) or ciltacabtagene autoleucel (Cilta-cel; CARVYKTI®).
  • the percentage of BCMA CAR T cells stained positive for phosphorylated STAT5 (pSTAT5) as determined by flow cytometry are shown.
  • FIGs. 25A –25F show STAT5 phosphorylation in BCMA CAR T cells induced by treatment with the BCMA-muIL2 CAR-engagers detailed in Table 9.
  • the CAR T cells contain the FDA-approved BCMA CAR constructs Ide-cel or Cilta-cel.
  • the percentage of BCMA CAR T cells stained positive for pSTAT5 as determined by flow cytometry are shown.
  • FIGs. 26A – 26B show dose-dependent activation of rested BCMA CAR T cells by treatment with the BCMA CAR-engagers detailed in Table 9.
  • the CAR T cells contain the FDA- approved BCMA CAR constructs Ide-cel or Cilta-cel.
  • FIGs. 27A – 28B show dose-dependent activation of rested BCMA CAR T cells after treatment with additional BCMA CAR-engagers detailed in Table 9.
  • the CAR T cells contain the FDA-approved BCMA CAR constructs Ide-cel or Cilta-cel.
  • FIGs.29A – 29C show that select CAR-engagers induce CAR T cell proliferation in vitro with reduced impact on non-transduced T cells as compared to wild-type IL-2.
  • FIG. 29A shows incubation of activated BCMA CAR T cells containing the Cilta-cel CAR construct, treated with increasing concentrations of BCMA CAR-engager, followed by assessment of CAR T cell proliferation using flow cytometry after 3 days.
  • the dashed line in FIG.29A represents the number of CAR T cells that received no treatment.
  • FIG.29B shows a repetition of the experiment show in FIG. 29A with additional CAR-engagers.
  • FIG. 29A shows incubation of activated BCMA CAR T cells containing the Cilta-cel CAR construct, treated with increasing concentrations of BCMA CAR-engager, followed by assessment of CAR T cell proliferation using flow cytometry after 3 days.
  • the dashed line in FIG.29A represents the number of CAR T cells that received no treatment
  • 29C shows a similar experiment evaluating the impact of CAR-engager on the cellular proliferation of normal, non-transduced T cells.
  • CAR- 98 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 engagers induce proliferation in non-transduced T cells significantly less than wild-type IL-2.
  • CAR- engagers containing the triple mutant IL-2 (V7, V9, Y2, and Y9) induced significantly less proliferation in normal T cells as compared to CAR-engagers containing the double-mutant IL-2 (U4).
  • FIG.42 shows that CAR-engagers induce CAR T cell proliferation in vitro with no little to no impact on non-transduced T cells.
  • BCMA CAR-engagers containing immune cell effector domains containing a U4, V7, Y2, V6, X12, X15, or U8 IL-2 variant were examined for their ability to induce proliferation of anti-BCMA CAR T cells or non-transduced T cell controls.
  • Wild-type IL- 2 a non-targeting CAR-engager with an immune cell effector domain containing E3 IL-2 variant (VHH-cH3-IL2), and a BCMA CAR-engager without an immune cell effector domain (BCMA- CH3) were used as controls.
  • FIGs. 30A – 30C show that the V7 CAR-engager exhibits lower affinity for IL-2R ⁇ as compared to wild-type IL-2 or the U4 CAR-engager.
  • BLI analyses of association and dissociation between wild-type IL-2 (Teceleukin®), BCMA-muIL2 CAR-engager (U4; double mutant IL-2), or BCMA-muIL2 CAR-engager (V7; triple mutant IL-2) molecules and IL-2R ⁇ are shown.
  • FIGs. 31A – 31B show the newly developed CAR-engager containing IL-2 variants induce proliferation of CAR-T cells in vivo, and that the proliferation levels correlate with in vitro pSTAT5 signaling.
  • FIG. 31A schematically illustrations the experimental setup; mice received OPM2 cancer cells, followed by 0.5 million CAR-T cells (Cilta-cel) one week later. Mice were bled for 5 weeks on days 11, 18, 28, 35, and 42 and CD4 and CD8 CAR T-cells were counted (FIG. 31B).
  • FIGs. 32A – 32C show that the V7 CAR-engager enhances CAR-T cell activity when administered 3 or even 14 days post-injection of CAR T cells.
  • Another mouse cohort received 6 doses of V7 CAR-engager starting from 3 days post-injection of CAR T cells, 99 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 administered on days 3, 6, 10, 14, 21, 28.
  • the third mouse cohort received 6 doses of CAR-engagers starting from day 14, which is the post-CRS window in patients, and on days 14, 18, 21, 28, 35, 42.
  • BLI imaging was performed on the indicated days to assess tumor growth in the different cohorts (FIG.32B). Mice were bled for 6 weeks, once per week starting from day 14, and CAR T cells were counted (FIG. 32C).
  • FIGs.43 – 45 show that CAR-engagers containing IL-2 variants induce in vitro pSTAT5 signaling (FIG. 43) and gene expression (FIGs. 44 and 45).
  • CAR T cells (about 60% of CAR + T cells) and non-transduced T cells were incubated with increasing concentrations of CAR-engagers containing an immune effector domain containing an IL-2 variant (including U4, V7, Y2, V6, X12, X15, U8) and controls (wild-type IL-2 and a CAR-engager without an immune effector domain (BCMA-CH3)).
  • pSTAT5 activity was assessed by flow cytometry after 30 minutes of CAR-engager treatment (FIG. 43).
  • CD69 expression was assessed by flow cytometric analysis after a 2-hour incubation of T cells with CAR-engagers, followed by a wash, and a further 22-hour incubation (FIG.44).
  • TNF- ⁇ and IFN- ⁇ production was assessed by multiplex ELISA via flow cytometry after a 2-hour incubation of T cells with CAR-engagers, followed by a wash, and a further 22-hour incubation and media harvesting (FIG.45). Each condition included 3 technical replicates. T cells were obtained from PBMCs from a single healthy donor.
  • FIGs.46A – 46B show that CAR-engagers containing IL-2 variants bind to IL-2R ⁇ and IL-2R ⁇ by biolayer interferometry.
  • a biosensor tip was coated with dimeric IL-2R ⁇ (FIG.46A) or IL-2R ⁇ (FIG.46B), and incubated with CAR-engagers containing an immune effector domain containing an IL-2 variant (including U4, V7, Y2, V6, X12, X15, U8) or controls (monomeric wild-type IL-2 (positive control), CAR-E with wild-type IL-2 (positive control), and a CAR-engager with monomeric V7 IL-2 variant)) for the indicated times.
  • the release rate was assessed by transferring the biosensor tip into the washing solution. Each condition included 3 technical replicates.
  • Example 12 CD19 CAR-E does not inhibit killing efficacy of CD19 CAR T cells
  • CAR-engagers that bind BCMA were observed to bind but not inhibit killing efficacy of BCMA CAR T cells (FIGs.2D – 2H).
  • a killing assay was conducted using CD19 CAR T cells 100 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 and patient-derived CD19 + leukemia cells in the presence of varying concentrations of the CD19 CAR-engager.
  • Live (PI-) Nalm6 cells were counted 48 hours later, with an N of 3 for each of the experiments.
  • the experimental findings disclosed herein with CD19 are consistent with the findings in BCMA cancer models and BCMA CAR-E, above.
  • Example 13 CAR-engagers effectively enhance CAR immune cell efficacy and persistence at low doses on initial treatment and tumor rechallenge [000361]
  • Mice were intravenously injected with OPM2 cells (1 ⁇ 10 6 , i.v.). A week later, CAR T cells (0.5 ⁇ 10 6 , i.v.) were administered. Two weeks post-injection of CAR T cells, the mice were divided into two cohorts, with mice receiving the CAR-E treatment or not. The treatment group received four doses of CAR-E treatment (4 mg/kg per dose) on days 14, 18, 21, and 28.
  • Cytokine assessments also revealed elevated levels of IFN- ⁇ in the CAR-E treatment cohort by sandwich ELISA on 1:40 diluted serum samples collected the same days when CAR-T counts were assessed as shown FIG.22E (FIG.35F).
  • One of the CAR-E treated mice (M5) showed liver relapse on day 60 (FIG. 35B). Regardless, all the five CAR-E treated mice underwent re-challenge, using 1 million of the liver metastasis-derived OPM2 cells, on day 60 to assess the generation of memory CAR-T cells. All mice showed considerably less signal compared to the na ⁇ ve control mice (FIG.35B; see day 66).
  • mice While the mice exhibited liver signals, none showed bone marrow signals, suggesting the presence 101 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 of functional memory CAR T cells in the bone marrow inhibiting tumor growth.
  • CAR-E treatment could contribute to re-expanding CAR T cells and controlling tumor growth in liver metastasis, the mice were re-treated with CAR-E (4 mg/kg) on days 68, 70, 74, 77, and 80. Impressively, all mice successfully cleared the liver metastasis, indicating that CAR-E could facilitate the re-expansion and trafficking of CAR T cells to eliminate tumor cells (FIG. 35B; see day 83).
  • mice that showed relapse on day 60 also cleared the tumor from liver after re-challenge, although it exhibited some signal on the last day of the experiment.
  • Example 14 CAR T cell expansion in the absence of tumor antigens is dose-dependent.
  • CAR T cell expansion occurs post-infusion in patients, with peak expansion observed around 10-14 days post-infusion (Rodriguez-Otero et al., N. Engl. J. Med.388(11):1002- 1014 (2023)). This expansion is driven by antigen availability and the tumor-killing process, which promotes CAR T cell proliferation (Turtle et al., J. Clin.
  • the CAR-E mechanism of action may be independent of tumor cells and antigens presented thereon and may thus expand CAR T cells in the absence of tumor cells (and therefore tumor antigen), addressing the critical clinical challenge of limited in vivo CAR T cell expansion post-infusion.
  • mice were injected solely with 0.25 million BCMA CAR T cells in the absence of tumor cells.
  • Organs were collected one-month post-injection of CAR T cells, and flow cytometric analyses were conducted to assess the presence of CAR T cells. [000369] Further, the results demonstrated that CAR-E impact on CAR T cells is dose-dependent (FIGs.36B – 36C).
  • TEM cells 103 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 are CD45RA- CD45RO + CCR7-; TEMRA cells are CD45RA + CD45RO + CCR7-; TSCM cells are CD45RA + CD45RO + CCR7 + ; TCM cells are CD45RA- CD45RO + CCR7 + ; TNaive cells are CD45RA + CD45RO- CCR7 + .
  • the experiment in panels FIGs. 36A – 36E utilized PBMCs from one donor. Overall, these findings demonstrate that CAR-E treatment induces the expansion of CAR T cells and enables the CAR T cells develop diverse memory phenotypes, regardless of the presence of tumor cells.
  • the antigen-only, BCMA-CH3 treatment cohort did not result in the expansion or persistence of CAR T cells compared to the CAR-E treatment cohort, further validating that both the ectodomain (e.g., BCMA antigen) component and the immune cell effector domain (e.g., low-affinity IL-2) component of the CAR-E molecule are necessary for its impact on CAR T cells (FIG.36F – 36G and 41A – 41E).
  • Mice received CAR T cells and different treatments (4 mg/kg) following a schedule similar to that shown in FIG. 36A.
  • the BCMA-CH3 antigen, VHH-muIL2, or the low-dose wild-type IL-2 treatments did not result in expansion or persistence of CAR T cells compared to the CAR-engager-treated cohort. Error bars represent mean with standard deviation.
  • the experiment in panels FIGs.36F – 36G utilized PBMCs from two donors. The Kruskal-Wallis test was used for each subset of CAR T cells and total T cells. Subsequently, post-hoc Dunn’s analysis was conducted to compare each group with the treatment group. The table in FIG.23G displays the adjusted p-values. This aligns with the in vitro and in vivo analyses disclosed herein.
  • Example 15 The efficacy of the CAR-E requires signaling through both the CAR and the IL-2R intracellular signaling domains [000373] To better understand the mechanism of action of CAR-E, it was investigated whether the effects on CAR T cells are solely mediated by anchoring the low-affinity IL-2 onto CAR T cells through BCMA-to-CAR binding or if it involves simultaneous engagement of both IL-2R and CAR intracellular signaling domains. For this purpose, BCMA CAR T cells were made using a BCMA CAR construct lacking the 41BB-CD3 ⁇ intracellular signaling domain but retaining an identical extracellular ectodomain (referred to as CAR-Intracellular domain deletion, or CAR-ICD- ⁇ ).
  • Dasatinib a lymphocyte cell-specific protein-tyrosine kinase (LCK) inhibitor, and Ruxolitinib, a Janus kinase (JAK) inhibitor, both individually and in combination, significantly inhibited the impact of the CAR-E molecule on CAR T cells (FIGs.33E – 33G), further suggesting that the CAR-E molecules engages both CAR and IL-2R receptors and activating their intracellular signaling pathways.
  • LCK lymphocyte cell-specific protein-tyrosine kinase
  • Ruxolitinib a Janus kinase (JAK) inhibitor
  • CAR-ICD- ⁇ T cells were treated with the BCMA-muIL2 CAR-E molecule. Treatments were removed after 2 hours to mimic in vivo conditions, and cells were subjected to bulk RNA-sequencing either 2 or 22 hours later. [000376] Remarkably, the CAR-E induced substantial transcriptomic changes, demonstrating quantitatively larger fold-changes compared to all other conditions, including wild-type IL-2 (FIGs.33J – 33N).
  • GSEA gene set enrichment analysis

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Abstract

Disclosed are novel weak affinity IL-2 variants and uses thereof, including adoptive cell therapy by enhancing the potency and duration of CAR-immune cells to treat cancer.

Description

VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 CAR-ENGAGER CONTAINING IL-2 VARIANTS TO ENHANCE THE FUNCTIONALITY OF CAR T CELLS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Nos. 63/664,020, filed June 25, 2024 and 63/537,660, filed September 11, 2023, each of which is incorporated herein by reference in its entirety. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 7, 2024, is named 046094_788001WO_ST.xml and is 133 KB bytes in size. BACKGROUND OF THE DISCLOSURE [0003] The cognate IL-2 receptor (IL-2R) includes three subunits, namely IL-2 subunit alpha (IL-2Rα; CD25), IL-2 subunit beta (IL-2Rβ; CD122), and the IL-2 subunit gamma (IL-2Rγ; CD132). [0004] IL-2 may also induce an alternative differentiation pathway of T cells, resulting in the generation of distinct “better” effector CD8+ T cells (Hashimoto et al., Nature 610(7930):173-181 (2022)). This process may rely, at least in part, on IL-2 binding to IL-2Rα. Additionally, IL-2Rβγ- biased agonists may drive T cells towards a terminally differentiated state (Codarri et al., Nature 610(7930):161-172 (2022)). [0005] Known IL-2 variants may have different affinities for different IL-2R subunits and may offer advantages in connection with therapies. For example, a weak affinity IL-2 (muIL2) that contains the amino acid substitutions of H16A and F42A, relative to wild-type IL-2, has increased selectivity for T cells and less toxicity through reduced global IL-2 binding and activation of all IL- 2R expressing cells. The binding affinity of muIL2 for human IL2Rα and IL2Rβ is decreased 110- and 3-fold, respectively, compared with wild-type IL-2 (Quayle et al., Clin. Cancer Res.26(8):1953- 1964 (2020)). Challenges persist with IL-2-based therapies in the clinic, with many clinical trials failing to meet their primary endpoints (Raeber et al., Ebiomedicine 90:104539 pp. 1-25 (2023)). 1 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 For example, the PIVOT IO-001 trial (NCT03635983), tested NKTR-214, a pegylated prodrug of recombinant IL2, versus NKTR-214 combined with Pembrolizumab in metastatic melanoma, but the trail failed its primary endpoints of ORR, PFS, and OS (Diab et al., J. Clin. Oncol.41(30):4756- 4767 (2023)). [0006] Chimeric antigen receptor (CAR) expressing T cells have revolutionized the treatment of blood-borne malignancies and have shown promising results in the treatment of hematopoietic cancers. Six CAR T cell therapies targeting two antigens, CD19 and BCMA, are currently FDA- approved. CD19 is a B-cell co-receptor expressed on B cells and a wide variety of blood-borne malignancies. CD19 CAR T cells were initially approved for the treatment of acute lymphoblastic leukemia (ALL) and have subsequently been approved for Burkitt’s Lymphoma and Mantle Cell Lymphoma. BCMA is a receptor expressed on the surface of B-cell lineage cells and a major marker of multiple myeloma (MM). MM is associated with an uncontrollable expansion of plasma cells in the bone marrow, which can progress to extra-medullary lesions forming elsewhere in the body. BCMA CAR T cell therapy has shown great promise against MM with studies showing an overall response rate of 80% even in patients with extra-medullary lesions (Gagelmann et al., Eur. J. Haematol.104(4):318-327 (2020)). [0007] However, challenges persist with CAR T cell therapy. A recent meta-analysis of 22 CAR T cell clinical studies highlighted its ineffectiveness in solid tumors with a poor average overall response rate of 9% (Hou et al., Dis. Markers 2019:3425291 pp. 1-11 (2019)). The duration of response, even in hematological cancers remains a challenge, with almost all BCMA CAR-treated MM patients ultimately relapsing (Gagelmann et al., Eur. J. Haematol. 104(4):318-327 (2020); Roex et al., J. Hematol. Oncol.13(1):164 (2020); Raje et al., N. Engl. J. Med.380(18):1726-1737 (2019)). In addition, treatments can have severe side effects including cytokine release syndrome (CRS) and neurotoxicity. [0008] CAR T cells need to home to the tumor location, expand, and persist in circulation, at least until they neutralize and kill the last remaining cancer cells. Therefore, approaches to enhance and prolong the activity of CAR T cells in a controlled way are critically needed. SUMMARY OF THE DISCLOSURE [0009] The presently disclosed IL-2 variants, chimeric antigen receptor (CAR)-engagers containing an IL-2 variant, and methods of enhancing activity of chimeric CAR immune cells are 2 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 expected to address the above needs. The IL-2 variants, per se or connected with another active moiety, such as an antibody or binding fragment thereof, augment anti-cancer therapy. The CAR- engagers containing an IL-2 variant enhance CAR immune cells, driving them toward generation of memory cells, and preventing exhaustion. Further, since CAR-engager binding to CAR is reversible, the CAR-engagers does not induce immune synapse formation of the CAR on the CAR-NK cell surface. Therefore, CAR-engagers do not block CAR-mediated killing of target cells. [00010] A first aspect of the present disclosure is directed to an IL-2 variant, which differs from wild type IL-2 (SEQ ID NO: 102) in terms of from three to eight amino acid substitutions selected from amino acid residues H16, D20, R38, F42, Y45, E62, L72, and V91 of SEQ ID NO: 1. In some embodiments, the three to eight amino acid substitutions are selected from H16A, H16R, H16S, D20A, D20Q, R38D, F42A, Y45A, E62N, L72G, and V91H. [00011] Related aspects include nucleic acids encoding the IL-2 variants, vectors containing the nucleic acid, cells transformed with the vector, methods of making the IL-2 variants, pharmaceutical compositions containing the IL-2 variants, and uses thereof to treat cancer. [00012] One such use involves enhancement of adoptive cell therapy such as CAR T therapy. Therefore, another aspect of the present disclosure is directed to a chimeric antigen receptor (CAR)- engager designed for use with CAR-immune cell therapy such as CAR-T therapy, wherein the CAR- engager contains a first moiety comprising that binds an epitope on an extracellular domain (ED) of the CAR connected to a second moiety comprising an immune effector domain comprising the IL- 2 variant. The ED includes one or more extracellular binding domains (EBDs) of the CAR and any other extracellular portions of the CAR, e.g., a linker that connects antibody fragments, or EBDs, etc. The connection between the first moiety and the second moiety may be peptidic or non-peptidic (covalent), such that the CAR-engager may be a continuous protein or polypeptide, or a moiety that contains two proteinaceous entities connected by a covalent bond. [00013] Another aspect of the present disclosure is directed to a heterodimeric CAR-engager, containing a first moiety that binds an epitope on an EB of the CAR connected to first dimerization domain, and a second moiety containing an immune effector domain which comprises the IL-2 variant connected to a second dimerization domain, wherein the first and the second dimerization domains bind to form a heterodimeric CAR-engager. [00014] Related aspects include nucleic acids that encode the CAR-engager (in the embodiments wherein the CAR-engager is a continuous protein), nucleic acids that encode a first moiety of the 3 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 heterodimeric CAR-engager protein, nucleic acids that encode the second moiety of the heterodimeric CAR-engager protein, vectors containing the nucleic acids encoding the CAR- engager protein, vectors containing the first and/or second proteins of the heterodimeric CAR- engager, cells transformed with the vector(s), pharmaceutical compositions that contain the CAR- engager and a pharmaceutically acceptable carrier, and methods of making the CAR-engager protein. [00015] A further aspect of the present disclosure is directed to a method of treating cancer. The method entails administering to a subject a first course of an effective amount of CAR-engager therapy. In some embodiments, the subject will have received a prior administration of immune cells that express a CAR that contains an extracellular domain that binds an antigen present on a cancer cell that contains the ectodomain, and the ectodomain of the CAR-engager, a transmembrane domain, and an intracellular domain comprising a stimulatory domain. [00016] Working examples, e.g., Example 11, disclosed herein demonstrate that CAR-engagers with different IL-2 variants enhance CAR immune cells that target BCMA and CD19, drives them toward generation of memory cells, and prevents exhaustion of the CAR-immune cells. The working examples also demonstrate that the timing and dosage amounts of the CAR-engager therapy may optimize its effects on the prior CAR immune cell therapy in terms enhancing CAR immune cells that target a cancer antigen by driving them toward generation of memory immune cells, and exhaustion-preventative proliferation of the CAR immune cells. And thus, the working examples present a hypothesis of a fundamental mechanism of action as between the CAR-engager and the CAR immune cells. More specifically, the working examples demonstrate that the two binding events, namely the binding between the immune effector domain of the CAR-engager and the cognate receptor on the immune cells, and the binding between the moiety of the CAR-engager that binds the EB of the CAR, produce a synergistic, molecular “cross-talk” between the intracellular domain (endodomain) of the cognate receptor and the endodomain stimulatory regions of the CAR, respectively, that results in production of IFN-γ and TNF-α, and ultimately the generation of memory immune cells, and exhaustion-preventative proliferation of the CAR immune cells. BRIEF DESCRIPTION OF THE DRAWINGS [00017] FIG. 1 schematically illustrates the domains of a CAR-engager according to some embodiments that contains a first moiety containing an antigen that is used as a target for a CAR, 4 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 such as the ectodomain of CD19 or BCMA on the surface of cancer cells, a dimerization domain, such as CH3, and a second moiety containing an immune cell effector domain (ICE). The CAR- engager may be a monomer, a dimer, or a multimer. [00018] FIGs.2A – 2I are a set of illustrations and line plots that show three CAR-engagers. FIG. 2A schematically illustrates a CAR-engager that contains a BCMA ectodomain and a Neo2/15 synthetic cytokine immune cell effector domain. FIG.2B schematically illustrates a CAR-engager that contains a BCMA ectodomain and two weak affinity mutated IL-2 (mIL2) synthetic cytokine immune cell effector domains. FIG. 2C schematically illustrates a CAR-engager that contains a BCMA ectodomain and a 4-1BBL immune cell effector domain. FIG.2D is a line plot that shows dose-dependent staining of CAR T cells that bind CD19 or non-transduced T cells (NT T cells) with CAR-engager or control proteins. FIG.2E is a line plot that shows dose-dependent staining of CAR T cells that bind BCMA or non-transduced T cells (NT T cells) with CAR-engager or control proteins. FIGs.2F and 2G are a line and bar plot, respectively, that together show dose-dependent activation of CAR T cells after CAR-engager treatment. FIG. 2H is a line plot showing that the BCMA-muIL2 CAR-engager does not block the killing efficacy of the CAR T cells. FIG. 2I is a line plot that shows phosphorylation of signal transducer and activator of transcription (STAT5) in the BCMA CAR T cells. [00019] FIGs.3A – 3B are a set of illustrations and line plots showing the effects of CAR-engagers on non-transduced T cells. FIG.3A schematically illustrates the experimental design. FIG.3B is a set of line plots that show T cell count and carboxyfluorescein succinimidyl ester (CFSE) staining of non-transduced, activated T cells treated with teceleukin, a CAR-engager containing an BCMA ectodomain and two mutated weak affinity IL-2 (muIL2), or CAR-engager containing an BCMA ectodomain and a Neoleukin domain. [00020] FIGs. 4A – 4C are a set of illustrations and line plots showing that CAR-engagers specifically activate CAR T cells. FIG.4A schematically illustrates the experimental design. FIG. 4B is a bar plot that shows the percentage of CD69+ anti-BCMA CAR-transduced, activated T cells after treatment with BCMA CAR-engager, BCMA CAR-engager without an immune cell effector domain control, or a non-antigen-specific CAR-engager control. FIG. 4C is a bar plot that shows the percentage of CD69+ anti-CD19 CAR-transduced, activated T cells after treatment with CD19 CAR-engager or a non-antigen-specific CAR-engager control. 5 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [00021] FIG.5 is a line plot showing that CAR-engagers do not inhibit BCMA CAR T cell killing and that the percentage of OPM2 target cell survival after incubation with CAR T cells and CAR- engagers (red) or non-transduced T cells (blue). [00022] FIGs. 6A – 6C are a set of illustrations and photographs showing that CAR-engagers reduce tumor burden in vivo. FIG.6A schematically illustrates the experimental design. FIGs.6B – 6C are a set of photographs that show tumor burden in mice before and after CAR T cell infusion and CAR-engager treatment. [00023] FIGs.7A – 7C are a set of flow cytometry plots showing the tumor burden in mice after CAR T cell infusion and CAR-engager treatment. FIG. 7A is a set of flow cytometry plots that shows OPM2 tumor burden in blood, spleen, and lymph nodes. FIG.7B is a set of flow cytometry plots that shows OPM2 tumor burden in bone marrow and lung. FIG.7C is a set of flow cytometry plots that shows OPM2 tumor burden in liver, kidney, and the eye tumor site. eGFP (OPM2 cells) is shown on the y-axis and PerCP signal control is shown on the x-axis. [00024] FIGs.8A – 8C are a set of flow cytometry plots showing human CD45+ and CAR- T cells in mice after CAR T cell infusion and CAR-engager treatment. FIG.8A is a set of flow cytometry plots that shows CAR T cells in blood, spleen, and lymph nodes. FIG.8B is a set of flow cytometry plots that shows CAR T cells in bone marrow and lung. FIG.8C is a set of flow cytometry plots that shows CAR T cells in the liver, kidney, and the eye tumor site. CD45 staining is shown on the y- axis and CAR-engager labeled with AF647 staining is shown on the x-axis. [00025] FIGs.9A –9E are a set of schematics, line plots, and box plots showing that CAR-engager treatment results in enhanced activity and persistence of CAR T cells in vivo. FIG.9A is a line plot that shows circulating half-life of the BCMA CAR-engagers. FIG.9B schematically illustrates the experimental design. FIGs. 9C and 9D are a set of flow cytometry plots and box plots that show selective expansion and persistence of BCMA CAR T cells. FIG. 9E is a box plot that shows the percentage of CD8+ CAR T cells after CAR-engager treatment. [00026] FIGs. 10A – 10J are a set of schematics, survival, line, bar, and t-distributed stochastic neighbor embedding (tSNE) plots and photographs showing that CAR-engager treatment lowers the required dose of CAR T cells. FIG.10A schematically illustrates the experimental design. FIG.10B is a set of photographs that shows tumor burden in mice before and after CAR T cell infusion and CAR-engager treatment. FIG.10C is a Kaplan-Meier plot that shows survival analysis. FIG.10D is a line plot that shows flow cytometric analyses of CAR T cells in blood samples. FIG.10E is a set 6 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 of bar plots that show generation of memory CAR T cells. FIGs. 10F and 10G are a set of flow cytometric plots and bar plots showing that a substantial number of CAR T cells two months post- CAR T cell injection. FIG.10H is a line plot showing that mice maintained consistent body weight throughout the experiment. FIG.10I is a set of flow cytometric plots that show CAR T cells from CAR-engager treated mice have a stem-cell memory phenotype. FIG. 10J is a set of tSNE plots displaying FlowSOM defined clusters among persisting BCMA CAR T cells. [00027] FIGs. 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. FIG. 11A schematically illustrates the experimental design. FIG.11B is a set of photographs that show tumor burden in mice before and after CAR T cell infusion and CAR-engager treatment. FIG.11C is a set of flow cytometric plots showing persistence of CAR T cells. FIG.11D is a set of bar plots showing in vitro killing assays of persistent T cells. FIG.11E is a set of t-SNE plots showing immune cell markers from CD8+ T cells. [00028] FIGs. 12A – 12B is a schematic and a set of bar plots showing that CAR-E treatment expands CAR T cells in vivo in the absence of tumor cells. FIG.12A schematically illustrates the experimental design. FIG. 12B is a set of bar plots that show counts of CAR T cells 30 days-post injection. [00029] FIGs.13A – 13B are a set of flow cytometry plots showing that neither the BCMA-muIL2 nor the VHH-muIL2 treatment exhibited binding to any specific population within human PBMCs. PBMCs were labeled with various markers to pre-gate B cells (CD20), T cells (CD3), or myeloid cells (CD11b). Cells were stained using different concentrations of the treatments followed by an anti-FLAG-Alexa647 secondary staining. FIG. 13A is a set of flow cytometry plots showing that BCMA-muIL2 does not bind to human PBMCs. FIG.13B is a set of flow cytometry plots showing that VHH-muIL2 does not bind to human PBMCs. [00030] FIGs. 14A – 14C are a set of photomicrographs and dot plots showing specific binding and gradual internalization of the BCMA-muIL2 in CAR T cells. FIG. 14A is a set of photomicrographs that show cells stained with CellTracker Blue CMAC, incubated with the indicated treatment, each treatment labeled with Alexa647 (BCMA-muIL2) or dsRed (VHH- muIL2) for 1 to 5 hours and imaged. Photomicrographs are representative of >100 cell images. FIG. 14B is a dot plot that shows quantitative analysis of the imaged cells. FIG. 14C is a dot plot that 7 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 shows the correlation between Alexa647 mean intensity (BCMA-muIL2) and dsRed mean intensity (VHH-muIL2). [00031] FIGs. 15A – 15C are a set of flow cytometry plots showing individual flow cytometric data corresponding to the pooled data presented in FIG. 9D. FIG. 15A is a set of flow cytometry results from mice treated solely with CAR T cells. FIG.15B is a set of flow cytometry results from mice treated with CAR T cells and VHH-muIL2. FIG.15C is a set of flow cytometry results from mice treated with CAR T cells and BCMA-muIL2. [00032] FIGs. 16A – 16C are a set of flow cytometry plots showing individual flow cytometric data of the mice shown in FIGs.10A –10J. FIG.16A is a set of flow cytometric results from mice treated solely with CAR T cells. FIG.16B is a set of flow cytometry results from mice treated with CAR T cells and VHH-muIL2. FIG.16C is a set of flow cytometry results from mice treated with CAR T cells and BCMA-muIL2. [00033] FIGs. 17A – 17C are a set of bar, line, and tSNE plots showing human T cell-derived cytokines in the serum of mice that received OPM2 cancer cells followed by a low dose of CAR T cells. FIG.17A is a bar plot that shows levels of IFNγ, GM-CSF, and TNFα. Serum samples were diluted at a ratio of 1:40. The same plates were used to incubate both the standard samples and the serum samples, and a standard curve was plotted for each cytokine. FIG.17B is a set of line plots that shows IFNγ levels between the BCMA-muIL2 group and the VHH-muIL2 group (error bars represent mean with standard deviation). FIG. 17C is a set of Flt-SNE mapping of CAR T cells derived from the PBS, BCMA-muIL2 and VHH-muIL2 treated mice showing the expression of ten immune cell markers. [00034] FIG.18 is a set of t-SNE mapping of CD4+ CAR+ T cells derived from the five BCMA- muIL2 CAR-E treated mice showing the expression of nine immune cell markers. [00035] FIGs. 19A – 19G are a set of flow cytometry, tSNE, bar, violin, and pie plots and heatmaps showing single-cell RNA sequencing analyses elucidate BCMA-muIL2 effect on CAR T cells. FIG.19A is a set of flow cytometry plots showing CAR+ cells analyzed 89 days after CAR-T administration. FIG. 19B is a tSNE plot that shows data after Harmony algorithm, showing proportion of CD4, CD8, and proliferating (CD4 and CD8) cells. FIG.19C is a tSNE plot that shows split between the groups treated with BCMA-muIL2 or VHH-muIL2 treatments. FIG.19D is a set of heatmaps of significantly differentially expressed genes in CD4+ CAR T cells and CD8+ CAT T cells after the indicated treatment. FIG.19E is a set of violin plots of the gene scores between CD8 8 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 and CD4 cells, the scores being constructed using the normalized expression of the different genes for each phenotype in FIG. 19D. FIG. 19F is a set of pie plots that shows the diversity of T-cell receptor (TCR) clonotypes. FIG.19G is a bar plot that shows clonotype diversity within a sample’s total cell count. [00036] FIGs. 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. FIG. 20A schematically illustrates the experimental design. FIG. 20B is a set of flow cytometry plots that show selective trafficking, expansion, and persistence of BCMA CAR T cells. [00037] FIGs.21A – 21C are a set of line and bar plots showing the effects of CAR-engagers on non-transduced T cells and CAR T cells. FIGs.21A and 21B are a line and bar plot, respectively, that together show dose-dependent activation of CAR T cells after CAR-engager treatment (FIG.21A) and that the CAR-engager does not activate non-transduced T cells (FIG. 21B). FIG.21C is a line plot showing that the CD19-muIL2 CAR-engager does not block the killing efficacy of the CD19 CAR T cells or non-transduced T cells (NT T cells). [00038] FIGs. 22A – 22B are a set a of line plots showing activation of CAR T cells by CAR- engager variants. FIG. 22A is a line plot showing that CAR-engager variant treatment results in STAT5 phosphorylation. FIG.22B is a line plot showing that CAR-engager variant treatment results in CD69 expression on CAR T cells. [00039] FIGs.23A – 23B are a set of line plots showing activation of rested CAR T cells by CAR- engager variants. FIG. 23A is a line plot showing that CAR-engager variant treatment results in STAT5 phosphorylation in rested CAR T cells. FIG.23B is a line plot showing that CAR-engager variant treatment results in CD69 expression on rested CAR T cells. [00040] FIGs.24A – 24B are a set of line plots showing the percentage of phosphorylated STAT5 (pSTAT5) positive BCMA CAR T Cells containing FDA-approved CAR constructs induced by BCMA CAR-engagers. FIG. 24A is a line plot that shows pSTAT5 positive BCMA CAR T cells containing the FDA-approved idecabtagene vicleucel (Ide-cel) construct. FIG. 24B is a line plot showing pSTAT5 positive BCMA CAR T cells containing the FDA-approved Ciltacabtagene autoleucel (Cilta-cel) construct. BCMA CAR T cells were subjected to the indicated treatments at varying doses for 30 minutes at 37 °C, followed by the assessment of pSTAT5 by cytometry (n=3 for each condition). 9 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [00041] FIGs.25A – 25F are a set of line plots showing the percentage of phosphorylated STAT5 (pSTAT5) positive BCMA CAR T Cells containing FDA-approved CAR constructs induced by BCMA-muIL2 CAR-engagers. FIGs.25A – 25C are a set of line plots that show pSTAT5 positive BCMA CAR T cells containing the FDA-approved Ide-cel construct. FIGs.25D – 25F are a set of line plots that show pSTAT5 positive BCMA CAR T cells containing the FDA-approved Cilta-cel construct. For incubation of 24 hours, CAR T cells were washed at 2 hours to remove unbound any CAR-engager. [00042] FIGs. 26A – 26B are a set of line plots showing BCMA CAR-engager dose-dependent activation of rested BCMA CAR T cells through CD69 expression. FIG. 26A is a line plot that shows pSTAT5 positive BCMA CAR T cells containing the FDA-approved Ide-cel construct. FIG. 26B is a line plot showing pSTAT5 positive BCMA CAR T cells containing the FDA-approved cilta-cel construct. CD69 serves as an activation marker for human T cells. [00043] FIGs. 27A – 27B are a set of line plots showing BCMA CAR-engager dose-dependent activation of rested BCMA CAR T cells through CD69 expression. FIG. 27A is a line plot that shows pSTAT5 positive BCMA CAR T cells containing the FDA-approved idecabtagene vicleucel construct. FIG. 27B is a line plot showing pSTAT5 positive BCMA CAR T cells containing the FDA-approved ciltacabtagene autoleucel (Cilta-cel) construct. [00044] FIGs. 28A – 28B are a set of line plots showing BCMA CAR-engager dose-dependent activation of rested BCMA CAR T cells through CD69 expression. FIG. 28A is a line plot that shows pSTAT5 positive BCMA CAR T cells containing the FDA-approved idecabtagene vicleucel construct. FIG. 28B is a line plot showing pSTAT5 positive BCMA CAR T cells containing the FDA-approved ciltacabtagene autoleucel construct. [00045] FIGs.29A – 29C are a set of line plots showing induction of CAR T cell proliferation in vitro from CAR-engager treatment with reduced impact on non-transduced T cells as compared to wild-type IL-2. FIGs. 29A – 29B are a set of line plots that show incubation of activated BCMA CAR T cells with the ciltacabtagene autoleucel CAR construct, with varying concentrations of BCMA CAR-engager treatment, followed by assessment of CAR T cell proliferation using flow cytometry after 3 days. The dashed line represents the number of CAR T cells without any treatment. FIG. 29C is a line plot that shows the impact of CAR-engagers on expanding normal, non- transduced T cells. CAR-engagers proliferate non-transduced T cells significantly less than treatment with wild-type IL-2. CAR-engagers containing the triple mutant IL-2 (V7, V9, Y2, and 10 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 Y9) induces significantly less proliferation in normal T cells compared to CAR-engagers containing the double-mutant IL-2 (U4) [00046] FIGs.30A – 30C are a set of line plots showing bio-layer interferometry (BLI) associate and dissociation between CAR-engagers containing the triple mutant IL-2 V7 or CAR-engagers containing the double-mutant IL-2 U4 exhibits lower affinity for IL-2Ra as compared to wild-type IL-2. FIG.30A is a line plot that shows BLI between wild-type IL-2 (Teceleukin) and IL-2Ra. FIG. 30B is a line plot that shows BLI between the V7 CAR-engager and IL-2Ra. FIG.30C is a line plot that shows BLI between the CAR-engager containing the U4 CAR-engager and IL-2Ra. [00047] FIGs.31A – 31B are a set of schematics and line plots showing that the CAR-engagers containing IL-2 variants expand CAR-T cells in vivo, and that the expansion levels correlate with in vitro pSTAT5 signaling. FIG.31A schematically illustrates the experimental design. Mice received OPM2 cancer cells, followed by 0.5×106 CAR-T cells (ciltacabtagene autoleucel) after one week. FIG.31B is a set of line plots that show CD4 and CD8 CAR T cells circulating in the blood. Mice were bled for 5 weeks on days 11, 18, 28, 35, 42 post injection, and CD4 and CD8 CAR T-cells were counted. All the three tested CAR-engagers expanded CAR T cells in vivo as compared to the control, PBS treatment group. V7 and Y2 CAR-engagers expanded CAR T cells more than X12 CAR-engagers. [00048] FIGs.32A – 32C are a set of schematics, photographs, and line plots showing BCMA-V7 CAR-engagers enhances CAR-T cell activity when administered at day 3 or even day 14 post- CAR T-cell injection. FIG. 32A schematically illustrates the experimental design. One cohort of mice only received CAR T-cells (n=4). Another cohort of mice received 6 doses of V7 CAR-engager treatment starting from day 3 post-injection of CAR-T cells, administered on days 3, 6, 10, 14, 21, 28. The third cohort of mice received 6 doses of treatment starting from day 14, which is the post- CRS window in patients, and on days 14, 18, 21, 28, 35, 42. FIG.32B is a set of photographs that shows BLI imaging on the indicated days to assess tumor growth in the different cohorts. FIG.32C is a line plot that shows CAR T cells circulating in the blood. Mice were bled for 6 weeks, once per week starting from day 14, and CAR T cells were counted. V7 CAR-engager treatment expanded CAR T cells in both cohorts that received treatment, compared to the PBS treatment group. [00049] FIGs. 33A – 33M are a set of schematics, line, bar, and dot plots showing substantial activation and transcriptomic changes in CAR T cells after CAR-E treatment. FIG. 33A is a line plot that shows CAR-E induction of pSTAT5 activity in CAR T cells with either the full CAR 11 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 construct or the CAR-ICD-∆ construct. FIGs.33B – 33D are a set of line plots that show CAR-E CD69 (FIG. 33B), IFN-γ (FIG.33C), and TNF-α (FIG. 33D) staining in BCMA CAR T cells and BCMA CAR-ICD-∆ T cells. FIGs.33E – 33G are a set of line plots that show CD69 (FIG.33E), IFN-γ (FIG. 33F), and TNF-α (FIG. 33G) staining after CAR-E treatment with dasatinib or ruxolitinib on CAR T cells. FIG.33H schematically illustrates the experimental design for in vivo assessment of the efficacy of CAR-E on BCMA CAR T cells and BCMA CAR-ICD-∆ T cells. FIG. 33I is a set of bar plots that show expansion and persistence of BCMA CAR T cells and BCMA CAR-ICD-∆ T cells in mouse organs 1 month after CAR T cell injection; **** P<0.0001. FIG.33J is a volcano Plot that shows the highest upregulated genes in CD8+ CAR T cells 4 hours after CAR- E treatment. FIG. 33K is a volcano plot that shows the highest upregulated genes in CD8+ CAR- ICD-∆ T cells 4 hours after CAR-E treatment. FIG. 33L is a heatmap that shows gene expression changes in CD8+ and CD4+ T cells after 4 hours of CAR-E treatment. FIG.33M is a heatmap that shows gene expression changes in CD8+ and CD4+ T cells after 2 and 24 hours of CAR-E treatment. [00050] FIG.34 is a set of bar plots showing transcriptome changes in CAR T cells after CAR-E treatment. [00051] FIGs.35A – 35G are a set of schematics, photographs, line, and pie graphs showing that lower doses of CAR-engagers enhance CAR T cell activity and promote functional memory. FIG. 35A schematically illustrates the experimental design. FIG.35B is a set of photographs that show bioluminescence imaging (BLI) of monitored tumor burdens. FIG. 35C is a survival analyses showing that all CAR-engager treated mice survived for the duration of the experiment. FIG.35D is a line graph that shows the quantification of BLI analyses from FIG.35B. FIG.35E is a line graph that shows flow cytometric analyses of CAR T-cell presence in blood. FIG.35F is a line graph that shows IFN-γ levels. FIG.35G is a set of pie graphs that show the CAR T cells in the bone marrow and spleen of mice treated with CAR-engager. [00052] FIGs.36A – 36G are a set of schematics, bar, and line graphs showing treatment of CAR- engager expands CAR T cells in in vivo in the absence of tumor cells in a dose-dependent manner. FIG. 36A schematically illustrates the experimental design. FIGs. 36B – 36C are a set of bar and line graphs showing flow cytometric analysis of spleen and bone marrow tissues harvested 30 days post-injection of CAR T cells. FIGs. 36D – 36E are a set of bar graphs that show analyses of persisting CAR T cells and different subsets of memory CAR T cells in the spleen and bone marrow 12 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 of the CAR-E-treated mice. FIG. 36F is a bar graph showing that both the antigen and the low- affinity IL-2 components of CAR-engager are essential for its impact. [00053] FIG. 37 a set of flow cytometry plots showing anti-human-CD45 and BCMA-CAR staining in blood samples of mice that received human CAR T cells with and without CAR-E treatment. [00054] FIGs. 38A – 38B are a set of flow cytometry plots showing anti-human-CD45 and BCMA-CAR staining in individual mice that received human CAR T cells with and without CAR- E treatment. FIG.38A is a set of flow cytometry plots of mouse organs collected at different time points after CAR T cell administration. FIG.38B is a set of flow cytometry plots of mouse organs collected at different time points after CAR T cell and CAR-E administration. [00055] FIGs. 39A – 39D are a set of heatmaps and tSNE plots showing CAR-E promotion of phyotypic diversity of bone marrow and splenocyte-derived CAR T cells. FIG. 39A is a heatmap that shows eight FLOWSOM-derived metaclusters in bone marrow samples. FIG.39B is a heatmap that shows eight FLOWSOM-derived metaclusters in spleen samples. FIG.39C is a tSNE plot that shows CAR T populations in bone marrow samples. FIG. 39D is a tSNE plot that shows CAR T populations in spleen samples. [00056] FIGs. 40A – 40D are a set of flow cytometry plots showing anti-human-CD45 and BCMA-CAR staining in individual mice that received human CAR T cells and CAR-E treatment. FIG.40A is a set of flow cytometry plots of mice that received CAR T cells and PBS control. FIG. 40B is a set of flow cytometry plots of mice that received CAR T cells and 2 mg/kg BCMA-muIL2. FIG.40C is a set of flow cytometry plots of mice that received CAR T cells and 4 mg/kg BCMA- muIL2. FIG.40D is a set of flow cytometry plots of mice that received CAR T cells and 8 mg/kg BCMA-muIL2. [00057] FIGs. 41A – 41E are a set of flow cytometry plots showing anti-human-CD45 and BCMA-CAR staining in individual mice that received human CAR T cells and CAR-E treatment. FIG. 41A is a set of flow cytometry plots of mice that received CAR T cells and BCMA-CH3 control. FIG.41B is a set of flow cytometry plots of mice that received CAR T cells and low dose IL-2. FIG.41C is a set of flow cytometry plots of mice that received CAR T cells and VHH-muIL2. FIG. 41D is a set of flow cytometry plots of mice that received CAR T cells and BCMA-muIL2. FIG.41E is a set of flow cytometry plots of mice that received CAR T cells only. 13 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [00058] FIG.42 is a set of line plots showing CAR-engagers each containing an immune cell effector domain containing an U4, V6, V7, or Y2 IL-2 variant and their selective and potent induction of proliferation in CAR T cells but not non-transduced T cells. [00059] FIG.43 is a set of line plots showing phosphorylation of STAT5 in CAR T cells and non- transduced T cells following CAR-E treatment. [00060] FIG. 44 is a set of line plots showing CD69 expression on CAR T cells and non- transduced T cells following CAR-E treatment. [00061] FIG.45 is a set of line plots showing TNF-α and IFN-γ secretion from CAR T cells and non-transduced T cells following CAR-E treatment. [00062] FIG.46A – 46B are a set of line plots showing CAR-E binding to IL-2Rα and IL-2Rβγ by Biolayer Interferometry. DETAILED DESCRIPTION OF THE DISCLOSURE [00063] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated to facilitate the understanding of the present disclosure. [00064] As used in the description and the appended claims, the singular forms “a”, “an”, and “the” mean “one or more” and therefore include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an inhibitor” includes mixtures of two or more such inhibitors, and the like. [00065] Unless stated otherwise, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term “about.” [00066] The term “approximately” as used herein refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise 14 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). [00067] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element or method step not specified in the claim (or the specific element or method step with which the phrase “consisting of” is associated). The transitional phrase “consisting essentially of” limits the scope of a claim to the specified elements and method or steps and “unrecited elements and method steps that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure. IL-2 variants [00068] In one aspect, the disclosure provides an IL-2 variant, which differs from wild type IL-2 (SEQ ID NO: 102) in terms of from three to eight amino acid substitutions selected from amino acid residues H16, D20, R38, F42, Y45, E61, E62, L72, and V91 of SEQ ID NO: 102. In some embodiments, the three to eight amino acid substitutions are selected from H16A, H16R, H16S, D20A, D20Q, R38D, F42A, Y45A, E61A, E62N, L72G, and V91H. The term “IL-2 variant” as used herein refers to non-naturally occurring variant of IL-2 capable of binding to the cognate IL-2 receptor(s) on an immune cell and initiating signal transduction through that receptor to achieve substantially the same effect as the naturally occurring cytokine. [00069] The amino acid sequence of wild-type IL-2 is set forth below (SEQ ID NO: 102): 1 aptssstkkt qlqlehllld lqmilnginn yknpkltrml tfkfympkka telkhlqcle 61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [00070] All descriptions of the IL-2 variants of the present disclosure are made in reference to SEQ ID NO:102. In some embodiments, the IL-2 variant includes a first amino acid substitution (of the three to eight amino acid substitutions), from N-terminus to C-terminus, is selected from H16A, H16R, H16S, D20A, or D20Q, a second amino acid substitution is selected from R38D, F42A, and V91H, and a third amino acid substitution is selected from Y45A and E62N. [00071] In some embodiments, the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S. In other embodiments, the IL-2 variant includes a first amino acid substitution of D20A or D20Q. 15 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [00072] In some embodiments, the IL-2 variant includes a second amino acid substitution of F42A. In some embodiments, the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S and a second amino acid substitution of F42A. In some embodiments, the IL-2 variant includes a first amino acid substitution of D20A or D20Q and a second amino acid substitution of F42A. [00073] In some embodiments, the IL-2 variant includes a third amino acid substitution of Y45A. In other embodiments, the third amino acid substitution is E62N. [00074] In some embodiments, the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, and a third amino acid substitution of Y45A. In some embodiments, the IL-2 variant includes a first amino acid substitution of D20A or D20Q, a second amino acid substitution of F42A, and a third amino acid substitution of Y45A. [00075] In some embodiments, the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, and a third amino acid substitution of Y45A. In some embodiments, the IL-2 variant includes a first amino acid substitution of D20A or D20Q, a second amino acid substitution of F42A, and a third amino acid substitution of Y45A. [00076] In some embodiments, the IL-2 variant includes a fourth amino acid substitution selected from R38D, E61A, L72G, and V91H. [00077] In some embodiments, the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, a third amino acid substitution of Y45A or E62N, and a fourth amino acid substitution selected from D20A, D20Q, R38D, Y45A, E61A, L72G, and V91H. [00078] In some embodiments, the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, a third amino acid substitution of Y45A or E62N, a fourth amino acid substitution selected from D20A, D20Q, E61A, L72G, and V91H, and a fifth amino acid substitution of R38D. [00079] In some embodiments, the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, a third amino acid substitution of Y45A or E62N, a fourth amino acid substitution selected from D20A, D20Q, E61A, and V91H, a fifth amino acid substitution of R38D, and a sixth amino acid substitution of L72G. [00080] In some embodiments, the IL-2 variant includes a first amino acid substitution of H16A, H16R, or H16S, a second amino acid substitution of F42A, a third amino acid substitution of Y45A, 16 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 a fourth amino acid substitution selected from D20A, D20Q, E61A, and V91H, a fifth amino acid substitution of R38D, a sixth amino acid substitution of L72G, and a seventh amino acid substitution of E62N. [00081] In some embodiments, the IL-2 variant has amino acid substitutions D20Q, F42A, and Y45A, and has the amino acid sequence set forth below (SEQ ID NO: 112). Amino acid substitutions relative to wild-type IL-2 for SEQ ID NOs: 112-123 are boxed in the sequences below. 1 aptssstkkt qlqlehlllq lqmilnginn yknpkltrml tafampkka telkhlqcle 61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [00082] In some embodiments, the IL-2 variant has amino acid substitutions H16A, F42A, and E62N, and has the amino acid sequence set forth below (SEQ ID NO: 113): 1 aptssstkkt qlqleallld lqmilnginn yknpkltrml takfympkka telkhlqcle 61 enlkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [00083] In some embodiments, the IL-2 variant has amino acid substitutions H16A, F42A, and Y45A, and has the amino acid sequence set forth below (SEQ ID NO: 114): 1 aptssstkkt qlqleallld lqmilnginn yknpkltrml takfampkka telkhlqcle 61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [00084] In some embodiments, the IL-2 variant has amino acid substitutions H16R, F42A, and E62N, and has the amino acid sequence set forth below (SEQ ID NO: 115): 1 aptssstkkt qlqlerllld lqmilnginn yknpkltrml takfympkka telkhlqcle 61 enlkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [00085] In some embodiments, the IL-2 variant has amino acid substitutions H16S, F42A, and Y45A, and has the amino acid sequence set forth below (SEQ ID NO: 116): 1 aptssstkkt qlqlesllld lqmilnginn yknpkltrml takfampkka telkhlqcle 61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [00086] In some embodiments, the IL-2 variant has amino acid substitutions D20Q, V91H, F42A, and E62N, and has the amino acid sequence set forth below (SEQ ID NO: 117 1 aptssstkkt qlqlehlllq lqmilnginn yknpkltrml takfympkka telkhlqcle 61 enlkpleevl nlaqsknfhl rprdlisnin hivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt 17 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [00087] In some embodiments, the IL-2 variant has amino acid substitutions H16A, F42A, Y45A, and L72G, and has the amino acid sequence set forth below (SEQ ID NO: 118): 1 aptssstkkt qlqleallld lqmilnginn yknpkltrml takfampkka telkhlqcle 61 eelkpleevl ngaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [00088] In some embodiments, the IL-2 variant has amino acid substitutions at H16R, D20Q, F42A, and E62N, and has the amino acid sequence set forth below (SEQ ID NO: 119): 1 aptssstkkt qlqlerlllq lqmilnginn yknpkltrml takfympkka telkhlqcle 61 enlkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [00089] In some embodiments, the IL-2 variant has amino acid substitutions at H16S, D20Q, F42A, and E62N, and has the amino acid sequence set forth below (SEQ ID NO: 120): 1 aptssstkkt qlqleslllq lqmilnginn yknpkltrml takfympkka telkhlqcle 61 enlkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [00090] In some embodiments, the IL-2 variant has amino acid substitutions at H16S, F42A, Y45A, and L72G, and has the amino acid sequence set forth below (SEQ ID NO: 121): 1 aptssstkkt qlqlesllld lqmilnginn yknpkltrml takfampkka telkhlqcle 61 eelkpleevl ngaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [00091] In some embodiments, the IL-2 variant has amino acid substitutions at H16A, D20A, R38D, F42A, and E62N, and has the amino acid sequence set forth below (SEQ ID NO: 122): 1 aptssstkkt qlqleallla lqmilnginn yknpkltdml takfympkka telkhlqcle 61 enlkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [00092] In some embodiments, the IL-2 variant has amino acid substitutions at H16R, D20Q, R38D, F42A, and E62N, and has the amino acid sequence set forth below (SEQ ID NO: 123): 1 aptssstkkt qlqlerlllq lqmilnginn yknpkltdml takfympkka telkhlqcle 61 enlkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt CAR-engager (“CAR-E”) [00093] The IL-2 variants of the present disclosure may be useful in adoptive cell therapy, and particularly CAR T therapy, as immune effector domains of therapeutic moieties called CAR- engagers (also CAR-enhancers), as disclosed in Applicant’s PCT Application PCT/US2024/013091, entitled “DEVELOPING A CAR-ENGAGER PLATFORM TO ENHANCE 18 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 THE FUNCTIONALITY AND/OR PERSISTENCE OF CAR T CELLS.” As disclosed therein, CAR-engagers may augment CAR immune cell (e.g., CAR T cell) functionality and persistence in vivo. They may also reduce the cellular dose needed for CAR immune cell therapy, which may result in reduced adverse side effects (e.g., cytokine release syndrome) caused by the larger doses typically used in the clinic and therefore, the CAR-engagers are also referred herein as CAR-enhancers or CAR-E. Further, since CAR-engager binding to CAR is reversible, the CAR engager does not induce immune synapse formation of CAR on the CAR immune cell surface. Therefore, CAR- engagers do not block CAR-mediated killing of cancer cells. CAR immune cells often do not persist in the body during minimal residual disease (MRD), which, as known in the art, is associated with limited cancer antigens. The disclosed CAR-engagers may support persistence, proliferation, and efficacy of CAR T cells during states of MRD. [00094] Accordingly, in another aspect, the disclosure provides a CAR-E which contains a first proteinaceous moiety and a second proteinaceous moiety. The first proteinaceous moiety binds an epitope on the extracellular domain (ED) of a CAR. In some embodiments the first proteinaceous moiety binds an epitope present on the extracellular binding domain (EBD) of the CAR that binds a cancer antigen on the surface of a cancer cell. The second proteinaceous moiety, which is an immune effector domain, comprises an IL-2 variant of the present disclosure. [00095] In some embodiments, CAR-engager is a contiguous protein, where the first proteinaceous entity and the proteinaceous entity are connected by a peptide bond. In some embodiments, the first proteinaceous entity and the second proteinaceous entity are connected by click chemistry. [00096] In some embodiments, the CAR-engager is formulated and administered as a monomeric protein or proteinaceous entity. In other embodiments, the CAR-engager is formulated and administered in the form of a dimer, either as a homodimer or a heterodimer protein or proteinaceous entity. CAR-E – first proteinaceous moiety [00097] The first proteinaceous moiety of the CAR-engager is designed to bind an epitope present on the extracellular binding domain of the CAR that targets an antigen on the surface of a cancer cell. In some embodiments, the first moiety of the CAR-engager is an ectodomain that binds an EBD of a CAR presented on an immune cell. As is known in the art, the ectodomain of a cancer antigen is the portion of the antigen on the surface of a cancer cell that binds a T cell receptor or a CAR on 19 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 an immune cell. In some embodiments, the CAR-engager may include the entire extracellular domain of a cancer antigen. Ectodomains may be derived from (e.g., identified in) a cancer antigen in accordance with standard techniques. See, e.g., and Gershoni et al., Biodrugs 21(3):145-156 (2007) and Francino-Urdaniz and Whitehead, RSC Chem. Biol. 2(6):1580-1589 (2021). The term “derived from” as used herein when referring to a protein and nucleic acid refers to a sequence that originates and is identified from the sequence of a parent (e.g., wild-type or endogenous) protein and nucleic acid, respectively. A sequence derived from a parent sequence may be a portion (fragment) of the parent sequence and/or may vary by at least one position from the parent amino acid or nucleotide sequence. Protein variants may include amino acid substitutions, insertions, and/or deletions. For example, an amino acid sequence derived from a parent sequence may constitute a fragment of the parent sequence and be identical for a specific range of amino acids of the parent but does not include amino acids outside that specific region. Nucleic acid variants may include substitutions or in-frame insertions or deletions (i.e., insertions or deletions that do not result in downstream frame shift of the nucleic acid codons). [00098] The amino acid sequences of representative cancer antigens that may be targeted by a CAR immune cell, and from which an ectodomain may be derived are provided at the NCBI Accession numbers set forth in Table 1, and are incorporated herein by reference. Table 1: Gene Name, Symbols, and NCBI Accession Numbers of Representative Cancer Antigens Gene Name Gene Symbols Protein Accession No(s). Al h f t t i AFP HPAFP FETA AFPD NP 001125 NP 001341646 , , , , , ,
Figure imgf000022_0001
20 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 CD5 molecule CD5, LEU1, T1 NP_001333385, NP_055022 CD7 molecule CD7, GP40, TP41, LEU-9, NP_006128 , , , , , , , , , , , , , , , , , , , , , , , , , ,
Figure imgf000023_0001
21 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 CD80 molecule B7, B7-1, B7.1, BB1, NP_005182 CD28LG, CD28LG1, CD80, , , , , , , , , , , , , , , , , , , , , , , , ,
Figure imgf000024_0001
22 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 Claudin 18.2 CLDN18.2 NP_001002026 Cytotoxic T-lymphocyte ALPS5, CD, CD152, NP_001032720, NP_005205 , , , , , , , , , , , , , , , , , , ,
Figure imgf000025_0001
23 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 NP_001369734, NP_001369735, NP_004439 9, , , , , , , , , , , , , , , , , , , , , , , , , , , , ,
Figure imgf000026_0001
24 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 NP_001193795, NP_001369698, NP_001369699, NP_001369700, 2, , , , , , , , , , , , , , , , , , ,
Figure imgf000027_0001
25 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 1/X, MUC1, MUC1/ZD, NP_001191222, NP_001191223, Mucin-1, PEM, PEMT, PUM NP_001191224, NP_001191225, 9, , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,
Figure imgf000028_0001
26 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 NP_001358335, NP_001358336, NP_001358337, NP_006008 , , , , , , , , , , , , , , , , , ,
Figure imgf000029_0001
27 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [00099] The ectodomains are not limited to known cancer antigens. Unique cancer antigens (neoantigens) may be determined by known methods. For example, cancer genomes can be compared with normal cell genomes to identify neoantigens. In some embodiments, caner transcriptomes are compared to normal cell transcriptomes. Computational methods may then be utilized to identify suitable binding sites for a CAR. Most often the CAR binds a portion of the extracellular domain of an antigen. In some embodiments, the CAR and the corresponding cancer antigen are known in the art. [000100] In some embodiments, the ectodomain of the CAR-engager contains the entire extracellular domain of a cancer antigen. In some embodiments, the CAR-engager contains a portion of the extracellular domain of a cancer antigen which is targeted by a CAR. [000101] In some embodiments, wherein the CAR targets BCMA, the ectodomain of the CAR- engager contains the extracellular domain of BCMA. The amino acid sequence of a representative CAR-engager that contains a BCMA extracellular domain is MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA (SEQ ID NO: 1). [000102] In some embodiments, the ectodomain of the CAR-engager contains two repetitions of the extracellular domain of BCMA. The amino acid sequence of a representative CAR-engager that contains two repetitions of the BCMA extracellular domain is set forth below (SEQ ID NO: 2): 1 mlqmagqcsq neyfdsllha cipcqlrcss ntppltcqry cnasvtnsvk gtnagggsgg 61 gsprgsgggs mlqmagqcsq neyfdsllha cipcqlrcss ntppltcqry cnasvtnsvk 121 gtn [000103] In some embodiments wherein the CAR targets CD19, the ectodomain of the CAR- engager contains a variant of the extracellular domain of CD19. The amino acid sequence of a representative CAR-engager that contains a variant of CD19 extracellular domain is set forth below (SEQ ID NO: 3): 1 peeplvvkve egdeawlpcl kgtsdgptqq ltwsresplk pflkvsfgvp glgvhvrpna 61 vslvisnvsq qmggfylcqp gppsekawqp gwtvnvegsg elfrwnvsdl gglgcglknr 121 ssegpsspsg klmspklyvw akdrpeiweg eppclpprds lnqslsrdmt vapgstlwls 181 cgvppdsvsr gplswthvhp kgpksllsle lkddrpardm wvtgtrlflp rataqdagky 241 ychrgnltms fhlevkarpv sahtklrtgg wk [000104] In some embodiments, the ectodomain has at least 85% sequence identity to SEQ ID NO: 3, at least 90% sequence identity to SEQ ID NO: 3, at least 95% sequence identity to SEQ ID NO: 3, at least 98% sequence identity to SEQ ID NO: 3, at least 99% sequence identity to SEQ ID NO: 3. 28 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000105] The amino acid sequence of a representative CAR-engager that contains a second variant of CD19 extracellular domain is set forth below (SEQ ID NO: 4): 1 peeplvvkve egdeawlpcl kgtsdgptqq ltwsresplk pflkvsfgvp glgvhvrpna 61 vslvisqvsq qmggfylcqp gppsekawqp gwtvnvegsg elfrwqvsdl gglgcglkqr 121 ssegpsspsg klmspklyvw akdrpeiweg eppclpprds lqqslsrdmt vapgstlwls 181 cgvppdsvsr gplswthvhp kgpksllsle lkddrpardm wvtgtrlflp rataqdagky 241 ychrgqltms fhlevkarpv sahtklrtgg wk [000106] In some embodiments, the ectodomain of the CAR-engager contains at least a portion of the extracellular domain of CD19. The amino acid sequence of a representative CAR-engager that contains a CD19 extracellular domain set forth below (SEQ ID NO: 5): 1 peeplvvkve egdnavlqcl kgtsdgptqq ltwsresplk pflklslglp glgihmrpla 61 iwlfifnvsq qmggfylcqp gppsekawqp gwtvnvegsg elfrwnvsdl gglgcglknr 121 ssegpsspsg klmspklyvw akdrpeiweg eppclpprds lnqslsqdlt mapgstlwls 181 cgvppdsvsr gplswthvhp kgpksllsle lkddrpardm wvmetglllp rataqdagky 241 ychrgnltms fhleitarpv lwhwllrtgg wk [000107] In some embodiments, the ectodomain of the CAR-engager contains a portion of the extracellular domain of CD19. In some embodiments, the ectodomain of the CAR-engager is KDRPEIWEGEPP (SEQ ID NO: 106), which corresponds to amino acid residues 142-153 of SEQ ID NO: 5. [000108] In some embodiments, wherein the CAR targets CD20, the ectodomain of the CAR- engager contains at least a portion of the extracellular domain of CD20. The amino acid sequence of a representative CAR-engager that contains a CD20 extracellular domain is KISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQS (SEQ ID NO: 6). [000109] In some embodiments, wherein the CAR targets CD22, the ectodomain of the CAR- engager contains at least a portion of the extracellular domain of CD22. The amino acid sequence of a representative CAR-engager that contains a CD22 extracellular domain is set forth below (SEQ ID NO: 7): 1 dsskwvfehp etlyawegac vwipctyral dgdlesfilf hnpeynknts kfdgtrlyes 61 tkdgkvpseq krvqflgdkn knctlsihpv hlndsgqlgl rmesktekwm erihlnvser 121 pfpphiqlpp eiqesqevtl tcllnfscyg ypiqlqwlle gvpmrqaavt stsltiksvf 181 trselkfspq wshhgkivtc qlqdadgkfl sndtvqlnvk htpkleikvt psdaivregd 241 svtmtcevss snpeyttvsw lkdgtslkkq ntftlnlrev tkdqsgkycc qvsndvgpgr 301 seevflqvqy apepstvqil hspavegsqv eflcmslanp lptnytwyhn gkemqgrtee 361 kvhipkilpw hagtyscvae nilgtgqrgp gaeldvqypp kkvttviqnp mpiregdtvt 421 lscnynssnp svtryewkph gaweepslgv lkiqnvgwdn ttiacaacns wcswaspval 481 nvqyaprdvr vrkikplsei hsgnsvslqc dfssshpkev qffwekngrl lgkesqlnfd 541 sispedagsy scwvnnsigq taskawtlev lyaprrlrvs mspgdqvmeg ksatltcesd 601 anppvshytw fdwnnqslpy hsqklrlepv kvqhsgaywc qgtnsvgkgr splstltvyy 661 spetigrr 29 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000110] In some embodiments, the ectodomain of the CAR-engager contains a portion of the extracellular domain of CD22 (SEQ ID NO: 7). In some embodiments, the ectodomain of the CAR- engager contains the Ig domains 2 and 3 (2-3) of CD22. The amino acid sequence of a representative CAR-engager that contains Ig domains 2-3 of CD22 is set forth below (SEQ ID NO: 103): 1 phiqlppeiq esqevtltcl lnfscygypi qlqwllegvp mrqaavtsts ltiksvftrs 61 elkfspqwsh hgkivtcqlq dadgkflsnd tvqpkleikv tpsdaivreg dsvtmtcevs 121 ssnpeyttvs wlkdgtslkk qntftlnlre vtkdqsgkyc cqvsndvgpg rseevflq [000111] In some embodiments, the ectodomain of the CAR-engager contains the Ig domain 3 of CD22. The amino acid sequence of a representative CAR-engager that contains Ig domain 3 of CD22 is set forth below (SEQ ID NO: 104): 1 pkleikvtps daivregdsv tmtcevsssn peyttvswlk dgtslkkqnt ftlnlrevtk 61 dqsgkyccqv sndvgpgrse evflq [000112] In some embodiments, the ectodomain of the CAR-engager contains the Ig domains 5 through 7 (5-7) of CD22. The amino acid sequence of a representative CAR-engager that contains Ig domains 5-7 of CD22 is set forth below (SEQ ID NO: 105): 1 pkkvttviqn pmpiregdtv tlscnynssn psvtryewkp hgaweepslg vlkiqnvgwd 61 nttiacaacn swcswaspva lnprdvrvrk ikplseihsg nsvslqcdfs sshpkevqff 121 wekngrllgk esqlnfdsis pedagsyscw vnnsigqtas prrlrvsmsp gdqvmegksa 181 tltcesdanp pvshytwfdw nnqslpyhsq klrlepvkvq hsgaywcqgt nsvgkgrspl 241 stlt [000113] In some embodiments wherein the CAR targets Claudin 18.2, the ectodomain of the CAR- engager contains at least a portion of an extracellular domain of Claudin 18.2. The amino acid sequence of a representative CAR-engager that contains a Claudin 18.2 first extracellular domain is set forth below (SEQ ID NO: 8): 1 dqwstqdlyn npvtavfnyq glwrscvres sgftecrgyf tllglpamlq avr [000114] The amino acid sequence of a representative CAR-engager that contains a Claudin 18.2 second extracellular domain is set forth below (SEQ ID NO: 9): 1 vtnfwmstan mytgmggmvq tvqtrytfga a [000115] In some embodiments wherein the CAR targets SLAMF7, the ectodomain of the CAR- engager contains at least a portion of the extracellular domain of SLAMF7. The amino acid sequence of a representative CAR-engager that contains a SLAMF7 extracellular domain is set forth below (SEQ ID NO: 10): 1 sgpvkelvgs vggavtfplk skvkqvdsiv wtfnttplvt iqpeggtiiv tqnrnrervd 61 fpdggyslkl sklkkndsgi yyvgiysssl qqpstqeyvl hvyehlskpk vtmglqsnkn 121 gtcvtnltcc mehgeedviy twkalgqaan eshngsilpi swrwgesdmt ficvarnpvs 181 rnfsspilar klcegaaddp dssm [000116] In some embodiments wherein the CAR targets PD-1, the ectodomain of the CAR- engager contains at least a portion of the extracellular domain of PD-1. The amino acid sequence of 30 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 a representative CAR-engager that contains a PD-1 extracellular domain is set forth below (SEQ ID NO: 11): 1 fldspdrpwn pptfspallv vtegdnatft csfsntsesf vlnwyrmsps nqtdklaafp 61 edrsqpgqdc rfrvtqlpng rdfhmsvvra rrndsgtylc gaislapkaq ikeslraelr 121 vterraevpt ahpspsprpa gqfqtlv [000117] In some embodiments, the ectodomain of the CAR-engager contains a variant of the extracellular domain of PD-1. In some embodiments, the ectodomain of the CAR-engager contains the N-loop of PD-1. The amino acid sequence of a representative CAR-engager that contains the N- loop of the PD-1 extracellular domain is LDSPDRPWNP (SEQ ID NO: 107), which corresponds to amino acid residues 2-11 of SEQ ID NO: 11. [000118] In some embodiments, the ectodomain of the CAR-engager contains the CD-loop of PD- 1. The amino acid sequence of a representative CAR-engager that contains the CD-loop of the PD- 1 extracellular domain is NQTDKLAAFPEDRSQPGQDCRFRVTQ (SEQ ID NO: 108), which corresponds to amino acid residues 51-76 of SEQ ID NO: 11. [000119] In some embodiments wherein the CAR targets KIT, the ectodomain of the CAR-engager contains at least a portion of the extracellular domain of KIT. The amino acid sequence of a representative CAR-engager that contains a KIT extracellular domain is set forth below (SEQ ID NO: 12): 1 qpsvspgeps ppsihpgksd livrvgdeir llctdpgfvk wtfeildetn enkqnewite 61 kaeatntgky tctnkhglsn siyvfvrdpa klflvdrsly gkedndtlvr cpltdpevtn 121 yslkgcqgkp lpkdlrfipd pkagimiksv krayhrlclh csvdqegksv lsekfilkvr 181 pafkavpvvs vskasyllre geeftvtcti kdvsssvyst wkrensqtkl qekynswhhg 241 dfnyerqatl tissarvnds gvfmcyannt fgsanvtttl evvdkgfini fpminttvfv 301 ndgenvdliv eyeafpkpeh qqwiymnrtf tdkwedypks enesniryvs elhltrlkgt 361 eggtytflvs nsdvnaaiaf nvyvntkpei ltydrlvngm lqcvaagfpe ptidwyfcpg 421 teqrcsasvl pvdvqtlnss gppfgklvvq ssidssafkh ngtveckayn dvgktsayfn 481 fafkgnnkeq ihphtlftp [000120] In some embodiments wherein the CAR targets TROP2, the ectodomain of the CAR- engager contains at least a portion of the extracellular domain of TROP2. The amino acid sequence of a representative CAR-engager that contains a TROP2 extracellular domain is set forth below (SEQ ID NO: 13): 1 htaaqdnctc ptnkmtvcsp dgpggrcqcr algsgmavdc stltskclll karmsapkna 61 rtlvrpseha lvdndglydp dcdpegrfka rqcnqtsvcw cvnsvgvrrt dkgdlslrcd 121 elvrthhili dlrhrptaga fnhsdldael rrlfreryrl hpkfvaavhy eqptiqielr 181 qntsqkaagd vdigdaayyf erdikgeslf qgrggldlrv rgeplqvert liyyldeipp 241 kfsmkrlt [000121] In some embodiments wherein the CAR targets CD38, the ectodomain of the CAR- engager contains at least a portion of the extracellular domain of CD38. The amino acid sequence 31 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 of a representative CAR-engager that contains a CD38 extracellular domain is set forth below (SEQ ID NO: 14): 1 vprwrqqwsg pgttkrfpet vlarcvkyte ihpemrhvdc qsvwdafkga fiskhpcnit 61 eedyqplmkl gtqtvpcnki llwsrikdla hqftqvqrdm ftledtllgy laddltwcge 121 fntskinyqs cpdwrkdcsn npvsvfwktv srrfaeaacd vvhvmlngsr skifdknstf 181 gsvevhnlqp ekvqtleawv ihggredsrd lcqdptikel esiiskrniq fsckniyrpd 241 kflqcvknpe dssctsei [000122] In some embodiments wherein the CAR targets mesothelin (MSLN), the ectodomain of the CAR-engager contains at least a portion of MSLN, which is a GPI-anchored protein and therefore the entire MSLN protein is extracellular. The amino acid sequence of a representative CAR-engager that contains MSLN is set forth below (SEQ ID NO: 15): 1 malptarpll gscgtpalgs llfllfslgw vqpsrtlage tgqeaapldg vlanppniss 61 lsprqllgfp caevsglste rvrelavala qknvklsteq lrclahrlse ppedldalpl 121 dlllflnpda fsgpqactrf fsritkanvd llprgaperq rllpaalacw gvrgsllsea 181 dvralgglac dlpgrfvaes aevllprlvs cpgpldqdqq eaaraalqgg gppygppstw 241 svstmdalrg llpvlgqpii rsipqgivaa wrqrssrdps wrqpertilr prfrrevekt 301 acpsgkkare ideslifykk weleacvdaa llatqmdrvn aipftyeqld vlkhkldely 361 pqgypesviq hlgylflkms pedirkwnvt sletlkalle vnkghemspq aprrplpqva 421 tlidrfvkgr gqldkdtldt ltafypgylc slspeelssv ppssiwavrp qdldtcdprq 481 ldvlypkarl afqnmngsey fvkiqsflgg aptedlkals qqnvsmdlat fmklrtdavl 541 pltvaevqkl lgphveglka eerhrpvrdw ilrqrqddld tlglglqggi pngylvldls 601 mqealsgtpc llgpgpvltv lalllastla [000123] In some embodiments, the ectodomain of the CAR-engager contains a portion of an extracellular domain of a cancer antigen. In some embodiments, the ectodomain of the CAR-engager contains a portion of the MSLN protein. In some embodiments, the ectodomain of the CAR-engager is IPXGYLVLDLSMQEALS (SEQ ID NO: 17), where X is any amino acid. In some embodiments, the ectodomain of the CAR-engager is YXVXDLSMQEL (SEQ ID NO: 18), where X is any amino acid. CAR-E first moiety: CAR-binding antibodies and derivatives thereof [000124] In some embodiments, the first moiety of the CAR-engager may be an antibody that binds an epitope on the ED of the CAR, or an ED-binding derivative thereof. Antibody derivatives include antibody fragments (e.g., a scFv and nanobody fragments). [000125] In some embodiments wherein the CAR targets CD19, the first moiety of the CAR- engager is an anti-anti-CD19 antibody binding moiety that binds an epitope on a CAR the EBD of which binds CD19. A representative anti-anti-CD19 binding moiety is set forth below (SEQ ID NO: 124): 32 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 1 qvqlqqpgae lvrpgasvkl scktsgysft rywmnwvkqr pgqglewigm ihpsdsetrl 61 nqkfkdkatl tvdnssstay mqlssptsed savyycasiy yeeawgqgtl vtvsaggggs 121 ggggsggggs diqmtqspas lsasvgetvt itcrasgnih nylawyqqkq gkspqllvyn aktladsvps rfsgsgsgtq yslkinslqp edfgsyycqh fwstpytfgg gtkleik [000126] In some embodiments, the first moiety of the CAR-engager binds an epitope on a portion of the ED that does not directly engage the cancer antigen, such as a linker (e.g., a linker between VH and VL regions of the ED). In some embodiments, for example, wherein the EBD of the CAR includes a linker containing a G4S motif, the first moiety of the CAR-engager may be an anti-(G4S) binding moiety that binds an epitope on a CAR the linker of which has at least two repeats of GGGGS (SEQ ID NO: 71). A representative heavy chain variable region (VH) of an anti-(G4S) binding moiety is set forth below (SEQ ID NO: 125): 1 qsvkesggrl vtpgtpltlt ctvsgfslss naidwvrqap gkglewigil grsgstyyas 61 wakgrftisr tssttvdlki tspttedtat yfcarhfylw gpgtlvtvss [000127] A representative light chain variable region (VL) of an anti-(G4S) binding moiety is set forth below (SEQ ID NO: 126): 1 aqvltqtasp vsaavggtvt incqasqsvy snylswyqqk pgqppkllma ttstlepgvp 61 srfkgsgsgt qftltisdle cddaatyyca ggysvdiwvf gggtevvvk [000128] In some embodiments, the first moiety of the CAR-engager is an anti-κ light chain antibody binding moiety that binds an epitope on a CAR the EBD of which is a κ light chain. A representative anti-κ light chain binding moiety is set forth below (SEQ ID NO: 110). 1 mkinkkllma alagaivvgg ganayaaeed ntdnnlsmde isdayfdyhg dvsdsvdpve 61 eeidealaka laeaketakk hidslnhlse takklakndi dsattinain divaradvme 121 rktaekeeae klaaaketak khidelkhla dktkelakrd idsattinai ndivaradvm 181 erktaekeea eklaaaketa kkhidelkhl adktkelakr didsattida indivaradv 241 merklseket pepeeevtik anlifadgst qnaefkgtfa kavsdayaya dalkkdngey 301 tvdvadkglt lnikfagkke kpeepkeevt ikvnlifadg ktqtaefkgt feeatakaya 361 yadllakeng eytadledgg ntinikfagk etpetpeepk eevtikvnli fadgkiqtae 421 fkgtfeeata kayayanlla kengeytadl edggntinik fagketpetp eepkeevtik 481 vnlifadgkt qtaefkgtfe eataeayrya dllakvngey tadledggyt inikfagkeq 541 pgenpgitid ewllknakee aikelkeagi tsdlyfslin kaktvegvea lkneilkaha 601 geetpelkdg yatyeeaeaa akealknddv nnayeivqga dgryyyvlki evadeeepge 661 dtpevqegya tyeeaeaaak ealkedkvnn ayevvqgadg ryyyvlkied kedeqpgeep 721 genpgitide wllknakeda ikelkeagis sdiyfdaink aktvegveal kneilkahae kpgenpgiti dewllknake aaikelkeag itaeylfnli nkaktvegve slkneilkah aekpgenpgi tidewllkna kedaikelke agitsdiyfd ainkaktieg vealkneilk 901 ahkkdeepgk kpgedkkped kkpgedkkpe dkkpgedkkp edkkpgktdk dspnkkkkak 961 lpkagseaei ltlaaaalst aagayvslkk rk [000129] In some embodiments, the first moiety of the CAR-engager is an anti-anti-mouse antibody binding moiety that binds an epitope on a CAR the EBD of which is derived from antibodies originating from a mouse. Representative anti-anti-mouse binding moieties are known in the art, 33 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 see, Kochenderfer et al., J. Immunother. 32(7):689-702 (2009) and Cheng et al., Cytometry A. 103(1):16-26 (2023). [000130] Additional antibodies and derivatives thereof that bind CAR EDs that may be useful are known in the art, see, e.g., U.S. Patent 9,701,758 and U.S. Patent Application Publication 2005/0287148, both of which are incorporated herein by reference in their entireties. CAR-E – Second Moiety: Immune cell effector domain [000131] The second moiety is an immune cell effector domain that comprises an IL-2 variant as described herein. The IL-2 variant binds the one or more of the cognate receptor subunits of IL-2Rα, IL-2Rβ, and IL-2Rγ on the immune cell (containing the CAR). This binding event modulates the activity of the CAR-immune cell. The terms “modulate(s),” and “modulation” as used herein embrace both activation and inhibition of the CAR immune cell. [000132] In some embodiments, the CAR-engager contains a plurality (i.e., two, three, or more) of immune cell effector domains, wherein at least one of which is an IL-2 variant described herein, and wherein any two or more of immune effector domains may be the same as or different from each other. In some embodiments, the CAR-engager contains two immune effector domains containing two IL-2 variants as disclosed herein. In some embodiments, the CAR-engager contains three immune effector domains containing three IL-2 variants as disclosed herein. [000133] In some embodiments, the second immune cell effector domain is a cytokine, an immune cell-activating moiety, or an immune cell-inhibiting moiety, and variants and fragments thereof, that bind to their cognate targets on the immune cells. The term “cytokine”, as is known in the art, includes low molecular weight extracellular polypeptides/glycoproteins that promote, modulate, and regulate the immune response (i.e., increase or decrease activity, differentiation, or proliferation). Representative examples of cytokines include chemokines, interferons (IFNs), interleukins (ILs), lymphokines and tumor necrosis factors (TNFs). The term “immune cell activating variant” of a cytokine as used herein refers to non-naturally occurring variant of a cytokine capable of binding to a cytokine receptor on an immune cell and initiating signal transduction through that receptor to achieve substantially the same effect as the naturally occurring cytokine. [000134] In some embodiments, the second immune cell effector domain is an immune cell activating moiety, e.g., immune cell activating cytokines and immune cell-activating variants and 34 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 fragments thereof. Immune cell activating moieties activate, promote, or maintain the activity of immune cells. [000135] In some embodiments, the second immune cell effector domain is derived from CD40, CD48, CD58, CD70, CD80, CD86, CD112, glucocorticoid-induced TNFR-related protein ligand (GITRL; TNFSF18), herpesvirus entry mediator (HVEM; TNFSF14), Semaphorin 3B (SEMAA; SEMA3B), Signaling lymphocytic activation molecule family member 1 (SLAM; SLAMF1; CD150), T cell immunoglobulin and mucin domain containing 4 (TIM4), TNF superfamily member 4 (TNFSF4; OX40L), TNF superfamily member 8 (TNFSF8; CD30L), interleukin-2 (IL-2), IL-7, IL-9, IL-10, IL-12, IL-15, IL-18, IL-21, IL-27, CCL21, 4-1BBL (also known as TNF superfamily member 9; TNFSF9), or an immune cell-activating variant thereof. [000136] The amino acid sequences of representative immune cell activating moieties (e.g., cytokines) are from which the second immune cell effector domain may be derived are provided at the NCBI Accession numbers set forth in Table 2, and are incorporated herein by reference. Table 2: Gene Name, Symbols, and NCBI Accession Numbers of Representative Immune Cell- Activating Proteins Gene Name Gene Symbols Protein Accession No(s). C-C motif chemokine 6Ckine CCL21 CKb9 ECL NP 002980 , , , , , , ,
Figure imgf000037_0001
35 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 Nectin cell adhesion CD112, HVEB, HveB, XP_047295125, NP_001036189, molecule 2 NECTIN2, Nectin-2, PRR2, NP_002847 , , , , , , , , , , , , , , ,
Figure imgf000038_0001
36 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 TNF superfamily 4-1BB-L, 4-1BBL, CD137L, NP_003802 member 9 TNFSF9, TNLG5A [ from
Figure imgf000039_0001
wild-type IL-2 but different from the IL-2 variants disclosed herein. The amino acid sequence of a representative IL-2 is provided at NCBI Accession No. NP_000577, incorporated herein by reference. In some embodiments, the immune cell effector domain contains the weak affinity variant of IL-2 (muIL2), having the amino acid sequence set forth below (SEQ ID NO: 19): 1 aptssstkkt qlqleallld lqmilnginn yknpkltrml takfympkka telkhlqcle 61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [000138] In some embodiments, the second immune cell effector domain is an IL-2 variant that has an H16A substitution (i.e., an alanine (A) at amino acid residue 16 in place of the histidine (H)) and/or an F42A substitution (i.e., an alanine (A) at amino acid residue 42 in place of the phenylalanine (F)), both substituted alanine residues shown as boxed amino acids in SEQ ID NO: 19). [000139] In some embodiments, the second immune cell effector domain contains at least a portion of the weak affinity IL-2 variant, having together, the amino acid sequence set forth below (SEQ ID NO: 20): 1 aptssstkkt qlqleallld lqmilnginn yknpkltrml takfympkka telkhlqcle 61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tltggggsgg ggsggggsgg ggsaptssst kktqlqleal lldlqmilng 181 innyknpklt rmltakfymp kkatelkhlq cleeelkple evlnlaqskn fhlrprdlis 241 ninvivlelk gsettfmcey adetativef lnrwitfcqs iistlt [000140] The natural (wild-type) sequence of human IL-2 (NCBI Accession No. NP_000577) has higher affinity for the IL-2 receptor (IL-2R) than SEQ ID NO: 19. In some embodiments, the second immune cell effector domain contains at least a portion of the wild-type IL-2, having the amino acid sequence set forth below (SEQ ID NO: 102): 1 aptssstkkt qlqlehllld lqmilnginn yknpkltrml tfkfympkka telkhlqcle 61 eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr 121 witfcqsiis tlt [000141] The muIL2 (SEQ ID NO: 19) second immune cell effector domain has a dissociation constant (KD) of about 1200 nM for IL-2Rα (CD25), representing a 110-fold decrease as compared to wild type IL-2, and a KD of about 610 nM for IL-2Rβ, representing a 3-fold decrease as compared to wild type IL-2. 37 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000142] In some embodiments, the second immune cell effector contains at least a portion of IL- 7. The amino acid sequence of a representative IL-7 is set forth below (SEQ ID NO: 21): 1 mfhvsfryif glpplilvll pvassdcdie gkdgkqyesv lmvsidqlld smkeigsncl 61 nnefnffkrh icdankegmf lfraarklrq flkmnstgdf dlhllkvseg ttillnctgq 121 vkgrkpaalg eaqptkslee nkslkeqkkl ndlcflkrll qeiktcwnki lmgtkeh [000143] In some embodiments, the second immune cell effector domain contains at least a portion of IL-15. The amino acid sequence of a representative IL-15 is set forth below (SEQ ID NO: 22): 1 mriskphlrs isiqcylcll lnshflteag ihvfilgcfs aglpkteanw vnvisdlkki 61 edliqsmhid atlytesdvh psckvtamkc fllelqvisl esgdasihdt venliilann 121 slssngnvte sgckeceele eknikeflqs fvhivqmfin ts [000144] In some embodiments, the second immune cell effector domain contains at least a portion of IL-18. The amino acid sequence of a representative IL-18 is set forth below (SEQ ID NO: 23): 1 maaepvednc infvamkfid ntlyfiaedd enlesdyfgk lesklsvirn lndqvlfidq 61 gnrplfedmt dsdcrdnapr tifiismykd sqprgmavti svkcekistl scenkiisfk 121 emnppdnikd tksdiiffqr svpghdnkmq fesssyegyf lacekerdlf klilkkedel 181 gdrsimftvq ned [000145] In some embodiments, the second immune cell effector domain contains at least a portion of IL-21. The amino acid sequence of a representative IL-21 is set forth below (SEQ ID NO: 24): 1 mrsspgnmer iviclmvifl gtlvhksssq gqdrhmirmr qlidivdqlk nyvndlvpef 61 lpapedvetn cewsafscfq kaqlksantg nneriinvsi kklkrkppst nagrrqkhrl 121 tcpscdsyek kppkeflerf ksllqkmihq hlssrthgse ds [000146] In some embodiments, the second immune cell effector domain contains at least a portion of IL-27. The amino acid sequence of a representative IL-27 is set forth below (SEQ ID NO: 25): 1 mgqtagdlgw rlsllllpll lvqagvwgfp rppgrpqlsl qelrreftvs lhlarkllse 61 vrgqahrfae shlpgvnlyl lplgeqlpdv sltfqawrrl sdperlcfis ttlqpfhall 121 gglgtqgrwt nmermqlwam rldlrdlqrh lrfqvlaagf nlpeeeeeee eeeeeerkgl 181 lpgalgsalq gpaqvswpql lstyrllhsl elvlsravre llllskaghs vwplgfptls 241 pqp [000147] In some embodiments, the second immune cell effector domain contains at least a portion of neoleukin-2/15 (Neo-2/15), which binds to the IL-2R-β, having the amino acid sequence set forth below (SEQ ID NO: 26). 1 gshmpkkkiq lhaehalyda lmilnivktn sppaeekled yafnfelile eiarlfesgd 61 qkdeaekakr mkewmkrikt tasedeqeem anaiitilqs wifs [000148] In some embodiments, the second immune cell effector domain contains two repeats of Neo-2/15, having the amino acid sequence of SEQ ID NO: 26. [000149] In some embodiments, the second immune cell effector domain contains at least a portion of 4-1BBL. 4-1BBL is also known as TNF ligand superfamily member 9 (TNFSF9). The amino acid sequence of a representative 4-1BBL is provided at NCBI Accession No. NP_003802, incorporated herein by reference. In some embodiments, the second immune cell effector domain 38 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 contains at least a portion of the extracellular domain of 4-1BBL. In some embodiments, the second immune cell effector domain contains a portion of the extracellular domain of 4-1BBL, having the amino acid sequence set forth below (SEQ ID NO: 27): 1 dpaglldlrq gmfaqlvaqn vllidgplsw ysdpglagvs ltgglsyked tkelvvakag 61 vyyvffqlel rrvvagegsg svslalhlqp lrsaagaaal altvdlppas searnsafgf 121 qgrllhlsag qrlgvhlhte ararhawqlt qgatvlglfr vtpeipa [000150] In some embodiments, the CAR-engager contains three immune cell effector domains, e.g., wherein the first immune cell effector domain is an IL-2 variant and the second and the third immune cell effector domains are the extracellular domain of 4-1BBL, each having the amino acid sequence of SEQ ID NO: 27. [000151] In some embodiments, the second immune cell effector domain may be a fragment, e.g., a single-chain variable antibody fragment (scFv), that binds and activates the CAR immune cell. In some embodiments, the second immune cell effector domain is an scFv that binds an epitope on 4- 1BB, CD2, CD27, CD28, CD30 (TNFRSF8), CD40L, CD226, CTLA4, GITR, IL-2R, LIGHT, OX40, PD-1, TIM2, SLAM, or TIM1. [000152] In some embodiments, the second immune cell effector domain is derived from a commercially available anti-CTLA4 antibody, antibody fragment, or derivative thereof, e.g., bavunalimab (formerly pavunalimab/XmAb 22841), botensilimab, cadonilimab, ipilimumab (Yervoy®), quavonlimab, tremelimumab (Imjudo®), volrustomig, vudalimab, or zalifrelimab. In some embodiments, the second immune cell effector domain is a scFv that binds CTLA4. The amino acid sequences of representative heavy and light chains of antibodies that bind CTLA4 are set forth in Table 3. Table 3: Amino acid Sequences of Representative anti-CTLA Antibody Heavy and Light Chains botensilimab heavy chain (SEQ ID NO: 28) 1 evqlvesggg lvkpggslrl scaasgftfs sysmnwvrqa pgkglewvss isssssyiyy s l d t t q s p t
Figure imgf000041_0001
Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 1 qvqlvesggg vvqpgrslrl scaasgftfs sytmhwvrqa pgkglewvtf isydgnnkyy 61 adsvkgrfti srdnskntly lqmnslraed taiyycartg wlgpfdywgq gtlvtvssas 121 tkgpsvfpla psskstsggt aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl s t t q p p l t g l l v q v s p t y t a a f e r s p t [
Figure imgf000042_0001
000153] In some embodiments, the second immune cell effector domain contains the VL having the amino acid sequence set forth below (SEQ ID NO 36): 1 eivltqspgt lslspgerat lscraqsvsr ylgwyqqkpg qaprlliyga stratgipdr 61 fsgsgsgtdf tltitrlepe dfavyycqqy gsspwtfgqg tkveik [000154] In some embodiments, the second immune cell effector domain contains the VH having the amino acid sequence set forth below (SEQ ID NO 37): 40 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 1 evqlvesggg lvkpggslrl scaasgftfs sysmnwvrqa pgkglewvss isssssyiyy 61 aesvkgrfti srdnaknsly lqmnslraed tavyycarvg lfgpfdiwgq gtlvtvss [000155] In some embodiments, the second immune cell effector domain binds OX40. In some embodiments, the second immune cell effector domain is derived from a commercially available anti-OX40 antibody, antibody fragment (e.g., scFv), or derivative thereof, e.g., tavolimab, or vonlerolizumab (Pogalizumab; MOXR 0916). The amino acid sequences of representative heavy and light chains of which are set forth in Table 4. Table 4: Amino acid Sequences of Representative anti-OX40 Antibody Heavy and Light Chains tavolimab heavy chain (SEQ ID NO: 38) 1 qvqlqesgpg lvkpsqtlsl tcavyggsfs sgywnwirkh pgkgleyigy isyngityhn s s g y e r s p t y t y v y k g s p t [
Figure imgf000043_0001
000 56] n some embod ments, t e mmune ce e ector doma n conta ns t e V av ng t e amino acid sequence set forth below (SEQ ID NO 42): 1 diqmtqspss lsasvgdrvt itcrasqdis nylnwyqqkp gkapklliyy tsrlrsgvps 61 rfsgsgsgtd ftltisslqp edfatyycqq ghtlpptfgq gtkveik [000157] In some embodiments, the immune cell effector domain contains the VH having the amino acid sequence set forth below (SEQ ID NO 43): 1 evqlvqsgae vkkpgasvkv sckasgytft dsymswvrqa pgqglewigd mypdngdssy 41 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 61 nqkfrervti trdtststay lelsslrsed tavyycvlap rwyfsvwgqg tlvtvss [000158] In some embodiments, the second immune cell effector domain binds PD-1. In some embodiments, the second immune cell effector domain is derived from a commercially available anti-PD-1 antibody, antibody fragment (e.g., scFv), or derivative thereof, e.g., atezolizumab, avelumab, bintrafusp alfa, cosibelimab, danburstotug, durvalumab (Imfinzi®), inbakicept, lodapolimab, pimivalimab, or socazolimab. The amino acid sequences of representative heavy and light chains of which are set forth in Table 5. Table 5: Amino Acid Sequences of Representative anti-PD-1 Antibody Heavy and Light Chains atezolizumab heavy chain (SEQ ID NO: 44) 1 evqllesggg lvqpggslrl scaasgftfs syimmwvrqa pgkglewvss iypsggitfy v t s
Figure imgf000044_0001
42 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 121 psdeqlksgt asvvcllnnf ypreakvqwk vdnalqsgns qesvteqdsk dstyslsstl 181 tlskadyekh kvyacevthq glsspvtksf nrgec pimivalimab heavy chain (SEQ ID NO: 50) [
Figure imgf000045_0002
ving the amino acid sequence set forth below (SEQ ID NO 52): 1 eivmtqspat lsvspgerat lscrasqsvs snlawyqqkp gqaprlliyg astratgipa 61 rfsgsgsgte ftltisslqs edfavyycqq ynnwprtfgq gtkveik [000160] In some embodiments, the second immune cell effector domain contains the VH having the amino acid sequence set forth below (SEQ ID NO 53): 1 qvqlvesggg vvqpgrslrl scaasgftfs sygmhwvrqa pgkglewvav iwydgsnkyy 61 adsvmgrfti srdnskntly lqmnslraed tavyycasng dhwgqgtlvt vss [000161] In some embodiments, the second immune cell effector domain is an immune cell- inhibiting moiety, representative types of which include immune cell inhibiting cytokines and immune cell-inhibiting variants and fragments thereof. Immune cell inhibiting moieties repress or block immune cell activity and function. In some embodiments, the immune cell-inhibiting moiety may be derived from CD80, CD86, CD112, CD155, CD276 (B7-H3), Ceacam-1, FGL1, galectin- 3, HLA-E, HVEM, PD-L1, PD-L2, VISTA, or VTCN1 (B7-H4). The amino acid sequences of representative immune cell-inhibiting proteins from which the second immune cell effector domain may be derived are provided at the NCBI Accession numbers set forth in Table 6, and are incorporated herein by reference. Table 6: Gene Name, Symbols, and NCBI Accession Numbers of Immune Cell-Inhibiting Proteins Gene Name Gene Symbols Protein Accession No(s).
Figure imgf000045_0001
43 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 CD86 molecule B7-2, B7.2, B70, BU63, NP_001193853, NP_001193854, CD28LG2, CD86, FUN-1, NP_008820, NP_787058, , , , , , , , , , , , , , , , , , , , , , , , , , , ,
Figure imgf000046_0001
44 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 Programmed cell death 1 B7-H, B7-H1, B7H1, CD274, XP_047279218, NP_001254635, ligand 1 HPD-L1, PD-L1, PDCD1L1, NP_001300958, NP_054862 , , , , , , , [
Figure imgf000047_0001
rtion of the extracellular domain of CD80. The amino acid sequence of a representative CD80 extracellular domain is set forth below (SEQ ID NO: 54): 1 vihvtkevke vatlscghnv sveelaqtri ywqkekkmvl tmmsgdmniw peyknrtifd 61 itnnlsivil alrpsdegty ecvvlkyekd afkrehlaev tlsvkadfpt psisdfeipt 121 snirriicst sggfpephls wlengeelna inttvsqdpe telyavsskl dfnmttnhsf 181 mclikyghlr vnqtfnwntt kqehfpdn [000163] In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of CD86. The amino acid sequence of a representative CD86 extracellular domain is set forth below (SEQ ID NO: 55): 1 aplkiqayfn etadlpcqfa nsqnqslsel vvfwqdqenl vlnevylgke kfdsvhskym 61 grtsfdsdsw tlrlhnlqik dkglyqciih hkkptgmiri hqmnselsvl anfsqpeivp 121 isnitenvyi nltcssihgy pepkkmsvll rtknstieyd gvmqksqdnv telydvsisl 181 svsfpdvtsn mtifciletd ktrllsspfs ieledpqppp dhip [000164] In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of CD155 (nectin-5; PVR). The amino acid sequence of a representative CD155 extracellular domain is set forth below (SEQ ID NO: 56): 1 wpppgtgdvv vqaptqvpgf lgdsvtlpcy lqvpnmevth vsqltwarhg esgsmavfhq 61 tqgpsysesk rlefvaarlg aelrnaslrm fglrvedegn ytclfvtfpq gsrsvdiwlr 121 vlakpqntae vqkvqltgep vpmarcvstg grppaqitwh sdlggmpnts qvpgflsgtv 181 tvtslwilvp ssqvdgknvt ckvehesfek pqlltvnltv yyppevsisg ydnnwylgqn 241 eatltcdars npeptgynws ttmgplppfa vaqgaqllir pvdkpinttl icnvtnalga 301 rqaeltvqvk egppsehsgi srn 45 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000165] In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of CD276 (B7-H3). The amino acid sequence of a representative CD276 extracellular domain is set forth below (SEQ ID NO: 57): 1 levqvpedpv valvgtdatl ccsfspepgf slaqlnliwq ltdtkqlvhs faegqdqgsa 61 yanrtalfpd llaqgnaslr lqrvrvadeg sftcfvsird fgsaavslqv aapyskpsmt 121 lepnkdlrpg dtvtitcssy qgypeaevfw qdgqgvpltg nvttsqmane qglfdvhsil 181 rvvlgangty sclvrnpvlq qdahssvtit pqrsptgave vqvpedpvva lvgtdatlrc 241 sfspepgfsl aqlnliwqlt dtkqlvhsft egrdqgsaya nrtalfpdll aqgnaslrlq 301 rvrvadegsf tcfvsirdfg saavslqvaa pyskpsmtle pnkdlrpgdt vtitcssyrg 361 ypeaevfwqd gqgvpltgnv ttsqmaneqg lfdvhsvlrv vlgangtysc lvrnpvlqqd 421 ahgsvtitgq pmtfppea [000166] In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of Ceacam-1. The amino acid sequence of a representative Ceacma-1 extracellular domain is set forth below (SEQ ID NO: 58): 1 kltiesmpls vaegkevlll vhnlpqhlfg yswykgervd gnslivgyvi gtqqatpgaa 61 ysgretiytn aslliqnvtq ndigfytlqv iksdlvneea tgqfhvyqen apglpvgava 121 g [000167] In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of FGL1. The amino acid sequence of a representative FGL1 extracellular domain is set forth below (SEQ ID NO: 59): 1 makvfsfilv ttaltmgrei saledcaqeq mrlraqvrll etrvkqqqvk ikqllqenev 61 qfldkgdent vidlgskrqy adcseifndg yklsgfykik plqspaefsv ycdmsdgggw 121 tviqrrsdgs enfnrgwkdy engfgnfvqk hgeywlgnkn lhflttqedy tlkidladfe 181 knsryaqykn fkvgdeknfy elnigeysgt agdslagnfh pevqwwashq rmkfstwdrd 241 hdnyegncae edqsgwwfnr chsanlngvy ysgpytaktd ngivwytwhg wwyslksvvm 301 kirpndfipn vi [000168] In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of galectin-3. The amino acid sequence of a representative galecin-3 extracellular domain is set forth below (SEQ ID NO: 60): 1 madnfslhda lsgsgnpnpq gwpgawgnqp agaggypgas ypgaypgqap pgaypgqapp 61 gaypgapgay pgapapgvyp gppsgpgayp ssgqpsatga ypatgpygap agplivpynl 121 plpggvvprm litilgtvkp nanrialdfq rgndvafhfn prfnennrrv ivcntkldnn 181 wgreerqsvf pfesgkpfki qvlvepdhfk vavndahllq ynhrvkklne isklgisgdi 241 dltsasytmi [000169] In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of HLA-E. The amino acid sequence of a representative HLA-E extracellular domain is set forth below (SEQ ID NO: 61): 1 gshslkyfht svsrpgrgep rfisvgyvdd tqfvrfdnda asprmvprap wmeqegseyw 61 dretrsardt aqifrvnlrt lrgyynqsea gshtlqwmhg celgpdgrfl rgyeqfaydg 121 kdyltlnedl rswtavdtaa qiseqksnda seaehqrayl edtcvewlhk ylekgketll 181 hleppkthvt hhpisdheat lrcwalgfyp aeitltwqqd
Figure imgf000048_0001
241 fqkwaavvvp sgeeqrytch vqheglpepv tlrwkpasqp tipi
Figure imgf000048_0002
46 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000170] In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of HVEM (CD270). The amino acid sequence set of a representative HVEM extracellular domain is forth below (SEQ ID NO: 62): 1 lpsckedeyp vgseccpkcs pgyrvkeacg eltgtvcepc ppgtyiahln glskclqcqm 61 cdpamglras rncsrtenav cgcspghfci vqdgdhcaac rayatsspgq rvqkggtesq 121 dtlcqncppg tfspngtlee cqhqtkcswl vtkagagtss shwv [000171] In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of nectin-2 (CD112, HVEB). The amino acid sequence of a representative nectin-2 extracellular domain is set forth below (SEQ ID NO: 63): 1 qdvrvqvlpe vrgqlggtve lpchllppvp glyislvtwq rpdapanhqn vaafhpkmgp 61 sfpspkpgse rlsfvsakqs tgqdteaelq datlalhglt vedegnytce fatfpkgsvr 121 gmtwlrviak pknqaeaqkv tfsqdpttva lciskegrpp ariswlssld weaketqvsg 181 tlagtvtvts rftlvpsgra dgvtvtckve hesfeepali pvtlsvrypp evsisgyddn 241 wylgrtdatl scdvrsnpep tgydwsttsg tfptsavaqg sqlvihavds lfnttfvctv 301 tnavgmgrae qvifvretpn tagagatgg [000172] In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of PD-L1. The amino acid sequence of a representative PD- L1extracellular domain is set forth below (SEQ ID NO: 64): 1 ftvtvpkdly vveygsnmti eckfpvekql dlaalivywe medkniiqfv hgeedlkvqh 61 ssyrqrarll kdqlslgnaa lqitdvklqd agvyrcmisy ggadykritv kvnapynkin 121 qrilvvdpvt seheltcqae gypkaeviwt ssdhqvlsgk ttttnskree klfnvtstlr 181 intttneify ctfrrldpee nhtaelvipe lplahppner [000173] In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of PD-L2. The amino acid sequence set of a representative PD-L2 extracellular domain is forth below (SEQ ID NO: 65): 1 lftvtvpkel yiiehgsnvt lecnfdtgsh vnlgaitasl qkvendtsph reratlleeq 61 lplgkasfhi pqvqvrdegq yqciiiygva wdykyltlkv kasyrkinth ilkvpetdev 121 eltcqatgyp laevswpnvs vpantshsrt peglyqvtsv lrlkpppgrn fscvfwnthv 181 reltlasidl qsqmeprthp t [000174] In some embodiments, the second immune cell effector domain contains at least a portion of the extracellular domain of VTCN1 (B7-H4). The amino acid sequence of a representative VTCN1 is set forth below (SEQ ID NO: 66): 1 liigfgisgr hsitvttvas agnigedgil sctfepdikl sdiviqwlke gvlglvhefk 61 egkdelseqd emfrgrtavf adqvivgnas lrlknvqltd agtykcyiit skgkgnanle 121 yktgafsmpe vnvdynasse tlrceaprwf pqptvvwasq vdqganfsev sntsfelnse 181 nvtmkvvsvl ynvtinntys cmiendiaka tgdikvtese ikrrshlqll nskas Dimerization domain 47 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000175] In some embodiments, the CAR-engager further includes a dimerization domain. In these cases, the CAR-engager forms and is administered in the form of a homodimer or a homo-multimer. The homodimer thus contains two CAR-engager entities. The order of the first moiety, the second moiety and the dimerization domain are not critical. In some embodiments, the dimerization domain is disposed between the first moiety and the second moiety. [000176] In some embodiments, the CAR-engager is in the form of a heterodimer, which contains a first moiety connected to a first dimerization domain and a second moiety connected to a second dimerization domain. In these embodiments, the first and second dimerization domains dimerize the first and second moieties to form a heterodimer. [000177] In some embodiments, the first and second dimerization domains contain a knob-in-hole configuration. One of the dimerization domains contains a protuberance (knob) and the other dimerization domain contains a cavity (hole) that is sterically compensatory to the protuberance, where the tertiary structure of the protuberance is positionable within the tertiary structure of the cavity. Dimerization domains with knob-in-hole configurations may have directed amino acid mutations where the protuberance is an amino acid that has a larger side chain volume than present on a dimerization domain derived from a natural source (e.g., IgA, IgD, IgG, IgM, or IgE) and the cavity is an amino acid that has a smaller side chain volume than present on a dimerization domain derived from a natural source. [000178] In some embodiments, the protuberance is an amino acid change from a threonine (T) to a lysine (K) and the corresponding cavity is an amino acid change from a leucine (L) to an aspartic acid (D) or a lysine (K). In some embodiments, the first dimerization domain contains two amino acid substitutions, for example, a threonine (T) to a lysine (K) and a leucine (L) to a lysine (K), while the second dimerization domain contains a leucine (L) to an aspartic acid (D) or a glutamic acid (E) and a tyrosine (Y) to a glutamic acid (E) or aspartic acid (D). [000179] In some embodiments, the knob-in-hole dimerization domains are based on opposed charges. In some embodiments, the first dimerization domain contains a positively charged amino acid and the second dimerization domain contains a negatively charged amino acid sterically opposable to the positively charged amino acid on the first dimerization domain. [000180] Additional protuberance and cavity arrangements are known in the art. See, e.g., U.S. Patents 5,821,333, 7,183,076, 8,642,745, 9,248,182, 9,309,311, 9,527,927, 9,562,109, 9,890,204, 48 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 10,138,303, and 11,168,344 and U.S. Patent Application Publications 2005/0079170, 2006/0025576, 2013/0089554, and 2014/0024111. [000181] In some embodiments, the dimerization domains may be derived from IgA, IgD, IgG, IgM, or IgE. The first and the second dimerization domains may contain the same or different amino acid sequences, provided that they bind each other. In some embodiments, the first and the second dimerization domains are the IgG1 constant heavy (CH) 3 domain. The amino acid sequence of a representative IgG1 CH3 domain is set forth below (SEQ ID NO: 67): 1 epkspksadk thtapqprep qvytlppsrd eltknqvslt clvkgfypsd iavewesngq 61 pennykttpp vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg 121 k [000182] In some embodiments, the first and the second dimerization domains are the IgG1 constant heavy CH2 domain. The amino acid sequence of a representative IgG1 CH2 domain is set forth below (SEQ ID NO: 68): 1 pcpapellgg psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna 61 ktkpreeqyn styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kak [000183] In some embodiments, the first and the second dimerization domains are the IgG1 CH2 and CH3 domains. The CH2 and CH3 domains may be interconnected by a linker. In some embodiments, the first, the second, or both the first and the second dimerization domains contain a fragment crystallizable region (Fc). In some embodiments the Fc contains L234A and L235A substitutions relative to wild-type Fc that abolishes binding of the Fc to (1) the Fc-γ receptor and (2) the complement component 1q (C1q), referred herein as a “silent Fc”. The silent Fc maintains binding to the neonatal Fc receptor (FcRn) (and therefore extending circulatory half-life of CAR-E which contains the silent Fc to several days). Silent Fc also provides a stabilizing effect to the CAR- E (comparable to the stabilizing effect of wild-type Fc). The two L234A and L235A substitutions are also commonly referred to as “LALA”. In some embodiments, the silent Fc also has a P329G substation; these three L234A, L235A, and P329G substitutions are also commonly referred to as “PG-LALA”. Linkers [000184] In some embodiments, the CAR-engager contains one or more linkers. A linker is “flexible” when the peptide bonds allow rotation of the amino acid residues within the linker and allow the first and the second moieties to move and bind to their respective cognate receptors on the 49 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 CAR-expressing immune cell or steric spacing (i.e., a spacer) between the first and the second moieties. [000185] A linker may be disposed between any two CAR-engager components (also referred to herein as domains, entities, moieties, portions). [000186] A linker may be disposed between the dimerization domain and the adjacent domain. In some embodiments, a linker may be disposed between the dimerization domain and the second moiety. In some embodiments, the CAR-engager contains two linkers, where a first linker is disposed between the first moiety and the dimerization domain, and a second linker is disposed between the dimerization domain and the second moiety. [000187] In some embodiments, the linker comprises an amino acid having the sequence GGGX, GGGGX (SEQ ID NO: 69), or GSSGSX (SEQ ID NO: 70), where X is any nucleotide, typically either cysteine (C) or serine (S), or repeating sequence thereof. In some embodiments, the linker has the amino acid sequence GGGGS (SEQ ID NO: 71), GSPRG (SEQ ID NO: 72), GGGGSGGGGS (SEQ ID NO: 73), GGGGSGGGGSGGGGS (SEQ ID NO: 74), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 75), GSPRGGGGSGGGGSGGGGS (SEQ ID NO: 76), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 77), KESGSVSSEQLAQFRSLD (SEQ ID NO: 78), EGKSSGSGSESKST (SEQ ID NO: 79), or GSAGSAAGSGEF (SEQ ID NO: 80). [000188] 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. Amino acid sequences of representative linkers are listed in Table 7. Table 7: Amino acid Sequences of Representative Linkers Linker Sequence A
Figure imgf000052_0001
50 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000189] In some embodiments, the CAR-engager is in the form of a fusion protein, where the components are linked by peptide bonds. In other embodiments, the CAR-engager contains proteinaceous entities that may be covalently connected interconnected by click chemistry, which is a type of chemical connection formed by a method of controlled chemical ligation. The connection may be an azide-alkyne connection, an oxime or hydrazine connection, a tetrazine-transcyclooctene connection, an azide-nitrone connection, a thiol-alkene connection, an alkene-tetrazole connection, an alkene-tetrazine connection, an alkene-azide connection, a conjugated diene-alkene connection, or an isonitrile-tetrazine connection. [000190] Additional controlled protein ligation chemistries, systems, and methods are known in the art. See, e.g., U.S. Patents 7,375,234, 7,763,736, 8,101,238, 8,372,986, 8,394,914, 8,877,170, 8,927,682, 8,927,736, 9,302,997, 9,896,547, 11,028,185, 11,091,588, and 11,352,460 and U.S. Patent Application Publication 2009/0069561. Methods of producing the CAR-E [000191] In some embodiments, the CAR-E or the IL-2 variant may be encoded in a nucleic acid which is expressed in a cell to produce the CAR-E. The term “nucleic acid” as used herein refers to a polymer of nucleotides, each of which are organic molecules consisting of a nucleoside (a nucleobase and a five-carbon sugar) and a phosphate. The term nucleotide, unless specifically stated or obvious from context, includes nucleosides that have a ribose sugar (i.e., a ribonucleotide that forms ribonucleic acid, RNA) or a 2’-deoxyribose sugar (i.e., a deoxyribonucleotide that forms deoxyribonucleic acid, DNA). Nucleotides serve as the monomeric units of nucleic acid polymers or polynucleotides. The four nucleobases in DNA are guanine (G), adenine (A), cytosine (C) and thymine (T). The four nucleobases in RNA are guanine (G), adenine (A), cytosine (C) and uracil (U). Nucleic acids are linear chains of nucleotides (e.g., at least 3 nucleotides) chemically bonded by a series of ester linkages between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar (i.e., ribose or 2’-deoxyribose) in the adjacent nucleotide. [000192] Given the number of nucleic acid sequences that encode wild-type IL-2 due to the degeneracy of the code and codon preferences, a skilled artisan would be enabled able to make nucleic acid sequences that encode the presently disclosed IL-2 variants, taking into account the specific amino acid substitutions relative to SEQ ID NO: 102, in accordance with standard techniques. The nucleic acid sequence of a representative wild-type IL-2 is set forth below (NCBI Accession No. NM_000586.4; SEQ ID NO: 111;): 51 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 1 ctatcaccta agtgtgggct aatgtaacaa agagggattt cacctacatc cattcagtca 61 gtctttgggg gtttaaagaa attccaaaga gtcatcagaa gaggaaaaat gaaggtaatg 121 ttttttcaga caggtaaagt ctttgaaaat atgtgtaata tgtaaaacat tttgacaccc 181 ccataatatt tttccagaat taacagtata aattgcatct cttgttcaag agttccctat 241 cactctcttt aatcactact cacagtaacc tcaactcctg ccacaatgta caggatgcaa 301 ctcctgtctt gcattgcact aagtcttgca cttgtcacaa acagtgcacc tacttcaagt 361 tctacaaaga aaacacagct acaactggag catttactgc tggatttaca gatgattttg 421 aatggaatta ataattacaa gaatcccaaa ctcaccagga tgctcacatt taagttttac 481 atgcccaaga aggccacaga actgaaacat cttcagtgtc tagaagaaga actcaaacct 541 ctggaggaag tgctaaattt agctcaaagc aaaaactttc acttaagacc cagggactta 601 atcagcaata tcaacgtaat agttctggaa ctaaagggat ctgaaacaac attcatgtgt 661 gaatatgctg atgagacagc aaccattgta gaatttctga acagatggat taccttttgt 721 caaagcatca tctcaacact gacttgataa ttaagtgctt cccacttaaa acatatcagg 781 ccttctattt atttaaatat ttaaatttta tatttattgt tgaatgtatg gtttgctacc 841 tattgtaact attattctta atcttaaaac tataaatatg gatcttttat gattcttttt 901 gtaagcccta ggggctctaa aatggtttca cttatttatc ccaaaatatt tattattatg 961 ttgaatgtta aatatagtat ctatgtagat tggttagtaa aactatttaa taaatttgat 1021 aaatataaa [000193] In some embodiments, the CAR-engager is encoded by two nucleic acids, e.g., the first moiety is encoded by a first nucleic acid and the second moiety is encoded by a second nucleic acid. [000194] In some embodiments, nucleic acid encoding the CAR-engager includes a signal peptide- encoding nucleic acid disposed 5’ to the nucleic acid encoding the first moiety. The term “signal peptide” as used herein refers to a short (e.g., 5-30 or 10-100 amino acids long) stretch of amino acids that directs the transport of the protein during translation. CAR-engagers containing a signal peptide will be secreted from the cell. Typically, the signal peptide is cleaved from the CAR-engager before secretion. The signal peptide may be connected to the nucleic acid encoding the first moiety or the nucleic acid encoding the second moiety. [000195] In some embodiments, the signal peptide may be derived from Ig-γ-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). Signal peptides may also be synthetic (i.e., non-naturally occurring). Amino acid sequences of representative signal peptides are listed in Table 8. Table 8: Amino acid Sequences of Representative Signal Peptides Signal peptide Sequence
Figure imgf000054_0001
52 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 CD33 (SEQ ID NO: 92) MPLLLLLPLLWAGALA EPO (SEQ ID NO: 93) MGVHECPAWLWLLLSLLSLPLGLPVLG
Figure imgf000055_0001
[000196] IL-2 variant-encoding nucleic acids and CAR-engager-encoding nucleic acids may be introduced into a cell by a suitable vector. In embodiments, wherein the first moiety and the second moiety are linked chemically, e.g., via click chemistry, the CAR-encoding nucleic acids may be introduced into one or more cells by separate vectors. A vector is configured so as to contain the elements necessary to effect transport into the immune cell and effect expression of the nucleic acid(s) after transformation. Such elements include an origin of replication, a poly-A tail sequence, a selectable marker, and one or more suitable sites for the insertion of the nucleic acid sequences, such as a multiple cloning site (MCS), one or more suitable promoters, each promoter operatively linked to the insertion sites of the nucleic acid sequences and the selectable marker, and additional optional regulatory elements. [000197] The term “promoter” as used herein refers to a nucleic acid sequence that regulates, directly or indirectly, the transcription of a corresponding nucleic acid coding sequence to which it is operably linked, which in the context of the present disclosure, is an IL-2 variant, and in other aspects, a CAR-engager protein that contains the variant. A promoter may function alone to regulate transcription, or it may act in concert with one or more other regulatory sequences (e.g., enhancers or silencers, or regulatory elements that may be present in the nucleic acid sequences or the vector). Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (toward the 5’ region of the sense strand). Promoters typically range from about 100-1000 base pairs in length. 53 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000198] The term “operatively linked” as used herein is to be understood that a nucleic acid sequence is spatially situated or disposed in the vector relative to another nucleic acid sequence, e.g., a promoter is operatively linked to drive the expression of a nucleic acid coding sequence (e.g., the CAR-engager-encoding nucleic acid sequence). [000199] In some embodiments, a single vector contains a single promoter operatively linked to the CAR-engager-encoding nucleic acid of the enhancer. In some embodiments, a single vector contains a single promoter operatively linked to the first moiety-encoding nucleic acid and the second moiety-encoding nucleic acid. In some of these embodiments, the nucleic acids are separated by a nucleic acid encoding a self-cleaving peptide or an internal ribosome entry site (IRES). In some embodiments, the single vector contains a first promoter operatively liked to the first moiety- encoding nucleic acid and a second promoter operatively liked to the first moiety -encoding nucleic acid. [000200] In some embodiments, two vectors are constructed. In some embodiments, a first vector contains a promoter operatively linked to the first moiety-encoding nucleic acid and a second vector contains a promoter operatively linked to the second moiety-encoding nucleic acid. [000201] In some embodiments, the vector contains a strong mammalian promoter, for example a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, synthetic promoters (e.g., RPBSA (synthetic, from Sleeping Beauty), or CAG (synthetic, CMV early enhancer element, chicken β-Actin, and splice acceptor of rabbit β-Globin)) or promoters derived from the β-actin, phosphoglycerate kinase (PGK), or factor EF1α genes. In some embodiments, the promoter may contain a core region located close to the nucleic acid coding sequence. In some embodiments, the promoter is modified to remove methylation sensitive motifs (e.g., a cytosine nucleotide is followed by a guanine nucleotide, or “CpG”), or by the addition of a regulatory sequence that binds transcriptional factors that repress DNA methylation. In some embodiments, the vector includes A/T-rich, nuclear matrix interacting sequences, known as scaffold matrix attachment regions (S/MAR), which enhance transformation efficiency and improve the stability of transgene expression. [000202] In some embodiments, the vector is a viral vector, for example, a retroviral vector, a lentiviral vector, an adenoviral vector, a herpesvirus vector, an adenovirus, or an adeno-associated virus (AAV) vector. The construction of lentiviral vectors has been described, for example, in U.S. Patents 5,665,577, 5,981,276, 6,013,516, 7,090,837, 8,119,119 and 10,954,530. 54 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000203] In other embodiments, the vector is a non-viral vector, representative examples of which include plasmids, mRNA, linear single stranded (ss) DNA or linear double stranded (ds) DNA, minicircles, and transposon-based vectors, such as Sleeping Beauty (SB)-based vectors and piggyBac(PB)-based vectors. In yet other embodiments, the vector may include both viral and non- viral elements. [000204] In some embodiments the vector is a plasmid. In addition to a promoter operatively linked to the nucleic acids, the plasmid may also contain other elements e.g., that facilitate transport and expression of the nucleic acid in an immune cell. The plasmid may be linearized with restriction enzymes, in vitro transcribed to produce mRNA, and then modified with a 5’ cap and a 3’ poly-A tail. In some embodiments, the vector multiple plasmids, a first plasmid encoding a first proteinaceous entity (e.g., the ectodomain of the CAR-engager) and a second plasmid encoding a second proteinaceous entity (e.g., the immune effector domain of the CAR-engager). Cells [000205] The IL-2 variants and CAR-Es may be expressed in a genetically modified (or transformed) cell containing a vector that contains a nucleic acid encoding an IL-2 variant, the CAR- E, or components of the CAR-E for the purpose of making and purifying protein. [000206] Cells useful for the cloning and other manipulations of these vectors are conventional. Cells from various strains of E. coli may be used for replication of the vectors and other steps in the construction of the CAR-engagers of this disclosure. [000207] Suitable host cells or cell lines for the expression of the nucleic acids encoding the IL-2 variants and the nucleic acid-encoding CAR-engagers include eukaryotic cells. In some embodiments, the cells are a mammalian cell line. In some embodiments, the cells are mammalian cells such as CHO (e.g., DG44, CHO-S), fibroblast cells (e.g., 3T3, COS), embryonic cells (e.g., PER.C6, HEK (e.g., HEK.293)), somatic cell hybrids (e.g., Sp2/0), and cancer cells, for example, myeloma cells (e.g., NS0 (NS zero)). In some embodiments, the nucleic acids encoding the CAR- engager is expressed in a CHO or a myeloma cell. Human cells may be used, thus enabling the expressed CAR-engager to be modified with human glycosylation patterns. The selection of suitable mammalian cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Green et al., eds., Molecular Cloning: A Laboratory Manual, 5th ed., Cold Spring Harbor Laboratory Press, New York, 2012. 55 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000208] In some embodiments, the cells are prokaryotic. Prokaryotic (i.e., bacterial) cells may prove useful as host cells suitable for the expression of the nucleic acids encoding CAR-engagers (see, e.g., Pluckthun, Immunol. Rev.130:151-188 (1992)). However, due to the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded form or in a non-glycosylated form, any CAR-engagers produced in a bacterial cell would be screened for retention of function (e.g., CAR binding ability of an expressed CAR-E). If the CAR-engager expressed by the bacterial cell was produced in a properly folded form, that bacterial cell would be a desirable host, or in alternative embodiments the CAR-engager may express in the bacterial host and then be subsequently re-folded. For example, various strains of E. Coli used for expression are well-known as host cells in the field of biotechnology. Various strains of B. Subtilis, Streptomyces, other bacilli and the like may also be employed. [000209] After expression in a cell, the IL-2 variant or the CAR-engager protein is isolated from the cell (e.g., cell lysates) or from the medium in which the cell is cultured. Protein isolation techniques are known in the art. Representative isolation techniques include chromatography, affinity chromatography, nickel- 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, e.g., U.S. Patents 516,9936, 6,267,958, 8,357,778, 9,630,165, 9,708,399, 10,023,608, 10,207,229, 11,369,703, and 11,390,668, U.S. Patent Application Publications 2008/0090995, 2012/0244075, 2017/0158760, 2019/0276492, and 2021/0206815, and Traunecker et al., Embo J.10(12):3655-9 (1991). Pharmaceutical compositions containing an IL-2 variant or CAR-E [000210] For the purposes of practicing the disclosed methods, the IL-2 variants and/or the CAR- engagers may be formulated in a pharmaceutically acceptable carrier. The term “effective amount” as used herein refers to a sufficient amount of an IL-2 variant or a CAR-engager to provide the desired effect, e.g., which may include any one or more of killing at least cancer cell, reversing, alleviating, ameliorating, inhibiting, diminishing, slowing down, arresting, stabilizing, achieving remission, or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a cancer. The amount of IL-2 variant and the CAR-engager administered to a subject will vary between wide limits, depending 56 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 upon the location, type, and severity of the cancer, the age, body weight, and condition of the individual to be treated, etc. A physician will ultimately determine appropriate doses to be used. The IL-2 variant and/or the CAR-engager in the pharmaceutical composition may be in the form of a monomer (in embodiments lacking a dimerization domain), homodimer, or heterodimer, as described herein. [000211] IL-2 variants of the present disclosure may be used alone or in combination with another active agents to treat cancer. See, e.g., Ren, et al., J. Clin. Invest.132(3):e153604 pp.1-13 (2022). High-dose IL-2 treatment can overcome regulatory T cell (Treg)-associated IL-2 trapping and allow extra IL-2 to activate tumor infiltrating lymphocytes (TILs) for treating cancer, for example, metastatic renal cell carcinoma and melanoma. However, patients who respond to high-dose IL-2 treatment frequently suffer from intolerable toxicities (See, e.g., Li et al., Nat. Commun.8(1):1762 (2017)), which has limited its clinical use. Accordingly, IL-2 variants of the present disclosure may constitute a more attractive alternative in terms of binding to IL-2Rα on Tregs (See, e.g., Mott et al., J. Mol. Biol.247(5):979-94 (1995)) or increasing the binding to IL-2Rβ on effector cells (See, e.g., Levin et al., Nature 484(7395):529-33 (2012) and Sun et al., Nat. Commun. 10(1):3874 pp. 1-12 (2019)). IL-2 variants of the present disclosure may be conjugated to other active moieties, such as antibodies. See, Ren, et al., supra. Since tumor infiltrating lymphocytes express more PD-1 than other cells, the IL-2 variants of the present disclosure may be fused to an anti–PD-1 antibody. These fusion proteins may show better intratumoral T cell binding, more potent antitumor effects, and may also overcome PD-L1 therapy resistance. The IL-2 variants may also be conjugated to anti-tumor antibodies. See, e.g., Sun et al., Nat. Commun.10(1):3874 pp.1-12 (2019). [000212] Compositions may be provided as sterile solid or liquid preparations. Solid preparations may be reconstituted and diluted into a liquid preparation before use, e.g., with carriers containing isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous solutions, which may be buffered to a selected pH. Liquid carriers include aqueous and non-aqueous carriers alike. Representative examples of liquid carriers include sterile water for injection, saline, Lactated Ringer Injection solution, phosphate buffered saline, soluble sugars (e.g., dextrose), dimethyl sulfoxide (DMSO), ethanol, and suitable mixtures thereof. In some embodiments, the liquid carrier includes a protein dissolved or dispersed therein, representative examples include serum albumin (e.g., human serum albumin, recombinant human albumin), gelatin, and casein. In some embodiments, the liquid carrier includes a water-miscible polyol (e.g., glycerol, propylene glycol, liquid 57 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 polyethylene glycol, and the like). The compositions are typically isotonic, i.e., they have the same osmotic pressure as blood. Citric acid, sodium chloride, sugars, polyalcohols, and isotonic electrolyte solutions (e.g., Plasma-Lyte®) may be used to achieve the desired isotonicity. Depending on the carrier, other excipients may be added, e.g., wetting, dispersing, or emulsifying agents, gelling and viscosity enhancing agents, preservatives and the like as known in the art. In some embodiments, the compositions include citric acid, ethylenediaminetetraacetic acid (EDTA), and polysorbate 20 with a pH range between about 6.8 to about 7.2. Cancer Subjects [000213] In some aspects, the present disclosure is directed to methods of treating a cancer in a subject. In some embodiments, the methods may entail administration of the IL-2 variant, per se, or in the form of a fusion with another active moiety, such as an antibody or binding fragment thereof, that binds a receptor on an immune cell, e.g., a TIL, or antibody that binds an epitope on an antigen present on a tumor cell. In some embodiments, the administration of the IL-2 variant is used to treat kidney cancer (e.g., metastatic renal cell carcinoma), melanoma, colon cancer, lung cancer, or ovarian cancer. [000214] In other aspects, the method entails administering to a subject in need thereof a pharmaceutical composition containing a CAR-engager described herein. Administration of the CAR-engagers may precede or succeed administration of CAR immune cells or be substantially simultaneous therewith. Cancers treatable in accordance with the disclosed methods broadly include hematopoietic cancers and cancers characterized by the presence of a solid tumor. [000215] The term “subject” (or “patient”) as used herein includes all members of the animal kingdom prone (or disposed) to or suffering from the indicated cancer. In some embodiments, the subject is a human. Therefore, a subject “having cancer” or “in need of” treatment according to the present disclosure broadly embraces subjects who have been positively diagnosed, including subjects having active disease who may have been previously treated with one or more rounds of therapy, and subjects who are not currently being treated (e.g., in remission) but who might still be at risk of relapse, and subjects who have not been positively diagnosed but who are predisposed to cancer (e.g., on account of the basis of prior medical history and/or family medical history, or who otherwise present with a one or more risk factors such that a medical professional might reasonably suspect that the subject was predisposed to cancer). 58 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000216] The terms “treat”, “treating”, and “treatment” as used herein refer to the aft-recognized indicia of therapeutic efficacy, intervention, process performed on, or the administration of an active agent to the subject in need thereof with the therapeutic objective (“therapeutic effect”) of reversing, alleviating, ameliorating, inhibiting, diminishing, slowing down, arresting, stabilizing, or preventing the onset, progression, development, metastases, severity or recurrence of a symptom, improvement in survival time, total/complete or partial remission, complication or condition, or biochemical indicia associated with cancer. Remission may include no detectable cancer cells, less tumor cells, smaller tumors, or a reduction in tumor cell number. [000217] In some embodiments, the cancer is a hematopoietic cancer. Representative hematological cancers include plasma cell neoplasm (e.g., myeloma, multiple myeloma, relapsed or refractory multiple myeloma, plasma cell myeloma, extramedullary multiple myeloma, monoclonal gammopathy of unknown significance (MUGS), asymptomatic smoldering multiple myeloma, or solitary plasmacytoma), lymphoma (e.g., Hodgkin’s lymphoma (HL), non-Hodgkin’s lymphoma, Burkitt lymphoma, Waldenstrom macroglobulinemia, plasmablastic lymphoma, plasmacytoid lymphoma, B-cell lymphoma, high-grade B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), primary mediastinal large lymphoma (PMBL), follicular lymphoma (FL), and mantle cell lymphoma (MCL)), and leukemia (e.g., plasma cell leukemia, relapsed or refractory acute B lymphocytic leukemia (ALL), relapsed or refractory acute lymphoblastic leukemia, chronic lymphoblastic leukemia, or chronic lymphocytic leukemia (CLL)). [000218] In some embodiments, the cancer is characterized by the presence of a solid tumor. In some embodiments, the cancer is a bladder cancer (e.g.,transitional cell carcinoma, also called urothelial carcinoma), kidney cancer (e.g., renal cell carcinoma (RCC), kidney renal clear cell carcinoma (KIRC), transitional cell cancer, or Wilms tumor), skin cancer (e.g., melanoma, skin cutaneous melanoma (SKCM), basal cell carcinoma, and squamous cell carcinoma of the skin), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC)), head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN) also called head and neck squamous cell carcinoma (HNSC), laryngeal and hypopharyngeal cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, oral and oropharyngeal cancer, and salivary gland cancer), colon or rectal cancer (e.g., colorectal carcinoma (CRC), colon adenocarcinoma (COAD), rectum adenocarcinoma (READ)), ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian 59 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 adenocarcinoma, epithelial ovarian carcinomas, fallopian tube cancer, and primary peritoneal cancer), breast cancer (e.g., triple-negative breast cancer (TNBC)), pancreatic cancer, liver cancer, brain cancer (e.g., astrocytoma and gliomas such as glioblastomas), gastric cancer, biliary cancer, uterine serous carcinoma, cholangiocarcinoma, neuroblastoma, sarcoma, endometrial cancer, cervical cancer (e.g., cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC)), prostate cancer (e.g., prostate adenocarcinoma (PRAD)), and stomach cancer (e.g., stomach adenocarcinoma (STAD)). [000219] In some embodiments, the disclosed methods treat malignant mesothelioma, ovarian cancer, breast cancer (e.g., TNBC), pancreatic cancer, lung cancer, liver cancer, glioblastoma, gastric cancer, endometrial cancer, cervical cancer, biliary cancer, uterine serous carcinoma, cholangiocarcinoma, neuroblastoma, sarcoma, or melanoma. [000220] In some embodiments, the disclosed methods further include an anti-CD19 immune cell therapy, a CAR-E that binds CD19, and are used to treat HL, non-Hodgkin’s lymphoma, ALL, CLL, chronic lymphocytic leukemia, Burkitt lymphoma, DLBCL, PMBL, high-grade B-cell lymphoma, FL, MCL, or multiple myeloma (MM). [000221] In some embodiments, the disclosed methods further include anti-BCMA immune cell therapy and a CAR-E that binds BCMA, and are used to treat MM, HL, non-Hodgkin’s lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), plasma cell leukemia, SLE, acute AMR, chronic AMR, or AL-amyloidosis. [000222] In some embodiments, the cancer is characterized as being in a state of minimal residual disease (MRD). MRD is a state at which a cancer patient has a small number of cancer cells that remain in the body after treatment. The number of remaining cells may be so small that they do not cause any physical signs or symptoms of the cancer, and often may not be detectable through traditional methods, such as viewing cells under a microscope and/or by tracking abnormal serum proteins in the blood. [000223] The amount of cancer antigens present in a subject in a state of MRD are limited. And this limited presence of the cancer antigen may not adequately support the proliferation and efficacy of CAR immune cells. The additional presence of a CAR-engager presents the CAR immune cells with not only additional cancer antigen, but also a supportive IL-2 variant, and in some embodiments, a second immune cell effector domain, that may modulate the activity of the CAR immune cell to promote proliferation, efficacy, and/or persistence. 60 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000224] In some embodiments, the subject receiving an administration of CAR-engager is in a state of MRD. In some embodiments, the method of treating cancer involves treatment of a state of minimal residual disease (MRD) in the subject. In some embodiments, the method of treating cancer involves elimination of MRD in the subject. [000225] To test for MRD, samples from either a blood draw or a bone marrow aspiration may be used. The most widely used tests to measure MRD are flow cytometry, polymerase chain reaction (PCR) and next-generation sequencing. Methods that may be suitable for use in measuring MRD are described in, e.g., U.S. Patents 8,124,353, 9,528,160, 10,280,462, 11,618,787, and 11,633,426, and U.S. Patent Application Publications 2011/0294148, and 2022/0380852. CAR immune cell therapy [000226] In some embodiments, the methods of treating cancer of the present disclosure entails administration of the IL-2 variant and/or the CAR-E to a subject having had CAR immune cell therapy. As known in the art, CAR immune cells contain a synthetic CAR molecule that binds a cancer antigen. Typically, CARs contain an extracellular domain to which the CAR-E binds, a transmembrane domain, and an intracellular domain comprising a stimulatory domain. [000227] The extracellular domain of the CAR that binds the ectodomain of a cancer antigen may contain an antibody fragment. In some embodiments, the CAR binds BCMA. CAR extracellular domains that bind to BCMA are known in the art. See, e.g., FDA-approved CAR-expressing immune cells ciltacabtagene autoleucel (Carvykti®, also referred to herein as “Cilta-cel”), and idecabtagene vicleucel (Abecma®, also referred to herein as “Ide-cel”), U.S. Patents 10,072,088, 10,683,369, 11,084,880, and 10,174,095, and U.S. Patent Application Publications 2016/0131655, 2017/0226216, 2018/0133296, 2019/0151365, 2019/0359727, 2019/0381171, 2020/0339699, 2020/0360431, 2020/0055948, and 2022/0064316. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-BCMA antibody, BCMA-binding fragment, or derivative thereof, e.g., belantamab (Blenrep®), linvoseltamab (REGN5458), pacanalotamab (AMG 420), pavurutamab (AMG 701), and teclistamab (Tecvayli®). In some embodiments, the extracellular domain of the CAR binds the BCMA ectodomain of the CAR-engager that has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. [000228] In some embodiments, the CAR extracellular domain contains a single variable heavy (VHH) or variant thereof. In some embodiments, the VHH has the amino acid sequence set forth below (SEQ ID NO: 127): 61 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 1 qvkleesggg lvqagrslrl scaasehtfs shvmgwfrqa pgkeresvav igwrdistsy 61 adsvkgrfti srdnakktly lqmnslkped tavyycaarr idaadfdswg qgtqvtvss [000229] In some embodiments, the VHH has the amino acid sequence set forth below (SEQ ID NO: 128): 1 evqlvesggg lvqaggslrl scaasgrtft mgwfrqapgk erefvaaisl sptlayyaes 61 vkgrftisrd nakntvvlqm nslkpedtal yycaadrksv msirpdywgq gtqvtvss [000230] In some embodiments, the CAR extracellular domain contains a VH that has the amino acid sequence set forth below (SEQ ID NO: 129): 1 divltqspps lamslgkrat iscrasesvt ilgshlihwy qqkpgqpptl liqlasnvqt 61 gvparfsgsg srtdftltid pveeddvavy yclqsrtipr tfgggtklei k [000231] In some embodiments, the CAR extracellular domain contains a VL that has the amino acid sequence set forth below (SEQ ID NO: 130): 1 qiqlvqsgpe lkkpgetvki sckasgytft dysinwvkra pgkglkwmgw intetrepay 61 aydfrgrfaf sletsastay lqinnlkyed tatyfcaldy syamdywgqg tsvtvss [000232] In some embodiments, the CAR binds CD19. CAR extracellular domains that bind to CD19 are known in the art. See, e.g., FDA-approved CAR-expressing immune cells lisocabtagene maraleucel (Breyanzi®), tisagenlecleucel (Kymriah®), brexucabtagene autoleucel (Tecartus®), and axicabtagene ciloleucel (Yescarta®), U.S. Patents 9,629,877, 10,273,300, and 10,533,055, and U.S. Patent Application Publications 2020/0392248, and 2021/0238253. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-CD19 antibody, anti-CD19- binding fragment, or derivative thereof, e.g., loncastuximab (Zynlonta®), tafasitamab (Monjuvi®), denintuzumab (SGN-CD19A), and inebilizumab (Uplizna®). In some embodiments, the extracellular domain of the CAR binds the CD19 ectodomain of the CAR-engager having the amino acid sequence of any one of SEQ ID NOs: 3-5, or 103. [000233] In some embodiments, the CAR extracellular domain contains a VH that has the amino acid sequence set forth below (SEQ ID NO: 131): 1 diqmtqttss lsaslgdrvt iscrasqdis kylnwyqqkp dgtvklliyh tsrlhsgvps 61 rfsgsgsgtd ysltisnleq ediatyfcqq gntlpytfgg gtkleit [000234] In some embodiments, the CAR extracellular domain contains a VL that has the amino acid sequence set forth below (SEQ ID NO: 132): 1 evklqesgpg lvapsqslsv tctvsgvslp dygvswirqp prkglewlgv iwgsettyyn 61 salksrltii kdnsksqvfl kmnslqtddt aiyycakhyy yggsyamdyw gqgtsvtvs [000235] In some embodiments, the CAR binds CD20. CAR extracellular domains that bind to CD20 are known in the art. See, e.g., U.S. Patents 10,189,903, 10,442,867, 10,934,363, 11,066,457, 11,160,833, and 11,439,665, and U.S. Patent Application Publication 2018/0187149. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-CD20 62 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 antibody, anti-CD20-binding fragment, or derivatives thereof, e.g., ofatumumab (Arzerra®, Kesimpta®), veltuzumab (IMMU-106), tositumomab (Bexxar®), and rituximab (Rituxan®, Riabni®, Truximab®). In some embodiments, the extracellular domain of the CAR binds the CD20 ectodomain of the CAR-engager having the amino acid sequence SEQ ID NO: 6. [000236] In some embodiments, the CAR binds CD22. CAR extracellular domains that bind CD22 are known in the art. See, e.g., U.S. Patents 9,139,649, 9,181,343, and 10,494,435, U.S. Patent Application Publications 2015/0175711, 2018/0086843, 2021/0047402, 2021/0095022, 2022/0220198, and 2022/0273710, and Fry et al., Nat. Med. 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 derivatives thereof, e.g., bectumomab, epratuzumab, inotuzumab, moxetumomab, and epratuzumab. In some embodiments, the extracellular domain of the CAR binds a CD22 ectodomain of the CAR-engager. In some embodiments, the CAR-engager contains a CD22 ectodomain that has the amino acid sequence SEQ ID NO: 7. In some embodiments, the CAR-engager contains a CD22 ectodomain that has any one of the amino acid sequences SEQ ID NOs: 104-107. [000237] In some embodiments, the CAR binds Claudin 18.2. CAR extracellular domains that bind Claudin 18.2 are known in the art. See, e.g., U.S. Patents 10,421,817, 11,098,118, 11,485,782, 11,541,127, and 11,713,346, and U.S. Patent Application Publications 2022/0073643, 2023/0192840, and 2023/0242877. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-Claudin 18.2 antibody, anti-Claudin 18.2-binding fragment, or derivatives thereof, e.g., osemitamab and zolbetuximab (Vyloy®). In some embodiments, the extracellular domain of the CAR binds a Claudin 18.2 ectodomain of the CAR-engager. In some embodiments, the CAR-engager contains a Claudin 18.2 ectodomain that has any one of the amino acid sequences SEQ ID NOs: 8-9. [000238] In some embodiments, the CAR binds SLAMF7. CAR extracellular domains that bind SLAMF7 are known in the art. See, e.g., U.S. Patent 10,799,536, and U.S. Patent Application Publications 2020/0024342, 2020/0283534, 2021/0230548, and 2021/0253729. In some embodiments, the CAR extracellular domain is derived from a commercially available anti- SLAMF7 antibody, anti-SLAMF7-binding fragment, or derivative thereof, e.g., elotuzumab (Empliciti®). In some embodiments, the extracellular domain of the CAR binds the SLAMF7 63 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 ectodomain of the CAR-engager. In some embodiments, the extracellular domain of the CAR binds the SLAMF7 ectodomain of the CAR-engager having the amino acid sequence SEQ ID NO: 10. [000239] In some embodiments, the CAR binds PD-1. CAR extracellular domains that bind to PD- 1 are known in the art. See, e.g., U.S. Patents 10,124,023 and 11,136,392, and U.S. Patent Application Publications 2021/0061877, 2020/0281974, and 2022/0064595. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-PD-1 antibody, anti- PD-1-binding fragment, or derivative thereof, e.g., balstilimab, budigalimab, cadonilimab, cemiplimab (Libtayo®), cetrelimab, dostarlimab (Jemperli®), izuralimab, nivolumab (Opdivo®), pacmilimab, pembrolizumab (Keytruda®), penpulimab, peresolimab, pidilizumab, retifanlimab, rosnilimab, sintilimab, spartalizumab, tislelizumab, toripalimab, volrustomig, vudalimab, zeluvalimab, and zimberelimab. In some embodiments, the extracellular domain of the CAR binds a PD-1 ectodomain of the CAR-engager. In some embodiments, the extracellular domain of the CAR binds the PD-1 ectodomain of the CAR-engager that has any one of the amino acid sequences SEQ ID NO: 11 and 107-108. [000240] In some embodiments, the CAR binds Mast/stem cell growth factor receptor Kit (KIT; also known as Receptor tyrosine kinase KIT proto-oncogene). CAR extracellular domains that bind KIT are known in the art. See, e.g., U.S. Patent Application Publications 2017/0335281, 2020/0048359, 2020/0071397, and 2021/0299177. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-KIT antibody, anti-KIT-binding fragment, or derivative thereof, e.g., barzolvolimab. In some embodiments, the extracellular domain of the CAR binds the KIT ectodomain of the CAR-engager. In some embodiments, the extracellular domain of the CAR binds the KIT ectodomain of the CAR-engager having the amino acid sequence SEQ ID NO: 12. [000241] In some embodiments, the CAR binds TROP2. CAR extracellular domains that bind TROP2 are known in the art. See, e.g., U.S. Patents 11,602,525, and 11,768,203, and U.S. Patent Application Publications 2018/0296689, 2021/0169852, and 2022/0204582. In some embodiments, the CAR extracellular domain is derived from a commercially available anti-TROP2 antibody, anti- TROP2-binding fragment, or derivatives thereof, e.g., datopotamab and sacituzumab. In some embodiments, the extracellular domain of the CAR binds a TROP2 ectodomain of the CAR-engager. In some embodiments, the CAR-engager contains a TROP2 ectodomain that has the amino acid sequence SEQ ID NO: 13. 64 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000242] In some embodiments, the CAR binds CD38. CAR extracellular domains that bind CD38 are known in the art. See, e.g., U.S. Patents 10,709,775, 10,799,536, 10,836,998, and 11,365,394 and U.S. Patent Application Publications 2017/0296623, 2019/0135894, 2019/0135937, 2020/0308541, 2021/0046118, and 2022/0202859 In some embodiments, the CAR extracellular domain is derived from a commercially available anti-CD38 antibody, anti-CD38-binding fragment, or derivative thereof, e.g., daratumumab (Darzalex®), isatuximab (Sarclisa®), and mezagitamab. In some embodiments, the extracellular domain of the CAR binds the CD38 ectodomain of the CAR- engager. In some embodiments, the extracellular domain of the CAR binds the CD38 ectodomain of the CAR-engager having the amino acid sequence SEQ ID NO: 14. [000243] In some embodiments, the CAR binds MSLN. CAR extracellular domains that bind MSLN are known in the art. See, e.g., U.S. Patents 10,550,179, 10,640,569, 10,730,954, 11,648,268, and 11,702,472, and U.S. Patent Application Publications 2020/0255803, 2021/0079057, and 2022/0112263 In some embodiments, the CAR extracellular domain is derived from a commercially available anti-MSLN antibody, anti-MSLN-binding fragment, or derivative thereof, e.g., amatuximab. In some embodiments, the extracellular domain of the CAR binds a MSLN ectodomain of the CAR-engager. In some embodiments, the extracellular domain of the CAR binds the MSLN ectodomain of the CAR-engager that has any one of the amino acid sequences SEQ ID NO: 15 and 17-18. [000244] The intracellular domain of the CAR contains a signaling domain that enables intracellular signaling and immune cell function. The signaling domain may include a primary signaling domain and/or a co-stimulatory signaling domain. In some embodiments, the intracellular domain is capable of delivering a signal approximating that of natural ligation of an ITAM- containing molecule or receptor complex such as a TCR receptor complex. [000245] In some embodiments, the signaling domain includes a plurality, e.g., 2 or 3, costimulatory signaling domains, e.g., selected from 4-1BB, CD3ζ, CD28, CD27, ICOS, and OX40. In some embodiments, the signaling domain may include a CD3ζ domain as a primary signaling domain, and any of the following pairs of co-stimulatory signaling domains from the extracellular to the intracellular direction: 4-1BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28-4-1BB; 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 co-stimulatory signaling 65 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 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 signal transducer ((HCST)), DAP12 (TYROBP), Dectin-1, FcαRI, FcγRI, FcγRII, FcγRIII, IL-2RB, ICOS, KIR2DS2, MyD88, OX40, and ZAP70. [000246] A representative CAR with a CD3ζ stimulatory signaling domain is the FDA-approved CAR-expressing immune cells tisagenlecleucel (Kymriah®). Representative CARs with CD3ζ and 4-1BB co-stimulatory signaling domains are the FDA-approved CAR-expressing immune cells idecabtagene vicleucel (Abecma®), lisocabtagene maraleucel (Breyanzi®), and ciltacabtagene autoleucel (Carvykti®). Representative CARs with CD28 and CD3ζ co-stimulatory signaling domains are the FDA-approved CAR-expressing immune cells brexucabtagene autoleucel (Tecartus®) and axicabtagene ciloleucel (Yescarta®). [000247] In some embodiments, the CAR immune cell is a T cell. In some embodiments, the CAR immune cell is a NK cell. Additional CAR immune cells are known in the art, e.g., U.S. Patents 5,906,936, 7,446,190, 7,741,465, 8,389,282, 8,399,645, 9,422,351, 9,790,267, 9,885,298, 10,124,023, 10,815,301, and 11,433,100, and U.S. Patent Application Publications 2019/0375815, 2020/0281973, 2021/0300986, 2022/0056101, and 2022/0193138. [000248] The CAR immune cells may be autologous or allogeneic. For example, immune cells or progenitors thereof can be isolated from a tissue of body fluid from one subject prior to administration to the same subject (autologous) or a different, compatible subject (allogeneic). Most typically, the CAR immune cells are administered once. [000249] The number of CAR immune cells administered to a subject will vary between wide limits, depending upon the location, type, and severity of the cancer, the age, body weight, and condition of the individual to be treated, etc. A physician will ultimately determine appropriate number of cells and doses to be used. Typically, the CAR immune cells will be given in a single, one-time dose. [000250] Dosage amounts (e.g., numbers) of CAR immune cells effective to treat cancer are known in the ar. In some embodiments, the effective number of the CAR immune cells is between about 1×104 to about 1×1010 cells per subject. In some embodiments, the effective number of the CAR immune cells is between about 1×105 to about 1×1010 cells per subject. In some embodiments, the effective number of the CAR immune cells is about the number of cells given in FDA-approved 66 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 CAR T cell therapies, which is between about 1×106 to about 1×1010 cells per kg of subject body weight. In some embodiments, the effective number of the CAR immune cells is between about 1×105 to about 6×108 cells per kg of subject body weight. [000251] Since the CAR-engager promotes functionality and persistence of CAR immune cells, CAR therapy that contemplates coordinate administration of the CAR-engager may entail use of fewer CAR immune cells compared to FDA-approved CAR T cell therapies. Therefore, the presently disclosed methods might require fewer cells, e.g., from about 1×104 to about 1×107 cells per kg of subject body weight. [000252] The CAR immune cells may be administered to a subject for the treatment of a cancer by any medically acceptable route. The CAR immune cells are typically delivered intravenously, although they may also be introduced into other convenient sites (e.g., to an affected organ or tissue) or modes, as determined by an attending physician. Administration of the CAR-E [000253] Broadly, the order in which the CAR-engager and CAR immune cells are administered during the same course of treatment may be varied, provided that they are able to interact in vivo and cause the desired effect. In some embodiments, the CAR-engager is co-administered substantially simultaneously to the subject with the CAR immune cells. In some embodiments, the CAR-engager is contacted with the CAR immune cells in vitro before co-administration to the subject. In some embodiments, the CAR-engager is administered to the subject subsequent to the administration of the CAR immune cells. In some embodiments, the CAR-engager is administered to the subject prior to the administration of the CAR immune cells. [000254] Broadly, the methods entail administration of an effective amount of the CAR-engager to a cancer patient who had received, is receiving, or will receive an administration of immune cells containing a CAR that contains an extracellular domain to which the CAR-engager binds, a transmembrane domain, and an intracellular domain comprising a stimulatory domain. The order in which the CAR-engager and CAR immune cells are administered during the same course of treatment may not be critical, provided that they are able to interact in vivo and cause the desired effect. In some embodiments, the CAR-engager is co-administered substantially simultaneously to the subject with the CAR immune cells. In some embodiments, the CAR-engager is contacted with 67 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 the CAR immune cells in vitro before co-administration to the subject. In some embodiments, the CAR-engager is administered to the subject prior to the administration of the CAR immune cells. [000255] In some embodiments, the CAR-engager is administered to the subject subsequent to the administration of the CAR immune cells, e.g., upon a determination that the CAR-immune cells have lost vitality or persistence in the subject. This determination may be made in accordance with known techniques. In some embodiments, for example, a sample is obtained from the subject after the administration of the immune cells. The concentration of immune cells present within the sample may be used to calculate the difference between the concentration of the immune cells administered to the subject and the concentration of the immune cell measured in the sample. The CAR-engager may be administered once the measured immune cell concentration is less than the administered immune cell concentration. In some embodiments, the CAR-engager is administered once the measured immune cell concentration 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 administered concentration of immune cells. [000256] In some embodiments, administration of the CAR-engager is conducted at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 2 months, at least, at least about 3 months, at least about 6 months, at least about 9 months, or at least about a year after administering the CAR-immune cells. [000257] In some embodiments, the CAR-engager is administered for at least one cycle, for example, administered once a week, once every two weeks, once every three weeks. The cycle may be repeated, for example, for 2 cycles, 3 cycles, 5 cycles, or8 cycles. In some embodiments, the CAR-engager is administered for a period of consecutive days before cyclic administration, for example, administered once a day for five days and once every three weeks thereafter. In some embodiments, the CAR-engager is administered once every 3 weeks (a 21-day cycle) as an infusion over about 30 to about 90 minutes. In some embodiments, the CAR-engager is administered for 5 consecutive days every 21 days and repeated for 8 cycles. [000258] In some embodiments, the CAR-engager is administered as an intravenous infusion over a period of time. Representative infusion times are 30 minutes, 60 minutes, and 90 minutes. In some embodiments, the infusion time is between 30 and 60 minutes. In some embodiments, the first administration is infused into a patient for 90 minutes and subsequent administrations are infused into a patient for 30 minutes. 68 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000259] In some embodiments wherein the CAR-engager is administered subsequent to the CAR immune cells, the treatment method may entail administration of a first course of CAR-engager therapy that is initiated up to about 6 months after administration of the CAR immune cell therapy. The term “effective amount” as used herein refers to a sufficient amount of CAR-engager to provide the desired effect, e.g., the amount of a CAR-engager to bind to a CAR-expressing immune cell. [000260] In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 4 years after the CAR immune cell therapy. In some embodiments, the first course of CAR- engager therapy is initiated at any time up to about 3 years after the CAR immune cell therapy. In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 2 years after the CAR immune cell therapy. In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 1 year after the CAR immune cell therapy. In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 9 months after the CAR immune cell therapy. In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 6 months after the CAR immune cell therapy. [000261] In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 5 months after the CAR immune cell therapy. [000262] In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 4 months after the CAR immune cell therapy. [000263] In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 3 months after the CAR immune cell therapy. [000264] In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 2 months after the CAR immune cell therapy. [000265] In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 1 month after the CAR immune cell therapy. [000266] In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 4 weeks after the CAR immune cell therapy. [000267] In some embodiments, the first course of CAR-engager therapy is initiated at any time up to about 3 weeks after the CAR immune cell therapy. [000268] In some embodiments, the first course of CAR-engager therapy is initiated about 2 weeks after the CAR immune cell therapy. 69 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000269] In some embodiments, the first course of CAR-engager therapy is initiated 2 weeks after the CAR immune cell therapy. [000270] In some embodiments, the first course of CAR-engager therapy entails administering a total of about 1 to about 6 doses (e.g., 2 doses, 3 doses, 4 doses, 5 doses, or 6 doses) of the CAR- engager. In some embodiments, the first course of the CAR-engager therapy entails administration of about 1 dose per week, about 2 doses per week, about 3 doses per week, or about 4 doses per week. [000271] In some embodiments, the first course of CAR-engager therapy is conducted over a period of time of about 1 to about 3 weeks with administration of about 1 to about 3 doses of CAR-engager per week. [000272] The dosage amounts of the CAR-engager administered during the first course of CAR- engager therapy may range from about 1 to about 8 mg/kg of patient body weight. In some embodiments, the dosage (effective amount) of the CAR-engager is about 1 mg/kg, 2 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, or about 8 mg/kg. [000273] In some embodiments, the present methods further include administration of a second, subsequent course of CAR-engager therapy to the subject. In these embodiments, the subject may have relapsed, or is at risk of relapse. The CAR-engager administered in the second course of CAR- engager therapy may be the same as or different from the CAR-engager administered in the first course of CAR-engager therapy. The CAR-engager administered in the second course of CAR- engager therapy may be administered within the same period of time after the CAR immune cell therapy as described above or in the same amounts of time described above, but after the first course of CAR-engager therapy. Combination Therapy [000274] In some embodiments, the present methods may include co-administration of another anti-cancer therapy. The term “co-administered” includes substantially contemporaneous administration, by the same or separate dosage forms, or sequentially, e.g., as part of the same treatment regimen or by way of successive treatment regimens. Thus, if given sequentially, at the onset of administration of the second therapy, the first of the two therapies may still be detectable at effective concentrations at the site of treatment. The sequence and time interval may be determined such that they can act together (e.g., synergistically to provide an increased benefit than if they were administered otherwise). For example, the therapeutics may be administered at the same 70 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 time or sequentially in any order at different points in time; however, if not administered at the same time, they may be administered sufficiently close in time so as to provide the desired therapeutic effect, which may be in a synergistic fashion. Thus, the terms are not limited to the administration of the active agents at exactly the same time. [000275] In some embodiments, the subject may also have had an additional anti-cancer therapy. The additional therapy may be administered (1) prior to CAR immune cell therapy, (2) after the CAR immune cell therapy but before the first course of CAR-E therapy, (3) after the first course of CAR-E therapy but before the second course of CAR-E therapy, or (4) after the second course of CAR-E therapy. In some embodiments, the additional anti-cancer therapy is chemotherapy, radiotherapy, immunotherapy, targeted therapy, pro-apoptotic therapy, or cell cycle regulation therapy, therapy with thalidomide, lenalidomide, bortezomib, and/or melphalan. [000276] Expansion and differentiation agents may also be provided prior to, during, or after administration of the CAR immune cells to increase differentiation, expansion, and/or persistence of the CAR immune cells (e.g., T cells and NK cells). [000277] Anti-cancer agents that may be used in combination with the IL-2 variants and/or chimeric antigen receptor (CAR)-engagers are known in the art. See, e.g., U.S. Patent No.9,101,622 (Section 5.2 thereof). An "anti-cancer" agent is capable of negatively affecting cancer in a subject, for example, by 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 a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of cancerous cells. This process may involve contacting the cancer cells with recipient cells and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cancer cells with a single composition or pharmacological formulation that includes both agents, or by contacting the cancer cells with two distinct compositions or formulations, at the same time, wherein one composition includes recipient cells and the other includes the second agent(s). [000278] In some embodiments, the IL-2 variants and/or CAR-engagers of the present disclosure are used in conjunction with or following prior therapies such as chemotherapeutic, 71 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 radiotherapeutic, immunotherapeutic intervention, targeted therapy, pro-apoptotic therapy, or cell cycle regulation therapy. [000279] In some embodiments, the IL-2 variants and/or CAR-engagers of the present disclosure are used in conjunction with high-dose chemotherapy prior to the administering of the genetically modified immune cells. In some embodiments, bone marrow cells or peripheral blood stem cells are administered subsequent to the high-dose chemotherapy. [000280] In some embodiments, the IL-2 variants and/or CAR-engagers of the present disclosure are used in conjunction with an effective amount of thalidomide, lenalidomide, bortezomib, or a combination thereof. [000281] Additional potentiating treatments that may be used in conjunction with the IL-2 variants and/or CAR-engagers of the present disclosure include melphalan. Melphalan (Alkeran®, Evomela®), an alkylating antineoplastic agent, is used for high-dose conditioning prior to hematopoietic stem cell transplant 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 regiments before bone marrow transplant, and in some cases, malignant melanoma. Melphalan may be administered in pill form by mouth. Typically, in 2 mg doses taken on an empty stomach. In some cases, Melphalan may be administered as an injection or intravenous infusion. Dosing depends on weight, height, disease and disease state, and the subject’s general health. Immunotherapy [000282] Immunotherapy, including immune checkpoint inhibitors may be employed to treat a diagnosed cancer. Immune checkpoint molecules include, for example, 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, e.g., U.S. Patent Application Publications 2012/0321637, 2014/0194442, and 2020/0155520. 72 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 Chemotherapy [000283] Anti-cancer therapies also include a variety of combination therapies with both chemical and radiation-based treatments. Combination chemotherapies include, for example, Abraxane®, altretamine, docetaxel, Herceptin®, methotrexate, Novantrone®, Zoladex®, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, Taxol®, gemcitabien, Navelbine®, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, or any analog or derivative variant of the foregoing and also combinations thereof. Radiotherapy [000284] Anti-cancer therapies also include radiation-based, DNA-damaging treatments. Combination radiotherapies include what are commonly known as gamma-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells which cause a broad range of damage on DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells and will be determined by the attending physician. [000285] Radiotherapy may include external or internal radiation therapy. External radiation therapy involves a radiation source outside the subject’s body and sending the radiation toward the area of the cancer within the body. Internal radiation therapy uses a radioactive substance sealed in needles, seeds, wires, or catheters that are placed directly into or near the cancer. [000286] These and other aspects of the present application will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain embodiments of the application but are not intended to limit its scope, as defined by the claims. EXAMPLES Example 1: Materials and Methods [000287] The cloning and expression of proteins were done following standard approaches. Other procedures, including flow cytometric analyses, BLI imaging, CAR T cells production, cell culture and animal handling were performed following standard protocols as briefly explained below. 73 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000288] 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 production of the products, stable HEK293 cell lines were generated. Accordingly, HEK293 cells were transfected with pPAX2, pVSVG (packaging vectors), and the lentivirus plasmid containing the sequence of interest. The lentivirus was harvested at 48, 72, and 96 hours (h) post transfection, sedimented at 20,000 x g for 2h, and resuspended in optiMEM media. A new batch of HEK293 cells was then subjected to three rounds of transduction with the virus. Cells were allowed to recover in DMEM complete media and were subjected to puromycin selection to retain only cells that integrated the lentivirus plasmid. Cells were then expanded in four 15 cm culture dishes until they reached confluency, washed carefully with PBS, and incubated in serum-free DMEM for 24 to 48h. The supernatant was harvested, and protein expression was confirmed via SDS-PAGE and immunoblotting. Proteins were purified by adsorption onto a nickel nitriloacetic acid (Ni-NTA) metal affinity column. Non- specifically bound proteins were removed by washing with 40 mM imidazole. The imidazole concentration was increased to 250 mM, allowing recovery of the protein of interest. The protein was further purified via size-exclusion chromatography and were stored in 50 mM HEPES buffer, pH 7.5 at -80 °C until use. [000289] Some CAR-engagers were isolated by passage through an affinity chromatography resin, typically in the presence of a neutral phosphate buffer. The affinity chromatography resin was then subjected to an acidic buffer with a pH of about 3 to about 4, thereby washing CAR-engager off of the affinity chromatography resin. A basic buffer may be used to neutralize the acidic buffer, then a tangential flow filtration of the neutralized buffer can be performed with a formulation buffer, to isolate a concentrated and purified solution containing the CAR-engager. [000290] Production of CAR T cells. The CAR construct that binds 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 that binds human BCMA contains an scFv derived from the anti-human BCMA antibody clone MSK54, followed by human 39BB and CD3ζ intracellular signaling domains. The human signaling CAR constructs were transduced into HeLa cells that stably produce gamma-retrovirus pseudotyped with the envelope of the feline endogenous virus (RD114), which has been shown to transduce human hematopoietic cells (HSC) with high efficiency (Ward et al., Mol. Ther. 8(5):804-12 (2003)). High viral titer clones were isolated by 74 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 limiting dilution. The high expression clone was seeded and grown in DMEM complete media containing 10% FBS until 80% confluency. Media was exchanged with RPMI complete media containing 10% FBS. After 24 hours, the virus-containing media was harvested, sterile-filtered using a 0.45 μm PES filter, and utilized for producing CAR T cells. [000291] The production of CAR T cells was adapted from previous studies. See, for example, Li et al., Methods Mol. Biol.1514:111-118 (2017). In brief, whole blood was obtained from apheresis leukoreduction collars of platelet healthy donors, due to a high number of viable white blood cells. The whole blood was subjected to centrifugation through a Ficoll gradient to isolate PBMCs. Whole PBMCs were utilized without selecting for CD8+ T cells. PBMCs were resuspended in RPMI media containing 10% Fetal Bovine Serum (FBS), 200 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 a cell concentration of 4 × 106 cells/mL in 3 mL of media per well in a 6-well plate. After 24 hours, cells are harvested, spun down, and resuspended in the same volume of fresh media with FBS, IL-2, IL- 15, and IL-7 in addition to media harvested from anti-human BCMA CAR gamma-retrovirus producing cells, resulting in PBMC inoculation with the gamma-retrovirus. The PBMCs were then plated at 4 × 106 cells/mL in 3mL into 6-well plates coated with 20 μg retronectin (coated with 1mL of 20 μg/mL retronectin in PBS for 24 hours at 4 °C). The PBMC underwent spinoculation in a centrifuge for 1 h at 2000 × g at 30 °C and cultured at 37 °C. The transduction step was repeated with fresh gamma-retrovirus containing media, cytokines, and spinoculation. Flow cytometry analysis was utilized to assess the transduction efficiency of the CAR transgene using the dsRed reporter gene and recombinant BCMA labeled with AlexaFlour-647. [000292] In vivo experiments. For all experiments NOD/SCID/Gamma (NSG; NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were used due to their immunocompromised status and ability to effectively engraft human cancer cell lines. Cells from the human multiple myeloma OPM2 cell line were used to establish a multiple myeloma mouse model in NSG mice. In vivo experiments were initiated by tail vein intravenous injection of 1 × 106 OPM2 cells expressing GFP and Firefly Luciferase followed by biweekly Bioluminescent Imaging (BLI). Upon effective engraftment after 3 weeks, mice were intravenously injected through the tail vein with CAR T cells. BLI was performed biweekly afterwards to assess tumor burden. Quantification was measured using photons/sec using Aura software. 75 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000293] Organ analysis. At the conclusion of an experiment, the surviving mice were sacrificed and spleen, bone marrow, blood, liver, kidney, and lungs were harvested and weighed. The liver, kidney, and lungs were minced, digested with collagenase (final concentration of 1 μg/mL collagenase), and incubated at 37 °C for 1 h. Spleens were crushed, and bone marrow was aspirated using a 30-gauge insulin needle. All processed cells were pushed through a 70 μm strainer to produce a single cell suspension of cells. Cells were resuspended in 1 mL of ammonium-chloride- potassium (ACK) lysis buffer to deplete the sample of red blood cells for 2 m at room temperature. The resulting single cell suspensions were washed with fluorescence-activated single cell sorting (FACS) buffer of PBS and 0.5% BSA, stained, and analyzed using flow cytometry. [000294] Cell lines and culture. The OPM2 cell line, which endogenously express BCMA, were engineered to express green fluorescent protein (GFP) and Firefly Luciferase. Peripheral Blood Mononuclear Cells (PBMC) were obtained by Ficoll gradient of apheresis leukoreduction collars of platelet healthy donors. HEK293T cells were cultured in complete DMEM (Gibco), 1% L-glutamine (Gibco), 1% non-essential amino acids (NEAA) (Gibco), 1% pyruvate (Gibco), and 1% penicillin and streptomycin (Cytiva), and 10% fetal bovine serum (FBS). OPM2, and PBMCs were cultured in complete 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 in 5% CO2, 95% air-humidified incubators at 37 °C. [000295] Mouse studies. All experiments adhered to the pertinent ethical and safety protocols. The studies were carried out under the oversight of the Dana-Farber Cancer Institute Institutional Animal Care and Use Committee (protocol no.20-006). The xenograft models utilized herein are described in Smith et al., Mol. Ther.26(6):1447-1456 (2018). Briefly, 8- to 12-week-old NOD-scid IL2Rγnull (NSG) and NOD-scid H2-K1null H2-Ab1null H2-D1null IL2Rgnull (NSG-MHC I/II double knockout (DKO)) were either purchased from Jackson Laboratory or bred in-house. All mice were sex and age matched into groups. Xenograft models were established by intravenous injection of 1 × 106 cells of OPM2 or Nalm6 expressing GFP and Luciferase in 200 mL of PBS. Mice received indicated treatments in 300 mL of PBS through intraperitoneal injections. Tumor burden was assessed using the IVIS® Lumina Series III (Perkin Elmer) after intraperitoneal injection of D-Luciferin (150 mg/kg, from a 15 mg/ml solution) at the indicated time. Each mouse was imaged in groups of up to five mice in the supine position at the same time points (5 min). BLI intensity was analyzed by Aura imaging analysis software (Spectral Instruments Imaging). Peripheral blood from mice was obtained 76 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 by submandibular bleeding in an EDTA-coated tube and analyzed for CAR T cell detection and expansion. In brief, volume of the blood was determined in order to calculate absolute values. Samples were then centrifuged, and serum harvested. Cell pellets were resuspended in 500-1000 mL of ACK Lysis Buffer (150 mM NH4Cl, 10mM KHCO3, 0.1 mM EDTA) for 1 minute. Cells were then washed twice in FACS buffer, PBS + 1% Bovine Serum Albumin (BSA). Samples were then stained with anti-CD45-PacificBlue (1:50, Biolegend), anti-CD4-PE/Dazzle594 (1:50, Biolegend), anti-CD8-FITC (1:50, Biolegend), anti-CCR7-AlexaFluor700 (1:50, Biolegend), anti- CD62L-PE (1:50, Biolegend), anti-CD45RO-PerCP/Cy5.5 (1:50, Biolegend), anti-CD45RA- APC/Fire750 (1:50, Biolegend), anti-PD1-BV605 (1:50, Biolegend), anti-HLA-DR-PE/Cy7 (1:50, Biolegend), anti-CD69-BV421 (1:50, Biolegend), and recombinant BCMA-AlexaFlour647 (made in-house). Samples were processed on a Sony SP6800 Spectral Analyzer. Flow rate and acquisition time were noted to calculate absolute values. All experiments were performed in a blinded and randomized fashion. Animals were euthanized at the end of the experiment or when they met prespecified endpoints according to the IACUC protocols. Upon endpoint, major immune organs (spleen and bone marrow) as well as essential organs where possible metastatic lesions can form (liver, lung, and kidney) were harvested, weighed, and analyzed. In brief, spleen was crushed using a plunger and passed through a 40 μm strainer to acquire a single cell suspension. Bone marrow was aspirated using a 30-gauge insulin needle. Liver, lung, and kidney were diced using surgical scissors in 3 mL of Digestion buffer (1mL RPMI + 2mL PBS). Collagenase, Type 1 (Worthington) was added at a final concentration of 100 mg/mL and incubated at 37 °C for 1 hour. The resulting samples were passed through a 40 μm strainer to acquire a single-cell suspension. Samples were then stained with same antibodies used to stain blood samples and analyzed using a Sony SP6800 Spectral Analyzer. Flow rate and acquisition time was noted to calculate absolute values. [000296] Microscopy. Cells were first stained with CellTracker Blue CMAC and seeded on poly- d-lysine-coated coverslips. Subsequently, the samples were incubated with the indicated treatment, labeled with Alexa647, at the specified time and temperature. After fixation using BD Cytofix buffer, the cells were imaged using the Leica THUNDER Imager. The intensity cut-off for the AlexaFluor647 channel was set at 2000 for all images, except for the VHH-muIL2 samples shown on the right (condition 4), for which, the image sensitivity was enhanced by 25-fold (intensity cut- off 80) to visualize the Alexa647 signal. Quantitative analysis of the fluorescence intensity was analyzed by aligning the base 2 logarithm of the ratio of integrated intensity of the membrane to the 77 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 cytoplasm of the cells (see y-axis of FIG.16B). For BCMA-muIL2 and BCMA-CH3 (conditions 1 and 2), only cells with a mean intensity to background ratio above 4 based on the dsRed channel were analyzed as they were identified as CAR+ T cells. For VHH-muIL2, cells with a mean intensity to background ratio greater than 2 based on the Alexa647 channel were selected to eliminate the background artifacts. Image quantification was performed using ImageJ software. [000297] ELISA. ELISA analyses were performed to measure the levels of human T cell-derived cytokines in the serum of mice that received OPM2 cancer cells followed by a low dose of CAR T cells, as shown in FIG. 19A. The cytokines analyzed with the ELISA MAX™ Standard Set (Biolegend) included IFN-γ, GM-CSF, and TNF-α following the manufacturer’s provided protein; however, only IFN-γ was detectable in the collected samples. Serum samples were diluted at a ratio of 1:40. The same plates were used to incubate both the standard samples and the serum samples, and a standard curve was plotted for each cytokine. [000298] pSTAT5 assay. Initially, CAR T were incubated in complete RPMI media without the presence of cytokines (rested) for 24 h. Cells were then stained with CellTrace™ Blue (Invitrogen) or CellTracker™ Red (Invitrogen) for 30 minutes at 37 °C for the indicated conditions. Cells were washed once with complete RPMI + 10% FBS media. CAR T cells stained with CellTracker™ Red were blocked using recombinant BCMA-CH3 (100 nM) for 20 minutes on ice while cells stained with CellTrace™ Blue were not blocked. Cells were then washed once with complete RPMI + 10% FBS media followed by seeding of approximately 2×105 cells (either the two separately stained cells co-cultured or separately cultured) per well of a 96-well plate in the presence of serial dilutions of treatments or cytokine controls at 37 °C. After 5 minutes of incubation, cells were immediately fixed with 1.5% formaldehyde in PBS for 10 minutes at room temperature. Cells were then permeabilized with ice cold 100% methanol for 20 minutes on ice 4 °C. Fixed and permeabilized cells were washed twice with FACS buffer, PBS + 1% Bovine Serum Albumin (BSA) and then incubated with anti- STAT5 pY694-PE/Cy7 (1:200, Biolegend) for 30 minutes on ice. Cells were then washed twice with FACS buffer and analyzed using the BD FACSCanto™ II (Becton Dickinson). Flow cytometry data was analyzed using FlowJo™ (Becton Dickinson) and dose-response curves were fitted to a logistic sigmoidal model and half-maximal effective concentration (EC50) and 95% confidence intervals were calculated using Prism data analysis software (GraphPad). [000299] scRNA-seq Analysis. Gene counts for each sample were obtained using the CellRanger multi-function through 10x Cloud computing and pooled using the CellRanger aggr function to 78 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 produce an .h5 file that could be loaded into R as a Seurat object. Seurat pipeline was performed for QC filtering (number of total counts < 20’000, molecular identifiers [nUMI] < 6,000 and ribosomal RNA <10% of the reads). Data was then scaled and normalized using scTransform, and original samples were traced back using the demultiplexing function HTODemux(). Phenotype of the cells was then determined using projection of our sample to the Seurat pbmc_multimodal dataset using the FindTransferAnchors() and FindQuery() workflow. The FindMarkers() function was used to find differentially expressed genes between chosen groups. The heatmaps were generated with the DoHeatmap() function with a downsampling of 500 cells. Gene Scores were obtained by creating a list of the genes of interest which was given as the feature for the function AddModuleScore(). [000300] Preparing samples for bulk RNA-sequencing. CAR T cells with and without the intracellular domain were incubated in complete RPMI media without the presence of cytokines for 24 hours. A 96 well plate was seeded with approximately 2×105 cells/well in the presence of 10 nM treatment or controls at 37 °C for 2 hours. Cells were washed with FACS buffer and left at 37 °C for 2 or 22 hours (4 hour and 24-hour timepoints). Cells were stained with anti-CD8-FITC (1:50, Biolegend), anti-CD4-PE/Dazzle594™ (1:50, Biolegend) and Alexa647 labeled BCMA and sorted on the Sony Sorter MA900.10,000 CD4 and 10,000 CD8 cells were sorted per condition. SMART- Seq mRNA library preparation kit (Takara Bio) was utilized to generate mRNA libraries, with each replicate tagged with a unique index. Libraries were pooled and sequenced through Novogene at a sequencing depth of 20 million reads per sample. [000301] Bulk RNA Seq-Analysis. Gene counts for the samples were obtained by trimming the fastQ files and transcript quantification using the RNAlysis software. Gene names were obtained from the homo sapiens ensembl database with biomaRt, and differential expression between different conditions was determined using the DESeq2 pipeline. Volcano plots were drawn using the EnhancedVolcano library, with a cutoffs at logFC > |2| and p-value > 10-6. Heatmaps were generated using the pheatmap library. GSEA was performed using the pipeline of the fgsea package with the ranking metric being -log10(p-value)*sign (fold change) and basing the computations on the hallmark pathways of the MSigDB collection. Example 2: BCMA-containing CAR-engager in vitro characterization. [000302] A Fusion protein consisting of the human BCMA ectodomain was fused with a two low- affinity mutated human IL-2 (muIL2) domains, and to improve pharmacokinetics and enhance stability, the CH3 domain (Feige et al., Trends Biochem. Sci.35(4):189-198 (2010)) of human IgG1 79 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 (approximately 14 kDa in size) was incorporated between the antigen and the muIL2 (FIG. 2B) (Quayle et al., Clin. Cancer Res.26(8):1953-1964 (2020)). Consequently, the BCMA-muIL2 CAR- engager preferentially delivers the low-affinity IL-2 to the surface of CAR T cells through antigen- to-CAR specific binding, minimizing effects on normal T cells, Tregs, or systemic toxicity. [000303] It was recently demonstrated that IL-2 induces an alternative differentiation pathway of T cells, resulting in the generation of distinct “better effector” CD8+ T cells (Hashimoto et al., Nature 610(7930):173-181 (2022)). This process may rely, at least in part, on IL-2 binding to IL- 2Rα. Additionally, IL-2Rβγ-biased agonists may drive T cells towards a terminally differentiated state (Codarri et al., Nature 610(7930):161-172 (2022)). CAR-engagers may be able to overcome the need for IL-2Rα in the alternative differentiation pathway by anchoring the low-affinity IL-2 on the surface of the CAR T cells via the antigen-to-CAR binding, thereby promoting the generation of memory CAR T cells. A potential synergistic effect between CAR signaling and IL-2 signaling may also exist. [000304] To assess the binding affinity of BCMA-containing CAR-engagers, flow cytometric analysis was performed by staining BCMA CAR T cells with varying concentrations of the BCMA CAR-engagers. An EC50 of about 0.21 nM was observed for the BCMA CAR-engager, which was comparable to that of a dimeric BCMA lacking the muIL2 (BCMA-CH3) (FIG. 2E), indicating binding was mainly due to the BCMA ectodomain rather than the muIL2. FIG.2E shows the dose- dependent staining of BCMA CAR T cells with the CAR-engager using flow cytometry (n=3 for each point), non-transduced T cells were used as controls, a secondary Alexa647-labeled anti-FLAG antibody was used for staining. Error bars represent mean with 95% confidence interval. Minimal binding of BCMA CAR-engager to non-transduced T cells was observed as well as minimal binding of VHH-muIL2, a control construct which replaces the BCMA ectodomain with an irrelevant nanobody (VHH) in the CAR-engager CH3-muIL2 construct. This observation further suggests that the binding of the CAR-engager to CAR T cells is primarily driven by the BCMA antigen, and that the muIL2 exhibits weak binding to both CAR T and non-transduced T cells. The BCMA-muIL2 CAR-engager did not exhibit binding to any immune cell populations in human peripheral blood mononuclear cells (PBMCs) (FIGs.15A – 15B). [000305] Next, the functional effects of BCMA CAR-engager on BCMA CAR T cells were evaluated. After a 24-hour resting period without cytokines, BCMA CAR T cells were incubated with varying concentrations of the BCMA-muIL2 CAR-engager for 24 h, followed by assessment 80 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 of the expression of the CD69 activation marker (Cibrián and Sánchez-Madrid, Eur. J. Immunol. 47(6):946-953 (2017)) using flow cytometric analysis. The results demonstrated a dose-dependent and selective increase in CD69 expression on CAR T cells (FIG.2F), while no effect was observed on non-transduced T cells (FIG. 2G). Moreover, BCMA CAR-engager treatment resulted in a significantly higher increase in CD69 expression compared to VHH-muIL2, BCMA-CH3, or their combination suggesting the observed effect is only evident when the low-affinity IL-2 is fused to the antigen. An unpaired t-test indicated a statistically significant (P ≤ 0.0001) increase in activation in the BCMA-muIL2 treatment group compared to VHH-muIL2, BCMA-CH3, or their combination control groups at a concentration of 0.1 nM or higher (error bars represent mean with 95% confidence interval) (FIG.2F). Zero treatment and CD3/CD28 activation in FIG.2G were used as negative and positive controls, respectively (error bars represent mean with 95% confidence interval; the difference between CD3/CD28 activation to BCMA-CH3-muIL2 treatment was p < 0.0001). [000306] BCMA CAR-E does not inhibit killing efficacy of BCMA CAR T-cells. Since both the CAR-engagers and cancer antigens bind the CAR, the potential inhibitory effect of BCMA CAR- engager on the killing activity of BCMA CAR T cells was investigated. To investigate, a killing assay was conducted using BCMA CAR T cells and patient-derived BCMA+ OPM2 cancer cells in the presence of varying concentrations of the BCMA CAR-engager. Remarkably, the results demonstrated no inhibition of killing even at the highest tested concentration (100 nM of the CAR- engager) (FIG.2H). OPM2 cells were co-incubated with BCMA CAR T cells (shown in red) or non- transduced T cells (shown in gray) (E:T ratio 1:1; 30,000 cells of each) in the presence of varying concentrations of the BCMA-muIL2 CAR-E treatment. Live (PI-) OPM2 cells were counted 48 hours later, with an N of 3 for each of the experiments. Error bars in FIG.2H represent mean with standard deviation. Without being bound by theory, this finding might be attributed to the reversibility of CAR-engager binding to CAR, while the killing process, which involves the clustering effect and synapse formation between CAR and cancer antigen, is an irreversible event. Moreover, the binding avidity of CAR to membrane-bound BCMA might surpass the CAR binding avidity to the soluble antigen, contributing to this result. Notably, multiple myeloma patients exhibit high levels of soluble BCMA in their circulation due to shedding caused by γ-secretase (Laurent et al., Nat. Commun. 6:73331-12 (2015)). Despite this, BCMA CAR T cells produced remarkable initial responses in patients suggesting that the soluble BCMA antigen does not inhibit the activity 81 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 of CAR T cells. The experimental findings disclosed herein are consistent with these earlier findings. [000307] The BCMA CAR-engager selectively induces STAT5 activity in CAR T cells through the cis-delivery of the low-affinity IL-2 to the same targeted CAR T cells. IL-2 is known to exhibit strong activity on T cells, and the phosphorylation of STAT5 both serves as a reliable indicator of IL-2/IL-2R engagement and correlates with downstream effects such as phenotypic marker expression and cell proliferation (Jones et al., J. Immunol.205(7):1721-1730 (2020)). To evaluate the impact of BCMA CAR-engager on STAT5 activity, BCMA CAR T cells were exposed to varying concentrations of the BCMA CAR-engager. Following a 5-minute incubation at 37 °C, cells were fixed and stained for pY694 STAT5. The results revealed that the BCMA-muIL2 CAR- engager induced phosphorylation of STAT5 in CAR T cells with an EC50 of ~0.014 nM (FIG.2I). In contrast, the VHH-muIL2 control required a higher concentration (EC50 = 3.9 nM) to induce STAT5 phosphorylation, indicating that the BCMA-mediated delivery of the low-affinity IL-2 to CAR T cell surface significantly enhances the sensitivity of the muIL-2 by over 200-fold. Wild-type IL-2 exhibited a lower EC50 (approximately 0.001 nM) suggesting a difference in signaling kinetics. The two-step process involved in STAT5 activity mediated by the BCMA-muIL2 CAR-engager involves: (i) binding of the antigen-to-CAR on T cell surfaces and (ii) subsequent interaction of the low-affinity IL-2 with nearby IL-2R, which, without being bound by theory, may be the reason for the measured difference. In contrast, wild-type IL-2 requires only binding to IL-2R, enabling it to more rapidly induce STAT5 activity. The VHH-muIL2 can activate STAT5 solely through the low- affinity IL-2, which may explain its requirement for higher concentrations to induce STAT5 activity in T cells. [000308] BCMA CAR T cells pre-blocked with BCMA-CH3 showed a significant decrease in pSTAT5 levels to the same degree as control VHH-muIL2, validating that the potency of the CAR- engager is mediated by antigen-to-CAR binding (FIG.2I). To determine whether the CAR-engager binding to target cell can result in STAT5 signaling in an adjacent cell (trans-activation), non- blocked and pre-blocked BCMA CAR T cells were co-cultured in the presence of varying concentrations of CAR-engager. Pre-blocked CAR T cells had lower pSTAT5 levels compared to their co-cultured non-blocked CAR T cells, indicating that CAR-engager affects the targeted CAR T cells (cis-activation) but not adjacent cells. BCMA CAR T cells were treated with the indicated treatments for 5 min at 37 °C followed by STAT5 phosphorylation assessment. For the pre-blocking 82 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 experiments, the BCMA CAR T cells were treated with BCMA-CH3 (100 nM) for 20 min at 4 °C prior to the 5 min exposure to the CAR-engager treatment at 37 °C (n=3 for each condition). Error bars in FIG. 2I represent mean with standard deviation. Taken together, the analysis of STAT5 activity supports the notion that the BCMA CAR-engager exerts its influence on targeted CAR T cells through the cis delivery of the low-affinity IL-2, with the effect mediated via antigen-to-CAR binding. Example 3: The CAR-engager immune cell effector domain stimulates T cells independent of a CAR. [000309] To show that CAR-engager immune cell effector domains stimulate immune cells, the following experiment was performed. In this experiment, the experimental setup of which is illustrated in FIG.3A, peripheral blood mononuclear cells (PBMCs) were stimulated with anti-CD3, anti-CD28, IL-2, IL-7, and IL-15 to produce activated T cells, which were not transduced with any exogenous transgenes. The activated T cells were treated with teceleukin (recombinant human IL-2 without glycosyl units), a CAR-engager that contains a N-terminal ectodomain that binds BCMA (~7 kDa), a CH3 domain (~14 kDa), and two repeats of the weak affinity variant of IL-2 immune cell effector domain, with the overall structure of BCMA-CH3-muIL2-muIL2 and referred herein as BCMA-muIL2, or a CAR-engager that contains an ectodomain that binds BCMA, a CH3 domain, and the Neo-2/15 immune cell effector domain, with the overall structure of BCMA-CH3-Neo-2/15, referred to herein as BCMA-Neo-2/15, for 4 days. After treatment, T cells were counted and stained with carboxyfluorescein succinimidyl ester (CFSE) and analyzed for mean fluorescence intensity (MFI) of CFSE to determine T cell division. [000310] T cells treated with the CAR-engagers BCMA-muIL2 or BCMA-Neo-2/15 effected T cell counts and division (CFSE staining), similar to teceleukin, which is known to activate T cells. However, the CAR-engager treatment resulted in less sensitivity as compared to the teceleukin treatment. Systemic administration of IL-2 is associated with severe side effects (Rosenberg, J. Immunol. 192(12):5451-5458 (2014); Dutcher et al., J. Immunother. Cancer. 2(1):26 pp. 1-23 (2014); Pachella et al., J. Adv. Pract. Oncol. 6(3):212-221 (2015)), including vascular leak syndrome and preferential expansion of CD4+CD25+ regulatory T (Treg) cells, that are known to result in immune suppression. The presently disclosed results indicate that the CAR-engagers do not activate normal T cells when used at low concentrations, and that the stimulatory immune cell 83 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 effector domains retain their normal function when attached to an ectodomain of a CAR-engager to activate T cells. Example 4: CAR-engagers activate CAR T cells specifically through the ectodomain [000311] To show that CAR-engager immune cell effector domains stimulate immune cells, the following experiment was performed. As illustrated in FIG.4A, PBMCs were stimulated with anti- CD3, anti-CD28, IL-2, IL-7, and IL-15 to produce activated T cells, which were then transduced with a vector containing a CAR. The activated CAR-expressing T cells (CAR T cells) were rested for 24 hours and then treated with CAR-engagers containing an immune cell effector domain or CAR-engagers lacking an immune cell effector domain as an ectodomain control. [000312] CAR-engagers containing BCMA ectodomain, a CH3 domain, and containing either 4- 1BBL (BCMA-41BBL), weak affinity IL-2 (BCMA-muIL2), or Neo-2/15 (BCMA-Neo-2/15) immune cell effector domains were tested for T cell activation. To test the ectodomain specificity of the CAR-engagers for CAR T cells, as a control, irrelevant nanobody that binds the intracellular protein UBC6E (VHH6E) fused to a CH3 domain, and either 4-1BBL (VHH6E-41BBL) or weak affinity IL-2 (VHH6E-muIL2) were tested for T cell activation. An additional ectodomain specificity control containing a nanobody that binds FN1 (clone NJB2, abbreviated NJB2-VHH) fused to a CH3 domain, and the Neo-2/15 stimulatory (NJB2-VHH-Neo-2/15) was tested for T cell activation. The ectodomain specificity controls have similar overall structure as the CAR-engagers used in this experiment (protein domain-CH3-muIL2-muIL2 or protein domain-CH3-Neo-2/15). Ectodomain specificity controls and CAR-engagers were incubated with CAR T cells for 10 hours, and the cells were stained for CD69 as an activation marker and measured by flow cytometry. [000313] All of the BCMA ectodomain CAR-engagers induced expression of CD69 in CAR T cells (FIG.4B). The BCMA-CH3-Neo-2/15 had the lowest threshold of induced expression of CD69 in CAR T cells (0.01 nM CAR-engager). The BCMA-CH3 protein (lacking an immune cell effector domain) had minimal effect on CAR T cell expression of CD69 at the highest concentration tested, 10 nM of BCMA-CH3 protein. None of the ectodomain specificity control proteins induced CAR T cell expression of CD69. These results indicate that CAR-engagers containing a cancer antigen ectodomain specifically activate CAR T cells that expresses a CAR that recognizes CAR-engager’s cancer antigen ectodomain. Example 5: CAR-engagers stimulate CAR T cell killing target cells 84 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000314] To show that CAR-engagers do not inhibit CAR T l cell killing, the following experiment was performed. CAR T cells were produced as described above and co-incubated with CAR- engagers and BCMA+ multiple myeloma cancer cells. CAR T cells were incubated with OPM2 BCMA+ cells at an E:T ratio of 1:1 for 1 day and analyzed for target cell survival as compared to target cells without T cell coincubation (FIG.5A). [000315] BCMA ectodomain CAR-engagers with either a muIL2 (BCMA-CH3-muIL2) or a 4- 1BBL (BCMA-CH3-41BBL) immune cell effector domain did not inhibit killing of OPM2 cells (FIG.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 extend CAR T cell persistence in vivo [000316] To show that CAR-engagers reduce tumor burden, extend CAR T cell in vivo persistence, and extend survival, the following experiment was performed. 1 × 106 OPM2 BCMA+ multiple myeloma cancer cells were intravenously (i.v.) injected into NOD-scid IL2Rγnull (NSG) mice 10 days before infusing a suboptimal dose of 5 × 105 anti-BCMA CAR T cells by i.v. injection. After CAR T cell infusion, mice were treated twice weekly for two weeks, followed by once weekly with 200 μg/mouse of CAR-engager containing a BCMA ectodomain, a CH3 domain, and two weak affinity IL-2 immune cell effector domains (BCMA-CH3-muIL2-muIL2) by intraperitoneal (i.p.) injection (FIG.6A). [000317] Mice were subjected to bioluminescent imaging (BLI) for luciferase (indicating tumor burden of luciferase+ OPM2 cells) on days indicated in FIGs.6B – 6D. Control mice that received OPM2 cells and no-CAR T cell infusion had progressively more tumor burden during the experiment and reached a humane end point on days 39 and 46. Mice that received OPM2 cells and the suboptimal dose of CAR T cells had controlled tumor growth until day 32, when they also experienced progressively more tumor burden during the experiment and reached a humane end point on day 46. Mice that received OPM2 cells, CAR T cells, and CAR-engager therapy had reduced tumor burden (FIGs. 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 growth, due to formation of a solid tumor close to the eye 85 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 and reached a humane end point on day 42. The remaining two mice completely cleared OPM2 tumor cells, as no signal was detected by imaging and survived the experiment. [000318] Next, the in vivo persistence of OPM2 and CAR T cells were analyzed in these mice by flow cytometry. One control mouse (OPM2 cells with no-CAR T cell infusion) was sacrificed on day 46, two CAR-only mice were sacrificed on day 46, and one CAR T cell and CAR-engager treated mouse was sacrificed on day 42. Sacrificed mice were analyzed for GFP+ OPM2 cells (FIGs. 7A-7C) and CD45+ CAR+ T cells (FIGs. 8A-8C) in the blood, spleen, lymph node, bone marrow and lung. The CAR T cell and CAR-engager treated mouse was also analyzed for GFP+ OPM2 cells and CD45+ CAR+ T cells in the eye tumor site. [000319] FIGs. 7A – 7C show flow cytometry with GFP on the y-axis. GFP+ OPM2 cells were detected at similar levels in the bone marrow, lung (FIG.7B), and liver (FIG.7C) of the no-CAR control mouse and the CAR-only mice. One CAR-only mouse had significant levels of OPM2 cells in the blood and spleen (FIG.7A). [000320] The one mouse that received CAR T cell and CAR-engager treatment that developed an eye tumor had no to little OPM2 cells in the blood or spleen (FIG.7A), bone marrow or lung (FIG 7B), and liver (FIG. 7C). This mouse had more OPM2 cells in the kidney (3.18% of GFP+ cells), and the majority of cells in the eye tumor site were OPM2 cells (96.5% of GFP+ cells). [000321] FIGs. 8A – 8C show flow cytometry with anti-CD45 on the y-axis and BCMA+-CH3 tagged with Alexa FlourTM 647 (AF647) on the x-axis. CD45+ CAR+ T cells only persisted in CAR T cell and CAR-engager treated mice. CAR-only treated mice had little to no CD45+ cells in all organs tested (FIGs.8A-8C). However, CAR T cell + CAR-engager treated mice had CD45+ cells that also stained positive for the BCMA cancer antigen (which is also the CAR binding target) tagged with AF647, as shown on the x-axis. CD45+ AF647+ double positive CAR T cells were detected in the blood, spleen, and lymph node (FIG. 8A), bone marrow and lung (FIG. 8B), and liver and kidney (FIG.8C). CD45+ single positive cells were only detected in large numbers in liver and kidney (FIG.8C). Little to no CD45+ AF647+ double positive CAR T cells were detected in the eye tumor site (FIG.8C). These results indicated that CAR-engagers reduce tumor burden, extend CAR T cell in vivo persistence, and extend survival. Example 7: CAR-engager fate [000322] The CAR-engagers bind CAR T cells at the cell surface at 4 °C, and slowly internalize at 37 °C. Internalization of CAR-engager was assessed using fluorescently labeled BCMA-muIL2 86 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 CAR-engager. BCMA CAR T cells were exposed to AlexaFluor647-labeled BCMA-muIL2, BCMA-CH3, or VHH-muIL2 at a concentration of 2 nM. The cells were incubated at either 4 °C or 37 °C for various time intervals, followed by fixation and subsequent microscopy imaging. It was observed that the control VHH-muIL2 underwent rapid internalization within 30 minutes at 37 °C, whereas the internalization of BCMA-muIL2 CAR-engager was significantly slower (FIGs.16A – 16C). The internalization rate of BCMA-CH3 was similarly slow, even slower than that of BCMA- muIL2 CAR-engager. In FIG. 2C, the CAR and dsRed transcripts were encoded within the transgene, and thus the dsRed signal reflects the expression level of CAR. All imaged cells with mean intensities higher than background were reported. For the dsRed channel, the cytoplasm mean intensity was reported, as the dsRed is expressed inside the cell, whereas for the AlexaFluor 647 channel, the mean intensity for the entire cell was measured. [000323] The CAR-engager rapidly clears from the circulation. Pulsing CAR T-cells with the CAR- engager treatment, where pulsing involves periods of stimulation followed by periods of resting, is superior to prolonged exposure to CAR-engagers as extended exposure can lead to exhaustion or the generation of terminally differentiated CAR T cells. A CAR-engager with a short circulation half-life can be more effective at expanding CAR T cells, driving generation of memory CAR T cells, decrease potential competition with tumor antigen for CAR binding, and enhanced safety profile in patients. Therefore, the CH3 domain of IgG1 was used in the CAR-engager platform. Pharmacokinetic studies illustrated that the circulatory half-life of the CAR-engager was short (1- 1.5 hours) (FIG. 9A). NSG mice were administered 8 mg/kg of BCMA-muIL2 CAR-engager (delivered i.p., N=3 mice). Blood samples were collected via tail-vein puncture at five different time points (30 min, 2 h, 8 h, 24 h, 48 h) post-administration. The sera were then obtained by centrifugation and used for the subsequent analysis. An ELISA was performed to determine the concentration of the treatments in the sera. The ELISA plates were coated with 5 μg/ml anti-His6 antibody overnight, followed by incubation with the sera for 2 h at room temperature. An anti-FLAG HRP antibody was next used for the detection; the CAR-engager was engineered to have FLAG and His6 tags at the C-terminus. Based on the collected five time points, the initial concentration of the treatment in the sera was estimated to be 20% higher than the first (30 min) collected time point. The BCMA CAR-E was >90% and >99% cleared from the circulation in 8 h and 24 h, respectively. The circulating half-life was estimated to be about 1.5 hours for the BCMA CAR-engager. Error bars represent mean with standard deviation. 87 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000324] The BCMA CAR-engager enhances activity and persistence of CAR T-cells in a multiple myeloma (MM) model. An MM xenograft mouse model with OPM2 cells engrafted in immunocompromised NOD-SCID IL-2Rγnull (NSG) mice was utilized. Accordingly, NSG mice were intravenously injected with OPM2 cells (human MM, 1 million cells) via the tail vein. Two weeks after OPM2 cell injection, freshly prepared BCMA CAR T cells (0.5 million CAR+ cells containing an 39BB-CD3 ^ CAR construct) were intravenously administered. A cohort of mice received the BCMA-muIL2 CAR-engager treatment (FIG.9B). NSG mice (n=5) were injected with OPM2 (human MM) cells followed by BCMA CAR (human) T cell administration according to the schedule. BCMA-muIL2 CAR-E treatment (200 μg) was administered twice per week for two weeks, followed by once per week until the endpoint. After one month or longer, mice were euthanized, and flow cytometric analyses were performed on the harvested organs. These results revealed a significant expansion of CAR T cells in the spleen and bone marrow of the BCMA- muIL2 CAR-engager-treated group, demonstrating over a 100-fold selective expansion of CAR T cells compared to non-treated animals that only received CAR T cells in the spleen (FIG.9C, left panel) and bone marrow (FIG.9C, right panel). These experiments were replicated multiple times with similar outcomes (FIG.9D and FIGs.17A – 17C; n=12 for CAR T cells only, n=22 for CAR T cells plus CAR-engager treatment). [000325] BCMA CAR T cells were detected by co-staining with an anti-human CD45 antibody and Alexa647-labeled BCMA antigen. Similar results were obtained in repeated experiments. Additional control cohorts received VHH-muIL2 treatment with the same dose and schedule as BCMA-muIL2. FIG.9D shows pooled data from these experiments. The difference between CAR + BCMA-muIL2 CAR-E and CAR + PBS or CAR + VHH-muIL2 was significant in the spleen and bone marrow (p ≤ 0.01 in the Spleen, p ≤ 0.01 between CAR + BCMA-muIL2 and CAR + PBS in the bone marrow, and p = 0.025 between CAR + BCMA-muIL2 and CAR VHH-muIL2 in the bone marrow). Data were analyzed by group mean comparisons using one-way ANOVA and subsequent Tukey post-hoc analysis. Individual flow graphs for the pooled data are shown in FIGs.17A – 17C. Error bars represent mean with standard deviation. [000326] The control group that received CAR T cells plus VHH-muIL2 treatment (n=7) did not exhibit significant expansion or persistence of CAR T cells compared to the control group that received only CAR T cells without treatment. These results show that the BCMA-muIL2 CAR- engager can expand CAR T cells in vivo. Further analysis revealed that the BCMA-muIL2 treatment 88 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 had a more pronounced effect on CD8+ CAR T cells specifically, resulting in an unexpected significant increase in their proportion from the initial ~30% to ~70% of the total CD4+ and CD8+ CAR T cell population (FIG.9E). The cohorts receiving only CAR T cells or CAR T cells with the VHH-muIL2 control treatment did not yield a sufficient number of persisting CAR cells for a similar analysis. Data were analyzed by group mean comparisons using one-way ANOVA and subsequent Tukey post-hoc analysis. Error bars represent mean with standard deviation. Example 8: BCMA CAR-engager treatment enables CAR T therapy with low-dose of CAR T cells [000327] To further demonstrate the effectiveness of CAR-engager treatment and clearance of tumor cells by CAR T cells, a similar protocol as described above was conducted. However, in this study, a lower dose of only 100,000 CAR T cells was utilized (FIG. 10A). All mice treated with BCMA-muIL2 CAR-engager achieved complete tumor clearance (5/5), whereas not a single control mouse receiving either only CAR T cells (n=4) or CAR T cells combined with VHH-muIL2 treatment (n=4) was able to eliminate the tumors (FIGs.10B-10C). [000328] Analysis of blood samples collected at various time points revealed a substantial expansion of CAR T cells in the circulation following CAR-engager treatment, with the peak expansion observed at week 4 (FIG. 10D). Flow cytometric analyses of blood samples revealed robust expansion of CAR T cells in the treatment group compared to PBS or VHH-muIL2 cohorts. In contrast, the VHH-muIL2 treatment, despite slightly enhancing the initial response, did not induce a significant expansion of CAR T cells. Data was analyzed with two-way ANOVA for day 7, 14 and 21. Once all mice in PBS cohort were euthanized, BCMA-muIL2 and VHH-muIL2 comparisons were performed with multiple Mann-Whitney tests on days 28 and 35. Error bars represent mean with S.E.M. This expansion correlated to the levels of IFN-γ detected in the circulation (FIGs. 19A – 19C). Additionally, the treatment facilitated the generation of memory CAR T cells, demonstrating long-lasting effects (FIG. 10E and FIGs. 19A – 19C). *P<0.05, **P<0.01. Error bars represent mean with S.E.M. [000329] The mice treated with CAR-engager exhibited no signs of toxicity based on clinical observations and weight measurements (FIG. 10H). Subsequent analysis conducted two months after CAR T cell injection demonstrated a substantial presence of CAR T cells, including memory CAR T cells, in the CAR-engager treated mice (FIGs.10F-10J, FIGs.18A – 18C, and FIGs.19A – 19C). In the CAR + PBS group, CAR T cells were detected in the spleen; however, these mice succumbed to tumor growth at around 20 days post-CAR T cell injection. The BCMA-muIL2 89 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 treatment had also increased the presence of CAR T cells in bone marrow compared to PBS or VHH-muIL2 cohorts, but the difference was less significant than spleen. Data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test. *P<0.05, ***P<0.001, ****P<0.0001. Individual flow data are shown in FIGs.18A – 18C. Error bars represent mean with S.E.M. Data in FIG.10I show that persisting CAR T cells persisted in the spleens of mice that received the BCMA- muIL2 CAR-engager and exhibited a CCR7+CD45RA+CD62L+ stem-cell memory phenotype, which was absent in the CAR + VHH-muIL2 or CAR + PBS cohorts. [000330] tSNE analysis was based on surface marker expression of CD8a, CD4, CD45, CD45RA, CD45RO, CD62L, CD69, PD-1, HLA-DR, CCR7, and BCMA-CAR and revealed the presence of distinct memory T cell populations. CAR T cells were detected in the bone marrow of the VHH- muIL2-treated group but not in the spleen. In the BCMA-muIL2 group, persisting CAR T cells were predominantly CD8 T cells, while the majority of bone marrow CAR T cells in the VHH-muIL2 group were CD4 T cells. Further analyses are shown in FIG. 19A – 19C. The Flt-SNE mapping shown in FIG. 19C is of CAR T cells derived from the PBS, BCMA-muIL2 and VHH-muIL2 treated mice as shown in FIG. 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 3 PBS mice, 3 BCMA-muIL2 mice and 4 VHH-muIL2 mice were analyzed by flow cytometry. CD45+, ⍺-BCMA-CAR+ immune cells from the mice were concatenated to form a total of ~9800 (PBS spleen), ~8100 (BCMA-muIL2 spleen), ~7600 (PBS bone marrow), ~14200 (BCMA-muIL2 bone marrow), ~1420 (VHH-muIL2 Bone Marrow). The entire high dimensional dataset was merged to create a single Flt-SNE map for each condition with the signal strength of various phenotypic markers defining specific immune phenotypes expressed with a blue-green-yellow-red continuous color scale. FltSNE was conducted with the following parameters: max iterations: 1000, theta: 0.5, learning rate: 200, perplexity: 20. There were inadequate numbers of CAR T cells in VHH-muIL2 cohort spleen to conduct Flt-SNE. To enhance visibility, the dots representing the VHH-muIL2 bone marrow samples were enlarged, as fewer cells were detectable in these mice. In the BCMA-muIL2 treated mice, the majority of CAR+ cells were CD8+ cells, while in the VHH-muIL2 samples, CD4+ cells constituted the majority of CAR+ cells. Notably, CAR+ cells in the CAR+PBS cohort exhibited low or no expression of CD45RA, CD45RO, 90 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 or CD62L, whereas the BCMA-muIL2 treated mice showed a CAR+ population with elevated expression levels of these memory markers. [000331] Thus, the treatment not only facilitates robust proliferation and eradication of tumor cells using low doses of CAR T cells but also promotes the development of long-lasting memory cells, demonstrating the efficacy of BCMA-muIL2 CAR-engager treatment in enhancing the clearance of tumor cells by CAR T cells, and generation of long-lasting memory cells. Example 9: Persisting CAR T cells treated with CAR-engager treatment remain functional three months post infusion [000332] Mice received 1 million OPM2 cells followed by 0.5 million BCMA CAR T cells (FIG. 11A). One group of mice received CAR-engager treatment, administered twice per week for two weeks, followed by once per week for an additional two weeks (6 doses, 200 μg per dose on days 4, 10, 14, 17, 21, and 28; n=5). The control group received VHH-muIL2 treatment at the same dosage and schedule (n=5), while an additional control cohort received only tumor cells (n=3). All mice receiving CAR T cells exhibited an initial response compared to control mice without CAR T cells (FIG.11B). All CAR-engager treated mice (5 out of 5) and 3 out of 5 mice in the VHH-muIL2 group survived for over three months, which encompassed the duration of the experiment. One VHH-muIL2 mouse died in about a month, and a second mouse succumbed to cancer cell relapse with liver metastasis (FIG. 11B, day 77). The surviving mice were euthanized three months post- injection of CAR T cells, and the splenocytes and bone marrow cells were analyzed to assess the presence of CAR T cells. Remarkably, CAR-engager-treated mice exhibited a significant abundance of CAR T cells homing and persisting in the bone marrow and spleen compared to mice receiving CAR T cells with VHH-muIL2 treatment (FIG.11C). Given the two-month period of no treatment before the mice were sacrificed, these results further suggest that the treatment facilitated the generation of memory cells among CAR T cells. [000333] To demonstrate the functionality of the persisting CAR T cells in CAR-engager-treated mice, an in vitro killing assay was performed using the BCMA CAR T cells harvested from bone marrow and spleen. Bone marrow and splenocytes were analyzed via flow cytometry to detect and quantify CAR-expressing T cells and the bone marrow cells or splenocytes were co-incubated with OPM2 target cells at various E:T ratios (1:1 and 2:1) based on CAR-expressing cells. Survival was determined 24, 48, and 72 hours later using flow cytometric analysis. The three-month-old CAR T cells demonstrated efficient killing of tumor cells and long-term functionality (FIG.11D). Only one 91 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 of the VHH-muIL2 treated mice exhibited sufficient CAR T cells to perform a similar killing assay, and while it exhibited tumor cell killing, the efficiency was lower than that observed in CAR- engager treated CAR T cells (FIG. 11D) (error bars represent mean with standard deviation). Therefore, CAR-engager treatment robustly expands and drives the persistence of CAR T cells while maintaining their killing potential. [000334] To further characterize the phenotype of these persistent CAR T cells, flow cytometric analyses were conducted to evaluate the expression of a series of T cell markers (CD45, BCMA CAR, CD4, CD8, CD62L, CD45RO, CD45RA, CD69, and PD-1). In order to facilitate interpretation, a t-SNE mapping of splenocytes, and bone marrow cells was generated (FIG.11E). 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) on splenocytes and bone marrow from the five BCMA-muIL2 treated mice were analyzed by flow cytometry. CD45+, CD8+, ⍺-BCMA- CAR+ cells from the five mice were concatenated to form a total of ~17600 (spleen) and ~10800 (bone marrow) cells. The entire high dimensional dataset (excluding the CD45, CD8, and CD4 parameters) was merged to create a single tSNE map with the signal strength of six phenotypic markers defining specific immune phenotypes expressed with a blue-green-yellow-red continuous color scale. tSNE analysis was performed using 1000 iterations, a perplexity of 30 and a learning rate of 1237 and 756 for spleen and bone marrow respectively. Population labeled as 1 appears to display a memory-like phenotype, expressing higher levels of CD45RO, CD62L and CD45RA. Population labeled as 2 appears to display an effector-like phenotype, expressing low levels of CD45RO, CD62L and CD45RA. [000335] The flow cytometric analyses revealed that the persisting CAR T cells consisted of both CD4+ and CD8+ populations. The CD8+ CAR cells appeared to exhibit two distinct populations: effector cells and CD45RA+CD62L+ memory cells. The memory population exhibited higher expression levels of BCMA CAR and CD45 (FIG.11E). Similarly, CD4+ CAR T cells demonstrated two populations of effector and memory cells (FIG. 20). An insufficient number of CAR T cells could be detected from the VHH-muIL2 treated mice to perform a similar flow cytometric analysis. Therefore, the CAR-engager treatment leads to generation of long-lasting memory CAR T cells. [000336] FIG. 20 shows t-SNE mapping of CD4+ CAR+ T cells derived from the five BCMA- muIL2 CAR-E treated mice as shown in FIGs. 11A – 11E. The expression of nine immune cell 92 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 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 the five BCMA-muIL2 treated mice were analyzed by flow cytometry. CD45+, CD4+, ⍺-BCMA-CAR+ immune cells from the five mice were concatenated to form a total of ~9000 (spleen) and ~6600 (bone marrow) cells. The entire high dimensional dataset (excluding the CD45, CD8, and CD4 parameters) was merged to create a single t-SNE map with the signal strength of six phenotypic markers defining specific immune phenotypes expressed with a blue-green-yellow-red continuous color scale. tSNE analysis was performed using 1000 iterations, a perplexity of 30 and a learning rate of 630 and 466 for spleen and bone marrow, respectively. The population labeled as “1” appears to display a memory-like phenotype, expressing higher levels of CD45RO, CD62L and CD45RA. The population labeled as “2” appears to display an effector-like phenotype, expressing low levels of CD45RO, CD62L and CD45RA. [000337] Single-cell RNA-sequencing (scRNAseq) analysis was performed on CAR+ T cells isolated from mice treated with either the BCMA-muIL2 or the VHH-muIL2 control. Despite the limited presence of CAR T cells in the VHH-muIL2-treated mice, a sufficient number of cells were obtained from one of the VHH-treated mice for the experiment (FIG.21A). CAR+ cells were sorted after staining with BCMA-AlexaFluor647 and TotalSeq-C hashing antibodies from BCMA-muIL2 or VHH-muIL2 treated mice as shown in the red and green boxes, respectively in FIG.21A.5000 CAR+ cells from BCMA-muIL2 mice bone marrow and spleen and 2500 CAR+ cells from VHH- muIL2 mice bone marrow and spleen were loaded onto the 10X channel. The scRNAseq analysis revealed that the predominant population of persistent CAR T cells in the BCMA-muIL2-treated mouse consisted of CD8+ T cells (FIGs. 21B – 21C), which exhibited an enrichment of genes associated with an activated T cell state (FIGs. 21D – 21E). Heatmaps in FIG. 21D show significantly differentially expressed genes between CAR-engager treatment and VHH conditions in CD8 and CD4 CAR T cells, split among different relevant conditions. Genes marked with an * are the significantly differentially expressed genes between BCMA-muIL2 and VHH-muIL2 treated mice in the subset of interest. This was evidenced by elevated expression levels of granzyme family genes, other cytotoxicity-associated genes, and MHC class II genes. No significant differences in activation markers were observed between CAR T cells obtained from the BCMA-muIL2 or VHH- muIL2 treated mice, as the mice had already cleared the tumors over 60 days prior. The BCMA- 93 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 muIL2 treatment did not induce upregulation of exhaustion markers, showing the treatment did not induce exhaustion in the persisting CAR T cells. [000338] Next, the diversity of T-cell receptor (TCR) clonotypes was evaluated in the BCMA- muIL2 and VHH-muIL2 treated mice (FIGs.21F – 21G). Both groups displayed similar diversity in clonotypes present, showing that the BCMA-muIL2 CAR-engager treatment could effectively facilitate the generation of a diverse TCR repertoire in persisting CAR T cells, as opposed to promoting the dominance of a restricted set of TCR clones. Pie plots in FIG.21F show the diversity of TCR clonotypes, with each slice of the pie chart representing the proportion of a different TCR clonotype present; colors were randomly assigned to different clonotypes. The clonotype diversity within each sample’s total cell count was visualized with a stacked bar plot (FIG. 21G), where similar clonotypes with counts below 50 were combined. To evaluate the diversity within each sample, the Simpson index was calculated, with higher values indicating greater diversity. Overall, the results showed that the BCMA CAR-engager could not only help CAR T cells to fully clear tumor cells, but also robustly induce generation of long-lasting and functional memory CAR T cells. Example 10: CAR-engager expands CAR T cells in the absence of tumor antigens [000339] CAR T cell expansion typically occurs following infusion in patients, with peak expansion observed around 10-14 days post-infusion (Rodriguez-Otero et al., N. Engl. J. Med. 388(11):1002-1014 (2023)). [000340] Eradication of minimal residual disease (MRD) facilitates long-lasting and complete responses. However, the limited presence of the corresponding antigen associated with MRD may not adequately support the proliferation and efficacy of conventional CAR T cells. To demonstrate efficacy in the absence of tumor antigen, NSG mice were solely injected with 0.25 million BCMA CAR T cells in the absence of tumor cells. These mice received two 25 μg doses of BCMA-muIL2 on days 1 and 8 post-injection of CAR T cells. The control group received VHH-muIL2 treatment (n=4 for each group). On day 30, the mice were euthanized, and their spleen and bone marrow were assessed for the presence of CAR T cells. The BCMA-muIL2 treated mice exhibited higher numbers of CAR+ T cells in the spleen (approximately 6.8-fold higher) and bone marrow (approximately 5.5- fold higher), showing that BCMA CAR-engager expanded CAR T cells in vivo, even without the presence of tumor cells (FIGs. 12A-12B); error bars represent mean with standard deviation. Overall, these findings demonstrate that the CAR-engager can expand CAR T cells, even in the 94 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 absence of tumor antigen. Additionally, the effectiveness of the treatment was evident even at lower doses and frequencies. Example 11: CAR-engager containing immune cell effector domain IL-2 variants [000341] The binding strength of the IL-2 variant to the CAR immune cell may have significant impacts on the efficacy of CAR-engager therapy. Thus, CAR-engagers IL-2 variant containing different IL-2 variants were generated and integrated into CAR-engagers and are summarized in Table 9. Amino acid substitutions are relative to the natural (wild-type) sequence of human IL-2 (NCBI Accession No. NP_000577; SEQ ID NO: 102). In addition, some of the CAR-engagers contain different dimerization domains, including CH3, and silent fragment crystallizable region (Fc). Silent Fc contains at least two mutations relative to wild-type Fc that abolish Fc-γ receptor and complement component 1q (C1q) binding, while maintaining the stabilizing effect and neonatal Fc receptor (FcRn) binding of wild-type Fc. The at least two mutations include L234A and L235A, and are commonly referred to as (“LALA”). CAR-engager variant E4 has a dimerization domain containing IgG1 with the silent Fc mutations of L234A and L235A (“LALA”) and further lacks an IL-2 variant. The CAR-engager variant E4 was developed and used as a control molecule. [000342] CAR-engagers containing a single IL-2 variant are listed in Table 9 as “One-IL-2” while CAR-engagers containing two repeats of IL-2 variants are listed in the below table as “IL-2.” For example, variants Q3, A8, A10, and A16 have two repeats of the indicated IL-2 variant on each molecule of CAR-engager. Furthermore, the CAR-engager may be present as a dimer due to the presence of a dimerization domain. CAR-engagers containing a wild-type CD19 (SEQ ID NO: 5) are listed in Table 9 as “CD19 wt,” for example, variants Y9 and Y3. CAR-engagers containing the variant of CD19 that has the amino acid sequence of SEQ ID NO: 3, are listed in Table 9 as “CD19.” Table 1: CAR-engager ICE Variants Variant CAR-engager structure IL-2 amino acid substitutions relative Activity
Figure imgf000097_0001
95 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 U10 BCMA-CH3-IL2 H16A-D20A-R38D-F42A-E62N ++ U11 BCMA-silent-Fc-IL2 H16R-D20Q-R38D-F42A-E62N +
Figure imgf000098_0001
[000343] FIG. 22A shows the percentage of BCMA CAR T cells stained positive for phosphorylated STAT5 (pSTAT5) after treatment by select CAR-engager ICE variants described in Table 9, as determined by flow cytometry. BCMA CAR T cells were subjected to the indicated treatments at varying doses for 10 minutes at 37 °C, followed by the assessment of pSTAT5 (n=3 for each condition). All of the newly developed IL-2 variants, with the exception of U7, exhibited the ability to induce STAT5 activity in BCMA CAR T cells after 10 min. [000344] FIG. 22B shows dose-dependent activation of rested BCMA CAR T cells after CAR- engager treatment after 24 hours. Rested BCMA CAR T cells were incubated of with varying concentrations of BCMA CAR-engager ICE variants and the subsequent assessment of CD69 expression was analyzed by flow cytometry after 24 hours. CD69 serves as an activation marker for human T cells. The dashed line at 0 nM represents the background expression of CD69 in the absence of any treatment or cytokines. The dashed line “+Cytokine” indicates the CD69 expression 96 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 level of non-rested CAR T cells incubated in the presence of IL-2, IL-7, and IL-15 cytokines. The dashed lines are included as additional negative and positive controls. [000345] FIG.23A shows STAT5 phosphorylation in BCMA CAR T cells after CAR-engager ICE variant treatment. The mean fluorescence intensity (MFI) of BCMA CAR T cells stained positive for pSTAT5 was determined by flow cytometry after treatment with varying doses of CAR-engager ICE variants for 3 hours at 37 °C. Before flow cytometry analysis the cells were washed to remove excess unbound CAR-engagers and pSTAT5 was assessed after 24 hours (n=3 for each condition). Several of the IL-2 variants exhibited the ability to induce STAT5 activity in BCMA CAR T cells. [000346] FIG. 23B shows the activation of rested BCMA CAR T cells by BCMA CAR-engager ICE variant in a dose-dependent manner. BCMA CAR T cells were incubated with varying concentrations of BCMA CAR-engager ICE variants for 3 h, followed by washing. CD69 assessment of CD69 was conducted using flow cytometry 24 h after washing. All CAR-engager ICE variants activated the BCMA CAR T cells as compared to the U5 negative control. [000347] FIGs. 24A –24B show STAT5 phosphorylation in BCMA CAR T cells induced by treatment with the BCMA CAR-engagers detailed in Table 9. The CAR T cells contain the FDA- approved BCMA CAR constructs idecabtagene vicleucel (Ide-cel; ABECMA®) or ciltacabtagene autoleucel (Cilta-cel; CARVYKTI®). The percentage of BCMA CAR T cells stained positive for phosphorylated STAT5 (pSTAT5) as determined by flow cytometry are shown. BCMA CAR T cells were subjected to the indicated treatments at varying doses for 30 minutes at 37 °C, followed by the assessment of STAT5 phosphorylation (n=3 for each condition). These results show constructs which specifically and effectively induce pSTAT5 activity in CAR T cells, but have no, or limited, impact on non-transduced T cells. [000348] FIGs. 25A –25F show STAT5 phosphorylation in BCMA CAR T cells induced by treatment with the BCMA-muIL2 CAR-engagers detailed in Table 9. The CAR T cells contain the FDA-approved BCMA CAR constructs Ide-cel or Cilta-cel. The percentage of BCMA CAR T cells stained positive for pSTAT5 as determined by flow cytometry are shown. BCMA CAR T cells were subjected to the indicated treatments at varying doses for 15 m, 2 hr, or 24 at 37 °C, followed by the assessment of STAT5 phosphorylation (n=3 for each condition). For the 24-hour analyses, CAR T- cells were washed at 2 hours to remove unbound CAR-engagers. These results show which constructs can specifically and effectively induce pSTAT5 activity in CAR T cells and for how long (monitored for 24 hours), but have no, or limited, impact on non-transduced T cells. 97 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000349] FIGs. 26A – 26B show dose-dependent activation of rested BCMA CAR T cells by treatment with the BCMA CAR-engagers detailed in Table 9. The CAR T cells contain the FDA- approved BCMA CAR constructs Ide-cel or Cilta-cel. The incubation of rested BCMA CAR T cells with varying concentrations of BCMA CAR-engager treatments and the subsequent assessment of CD69 expression by flow cytometry after 24 hours is shown. CD69 serves as an activation marker for human T cells. These results show which constructs specifically and effectively activate CAR T cells, but have no, or limited, impact on non-transduced T cells. [000350] FIGs. 27A – 28B show dose-dependent activation of rested BCMA CAR T cells after treatment with additional BCMA CAR-engagers detailed in Table 9. The CAR T cells contain the FDA-approved BCMA CAR constructs Ide-cel or Cilta-cel. The incubation of rested BCMA CAR T cells treated with varying concentrations of BCMA CAR-engagers containing immune cell effector domains containing a U4, X5, X6, or U5 Il-2 variant immune cell effector domain, and the subsequent assessment of CD69 expression by flow cytometry after 24 hours are shown. These results show which constructs specifically and effectively activate CAR T cells, but have no, or limited, impact on non-transduced T cells. [000351] Similarly, the incubation of rested BCMA CAR T cells treated with varying concentrations of BCMA CAR-engagers containing immune cell effector domains containing a U4, Y2, V7, X12, X15 or U5 IL-2 variant immune cell effector domain, and the subsequent assessment of CD69 expression or STAT5 phosphorylation by flow cytometry after 24 hours are also shown. These results show which constructs specifically and effectively activate CAR T cells and for how long (monitoring at 24 hours), but have no, or limited, impact on non-transduced T cells. IL-2 (Teceleukin®) and a CAR-engager without an immune cell effector domain (BCMA-CH3) were used as controls. [000352] FIGs.29A – 29C show that select CAR-engagers induce CAR T cell proliferation in vitro with reduced impact on non-transduced T cells as compared to wild-type IL-2. FIG. 29A shows incubation of activated BCMA CAR T cells containing the Cilta-cel CAR construct, treated with increasing concentrations of BCMA CAR-engager, followed by assessment of CAR T cell proliferation using flow cytometry after 3 days. The dashed line in FIG.29A represents the number of CAR T cells that received no treatment. FIG.29B shows a repetition of the experiment show in FIG. 29A with additional CAR-engagers. FIG. 29C shows a similar experiment evaluating the impact of CAR-engager on the cellular proliferation of normal, non-transduced T cells. CAR- 98 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 engagers induce proliferation in non-transduced T cells significantly less than wild-type IL-2. CAR- engagers containing the triple mutant IL-2 (V7, V9, Y2, and Y9) induced significantly less proliferation in normal T cells as compared to CAR-engagers containing the double-mutant IL-2 (U4). [000353] FIG.42 shows that CAR-engagers induce CAR T cell proliferation in vitro with no little to no impact on non-transduced T cells. BCMA CAR-engagers containing immune cell effector domains containing a U4, V7, Y2, V6, X12, X15, or U8 IL-2 variant were examined for their ability to induce proliferation of anti-BCMA CAR T cells or non-transduced T cell controls. Wild-type IL- 2, a non-targeting CAR-engager with an immune cell effector domain containing E3 IL-2 variant (VHH-cH3-IL2), and a BCMA CAR-engager without an immune cell effector domain (BCMA- CH3) were used as controls. Only wild-type IL-2 induced robust proliferation in non-transduced T cells and four CAR-engagers containing an IL-2 variant induced proliferation in anti-BCMA CAR T cells (FIG.42). [000354] FIGs. 30A – 30C show that the V7 CAR-engager exhibits lower affinity for IL-2Rα as compared to wild-type IL-2 or the U4 CAR-engager. BLI analyses of association and dissociation between wild-type IL-2 (Teceleukin®), BCMA-muIL2 CAR-engager (U4; double mutant IL-2), or BCMA-muIL2 CAR-engager (V7; triple mutant IL-2) molecules and IL-2Rα are shown. A dimeric IL-2Rα molecule was used for the assessments. [000355] FIGs. 31A – 31B show the newly developed CAR-engager containing IL-2 variants induce proliferation of CAR-T cells in vivo, and that the proliferation levels correlate with in vitro pSTAT5 signaling. FIG. 31A schematically illustrations the experimental setup; mice received OPM2 cancer cells, followed by 0.5 million CAR-T cells (Cilta-cel) one week later. Mice were bled for 5 weeks on days 11, 18, 28, 35, and 42 and CD4 and CD8 CAR T-cells were counted (FIG. 31B). All the three tested CAR-engagers (Y2, V7, and X12) induced CAR T cells proliferation in vivo compared to the control PBS treatment group. The V7 and Y2 CAR-engagers induced more CAR T cell proliferation than X12 CAR-engagers. [000356] FIGs. 32A – 32C show that the V7 CAR-engager enhances CAR-T cell activity when administered 3 or even 14 days post-injection of CAR T cells. FIG. 32A schematically illustrates the experimental setup; mice received OPM2 cancer cells, followed by 0.5 million CAR-T cells (Cilta-cel) one week later. One mouse cohort received only CAR T cells (n=4). Another mouse cohort received 6 doses of V7 CAR-engager starting from 3 days post-injection of CAR T cells, 99 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 administered on days 3, 6, 10, 14, 21, 28. The third mouse cohort received 6 doses of CAR-engagers starting from day 14, which is the post-CRS window in patients, and on days 14, 18, 21, 28, 35, 42. BLI imaging was performed on the indicated days to assess tumor growth in the different cohorts (FIG.32B). Mice were bled for 6 weeks, once per week starting from day 14, and CAR T cells were counted (FIG. 32C). Treatment with the V7 CAR-engager induced CAR T cell proliferation in both cohorts that received treatment, compared to the control PBS treatment group. [000357] FIGs.43 – 45 show that CAR-engagers containing IL-2 variants induce in vitro pSTAT5 signaling (FIG. 43) and gene expression (FIGs. 44 and 45). CAR T cells (about 60% of CAR+ T cells) and non-transduced T cells were incubated with increasing concentrations of CAR-engagers containing an immune effector domain containing an IL-2 variant (including U4, V7, Y2, V6, X12, X15, U8) and controls (wild-type IL-2 and a CAR-engager without an immune effector domain (BCMA-CH3)). pSTAT5 activity was assessed by flow cytometry after 30 minutes of CAR-engager treatment (FIG. 43). CD69 expression was assessed by flow cytometric analysis after a 2-hour incubation of T cells with CAR-engagers, followed by a wash, and a further 22-hour incubation (FIG.44). [000358] TNF-α and IFN-γ production was assessed by multiplex ELISA via flow cytometry after a 2-hour incubation of T cells with CAR-engagers, followed by a wash, and a further 22-hour incubation and media harvesting (FIG.45). Each condition included 3 technical replicates. T cells were obtained from PBMCs from a single healthy donor. [000359] FIGs.46A – 46B show that CAR-engagers containing IL-2 variants bind to IL-2Rα and IL-2Rβγ by biolayer interferometry. To measure CAR-engager binding rate, a biosensor tip was coated with dimeric IL-2Rα (FIG.46A) or IL-2Rβγ (FIG.46B), and incubated with CAR-engagers containing an immune effector domain containing an IL-2 variant (including U4, V7, Y2, V6, X12, X15, U8) or controls (monomeric wild-type IL-2 (positive control), CAR-E with wild-type IL-2 (positive control), and a CAR-engager with monomeric V7 IL-2 variant)) for the indicated times. The release rate was assessed by transferring the biosensor tip into the washing solution. Each condition included 3 technical replicates. Example 12: CD19 CAR-E does not inhibit killing efficacy of CD19 CAR T cells [000360] CAR-engagers that bind BCMA were observed to bind but not inhibit killing efficacy of BCMA CAR T cells (FIGs.2D – 2H). To investigate the impact of CAR-E containing other cancer antigens on antigen-specific CAR T cells, a killing assay was conducted using CD19 CAR T cells 100 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 and patient-derived CD19+ leukemia cells in the presence of varying concentrations of the CD19 CAR-engager. Remarkably, the results demonstrated no inhibition of killing even at the highest tested concentration (1000 nM of the CAR-engager) (FIG.2H). Nalm6 cells were co-incubated with CD19 CAR T cells (filled) or non-transduced T cells (open) (E:T ratio 1:1; 30,000 cells of each) in the presence of varying concentrations of the CD19-muIL2 CAR-E treatment. Live (PI-) Nalm6 cells were counted 48 hours later, with an N of 3 for each of the experiments. The experimental findings disclosed herein with CD19 are consistent with the findings in BCMA cancer models and BCMA CAR-E, above. Example 13: CAR-engagers effectively enhance CAR immune cell efficacy and persistence at low doses on initial treatment and tumor rechallenge [000361] Next, an experiment to investigate the efficacy of a reduced frequency of CAR-E treatment initiated two weeks after CAR T cell injection was conducted. Mice were intravenously injected with OPM2 cells (1×106, i.v.). A week later, CAR T cells (0.5×106, i.v.) were administered. Two weeks post-injection of CAR T cells, the mice were divided into two cohorts, with mice receiving the CAR-E treatment or not. The treatment group received four doses of CAR-E treatment (4 mg/kg per dose) on days 14, 18, 21, and 28. Surviving mice underwent re-challenge with 1×106 of OPM2 cells on day 60, followed by 4 mg/kg CAR-engager treatment on days 68, 70, 74, 77, and 80 (FIG.35A). [000362] Remarkably, bioluminescence imaging (BLI) analyses demonstrated that all mice (5/5) receiving CAR-E cleared tumors, while none of the control mice (0/4) achieved tumor clearance; the same BLI quantification scale is used for all images (photons/sec) (FIGs. 35B – 35D). Blood analyses indicated that the four doses of CAR-E treatment were sufficient to cause robust expansion of the CAR T cells in all the treated mice compared to the control cohort (FIGs. 35E and 37). Cytokine assessments also revealed elevated levels of IFN-γ in the CAR-E treatment cohort by sandwich ELISA on 1:40 diluted serum samples collected the same days when CAR-T counts were assessed as shown FIG.22E (FIG.35F). [000363] One of the CAR-E treated mice (M5) showed liver relapse on day 60 (FIG. 35B). Regardless, all the five CAR-E treated mice underwent re-challenge, using 1 million of the liver metastasis-derived OPM2 cells, on day 60 to assess the generation of memory CAR-T cells. All mice showed considerably less signal compared to the naïve control mice (FIG.35B; see day 66). While the mice exhibited liver signals, none showed bone marrow signals, suggesting the presence 101 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 of functional memory CAR T cells in the bone marrow inhibiting tumor growth. To explore whether CAR-E treatment could contribute to re-expanding CAR T cells and controlling tumor growth in liver metastasis, the mice were re-treated with CAR-E (4 mg/kg) on days 68, 70, 74, 77, and 80. Impressively, all mice successfully cleared the liver metastasis, indicating that CAR-E could facilitate the re-expansion and trafficking of CAR T cells to eliminate tumor cells (FIG. 35B; see day 83). The mouse that showed relapse on day 60 (M5, FIG.35B) also cleared the tumor from liver after re-challenge, although it exhibited some signal on the last day of the experiment. Blood analyses confirmed that the CAR-E had caused robust re-expansion of the CAR T cells in the circulation in all mice (FIG.35E). [000364] On day 90 post-injection of CAR T cells, the mice were euthanized, and organ analysis via flow cytometry revealed that all CAR-E treated mice harbored a significant amount of persisting CAR-T cells with diverse memory phenotypes (FIGs.35G, 38A, and 38B). For analysis, the human CD45+ BCMA-CAR+ cells from the bone marrow and spleen of the mice were gated and concatenated, then FLOWSOM analysis was conducted on the pooled populations to identify eight major phenotypic metaclusters. A heatmap representing the Mean Fluorescence Intensity (MFI) of each marker within each metacluster was used to qualitatively describe each cluster (FIGs. 35A - 35D). In FIG.35G, the proportion of each metacluster within the bone marrow and spleen of each mouse is depicted. The no-treatment “CAR-T only” cohort did not have enough persisting CAR T cells in the bone marrow or spleen to allow for a similar analysis. This experiment utilized PBMCs from one donor. [000365] In summary, these results indicate that BCMA CAR-E treatment not only facilitated the complete clearance of tumor cells by the CAR T cells but also significantly promoted the formation of functional memory CAR T cells. Notably, even a few doses of CAR-E administered two weeks after CAR T cell administration were effective in expanding CAR T cells and fostering the development of functional memory CAR T cells. The persisting CAR T cells retained the capacity to re-expand in response to CAR-E treatment. [000366] CAR-E treatment resulted in expansion and persistence of the CAR T cells in spleen and bone marrow (FIGs.36B – 36C and 40A – 40D). These results revealed that CAR-engager treatment led to dose-dependent expansion of the CAR T cells. Error bars shown in FIGs.36B – 36C are mean ± standard deviation and displays column bars indicating the absolute number of detected CAR T cells in each condition and statistical analyses that demonstrate that CAR-engager treatment led to 102 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 dose-dependent expansion of CAR T cells. The significance between the group of mice that received PBS and the group of mice received the lowest concentration CAR-engager treatment (2 mg/kg) was measured using the Mann-Whitney test. Additionally, a simple linear regression was conducted to demonstrate the dose-dependent effect of the treatment; the error bars on the graph represent a 95% confidence interval. Example 14: CAR T cell expansion in the absence of tumor antigens is dose-dependent. [000367] Typically, CAR T cell expansion occurs post-infusion in patients, with peak expansion observed around 10-14 days post-infusion (Rodriguez-Otero et al., N. Engl. J. Med.388(11):1002- 1014 (2023)). This expansion is driven by antigen availability and the tumor-killing process, which promotes CAR T cell proliferation (Turtle et al., J. Clin. Invest.126(6):2123-38 (2016), Gardner et al., Blood 129(25):3322-3331 (2017), Lee et al., Leukemia 35(1):255-258 (2021), Hossain et al., Blood 132(Suppl_1):490-490 (2018)). However, patients who exhibit limited CAR T cell expansion post-infusion show poor responses (Fraietta et al., Nat. Med.24(5):563-571 (2018)). Additionally, achieving long-lasting complete responses necessitates the eradication of minimal residual disease. Yet, the limited presence of the corresponding antigen associated with minimal residual disease may not sufficiently support the proliferation and efficacy of CAR T cells. [000368] Without being bound by theory, the CAR-E mechanism of action may be independent of tumor cells and antigens presented thereon and may thus expand CAR T cells in the absence of tumor cells (and therefore tumor antigen), addressing the critical clinical challenge of limited in vivo CAR T cell expansion post-infusion. To test this hypothesis, and assess the dose-dependent nature of CAR-E treatment, mice were injected solely with 0.25 million BCMA CAR T cells in the absence of tumor cells. As illustrated in FIG.36A, NSG-DKO mice treated with 0.25×106 BCMA CAR T cells and were assigned to different cohorts receiving varying doses of BCMA-muIL2 CAR-E treatment (2 mg/kg, 4 mg/kg, 8 mg/kg, or no CAR-E treatment; twice per week for four weeks; n=5 for each cohort). Organs were collected one-month post-injection of CAR T cells, and flow cytometric analyses were conducted to assess the presence of CAR T cells. [000369] Further, the results demonstrated that CAR-E impact on CAR T cells is dose-dependent (FIGs.36B – 36C). Interestingly, even at the lowest tested dose of 2 mg/kg, there was substantial persistence of CAR T cells compared to the no-treatment cohort, where very few to nearly no CAR T cells were detected one month post CAR-T injection (FIGs. 36B – 36C). Additional analyses indicated that CAR-E induced the generation of memory CAR T cells (FIGs.36D – 36E). TEM cells 103 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 are CD45RA- CD45RO+ CCR7-; TEMRA cells are CD45RA+ CD45RO+ CCR7-; TSCM cells are CD45RA+ CD45RO+ CCR7+; TCM cells are CD45RA- CD45RO+ CCR7+; TNaive cells are CD45RA+ CD45RO- CCR7+. The experiment in panels FIGs. 36A – 36E utilized PBMCs from one donor. Overall, these findings demonstrate that CAR-E treatment induces the expansion of CAR T cells and enables the CAR T cells develop diverse memory phenotypes, regardless of the presence of tumor cells. [000370] To further investigate CAR-E’s capacity to expand CAR T cells and to confirm the essential role of the low-affinity IL-2 component of BCMA-muIL-2 CAR-E in influencing CAR T cells, a similar experiment, as described above, was repeated using the 4 mg/kg dose and 0.25×106 CAR T cells. Mice received either the BCMA-muIL2 CAR-E treatment, treatment with the control BCMA-CH3 molecule, which contains only the BCMA antigen and not the low-affinity mutated IL-2 component, or no treatment. As anticipated, the antigen-only, BCMA-CH3 treatment cohort did not result in the expansion or persistence of CAR T cells compared to the CAR-E treatment cohort, further validating that both the ectodomain (e.g., BCMA antigen) component and the immune cell effector domain (e.g., low-affinity IL-2) component of the CAR-E molecule are necessary for its impact on CAR T cells (FIG.36F – 36G and 41A – 41E). Mice received CAR T cells and different treatments (4 mg/kg) following a schedule similar to that shown in FIG. 36A. The BCMA-CH3 antigen, VHH-muIL2, or the low-dose wild-type IL-2 treatments did not result in expansion or persistence of CAR T cells compared to the CAR-engager-treated cohort. Error bars represent mean with standard deviation. The experiment in panels FIGs.36F – 36G utilized PBMCs from two donors. The Kruskal-Wallis test was used for each subset of CAR T cells and total T cells. Subsequently, post-hoc Dunn’s analysis was conducted to compare each group with the treatment group. The table in FIG.23G displays the adjusted p-values. This aligns with the in vitro and in vivo analyses disclosed herein. [000371] To compare the efficacy of CAR-E treatment with a low-dose wild-type IL-2, which is used in the clinic in combination with CAR T therapy, an additional cohort was included that received low-dose wild-type IL-2 (SEQ ID NO: 102) (4.5 µg per mouse, for 14 days starting from day 1, and then twice per week for an additional two weeks). The low-dose IL-2 group failed to result in substantial persisting CAR-T cells compared to the CAR-E treatment cohort (FIGs.36F – 36G). Clinical studies that have used low-dose IL-2 in combination with CAR-T have not resulted in significant benefits, and in some cases, low-dose IL-2 was stopped due to IL-2-associated 104 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 toxicities (Katz et al., Clin. Cancer Res.21(14):3149-59 (2015)). Furthermore, as expected, the non- targeted, low-affinity IL-2 (VHH-muIL2) CAR-E did not lead to the expansion or persistence of CAR T cells (FIGs.36F – 36G), consistent with the prior findings disclosed herein. [000372] In summary, these findings demonstrate that the CAR-E molecule can cause expansion of CAR T cells and robust promotion of the development of diverse memory phenotypes, regardless of the presence of tumor cells. Both the ectodomain (e.g., BCMA antigen) component and the immune cell effector domain (e.g., low-affinity IL-2) component of the CAR-E molecule are necessary for the observed impact on CAR T cells. Additionally, the effectiveness of the CAR-E treatment was evident even at lower doses. Example 15: The efficacy of the CAR-E requires signaling through both the CAR and the IL-2R intracellular signaling domains [000373] To better understand the mechanism of action of CAR-E, it was investigated whether the effects on CAR T cells are solely mediated by anchoring the low-affinity IL-2 onto CAR T cells through BCMA-to-CAR binding or if it involves simultaneous engagement of both IL-2R and CAR intracellular signaling domains. For this purpose, BCMA CAR T cells were made using a BCMA CAR construct lacking the 41BB-CD3ζ intracellular signaling domain but retaining an identical extracellular ectodomain (referred to as CAR-Intracellular domain deletion, or CAR-ICD-Δ). In vitro analyses demonstrated that the BCMA-muIL2 CAR-E molecule induced pSTAT5 in CAR- ICD-Δ T cells after a 30-minute incubation with CAR-E (n=3), similar to full CAR T cells (FIG. 33A), suggesting that the effects of low-affinity IL-2 are similar in both CAR constructs and are mediated via antigen-to-CAR binding. [000374] However, interestingly, while CAR-E robustly activated full CAR T cells, as evidenced by elevated CD69 expression (FIG.33B) and increased production of IFN-γ (FIG.33C) and TNF- α (FIG.33D), its impact on CAR-ICD-Δ CAR T cells was negligible. Additional control conditions included the non-targeted VHH-muIL2 and the antigen-only BCMA-CH3 (n=3 for each condition). error bars represent mean with standard deviation and the experiment utilized PBMCs from one donor. Dasatinib, a lymphocyte cell-specific protein-tyrosine kinase (LCK) inhibitor, and Ruxolitinib, a Janus kinase (JAK) inhibitor, both individually and in combination, significantly inhibited the impact of the CAR-E molecule on CAR T cells (FIGs.33E – 33G), further suggesting that the CAR-E molecules engages both CAR and IL-2R receptors and activating their intracellular signaling pathways. CAR T cells were treated with varying doses of CAR-E treatment and the 105 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 individual inhibitor or their combination, with assessments conducted 24 hours later (n=3 for each condition); error bars represent mean with standard deviation. Subsequently, the in vivo impact of CAR-E on full CAR T cells and CAR-ICD-Δ T cells was compared. Mice injected with 0.25×106 CAR T cells in the absence of tumor cells received CAR-E treatments (4 mg/kg twice per week for four weeks; FIG.33H). After a month, animals were euthanized, and organs were analyzed via flow cytometry. Remarkably, while CAR-E robustly expanded CAR T cells with the full CAR construct consistent with the previous results, it did not result in expansion or persistence of CAR-ICD-Δ T cells (FIG. 33I). The experiment utilized PBMCs from one donor; statistical analyses were performed using an unpaired T-test. [000375] To gain additional insights into the mechanism of action of CAR-E, their impact on the transcriptome of CAR T cells was investigated. Various treatments, including the BCMA-muIL2 CAR-E molecule, the non-targeted low-affinity IL-2 (VHH-muIL2), BCMA-CH3 antigen, and wild-type IL-2, were added to CAR T cells. As an additional control, CAR-ICD-∆ T cells were treated with the BCMA-muIL2 CAR-E molecule. Treatments were removed after 2 hours to mimic in vivo conditions, and cells were subjected to bulk RNA-sequencing either 2 or 22 hours later. [000376] Remarkably, the CAR-E induced substantial transcriptomic changes, demonstrating quantitatively larger fold-changes compared to all other conditions, including wild-type IL-2 (FIGs.33J – 33N). The differences in gene upregulation of the BCMA-muIL2 compared to wild- type IL-2, VHH-muIL2, and BCMA-CH3, as well as GSEA of these different conditions (FIG.34) show that while these control treatments have an effect on their own, the stimulation induced by the CAR-E molecule is greatly superior (FIGs.33L – 33M). The impact of CAR-E on CAR-ICD-∆ T cells was mild, further emphasizing the significant role of the CAR-intracellular signaling domain in the mechanism of action of the CAR-E molecule (FIGs.33J – 33K). CAR-T cells underwent a 2- hour incubation with 10 nM of the BCMA-muIL2 CAR-E molecule or control molecules, followed by treatment removal through washing. Subsequently, RNA-sequencing is performed 2 and 24 hours later. The gene set enrichment analysis (GSEA) shown in FIG. 34 was performed with the HALLMAKR dataset of the molecular signatures database (MSigDB) and indicates that treatment with both control and CAR-E molecules activated the expected pathways. Similar pathways appear to be induced between the controls and the CAR-E treatment, with a much smaller impact in the controls as depicted in FIGs.33J – 33M. 106 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 [000377] In summary, these findings indicate that the mechanism of action of the CAR-E molecule goes beyond merely delivering low-affinity IL-2 to CAR T cells. Instead, it operates by engaging and, importantly, bridging the intracellular signaling domains of the CAR and IL-2R receptors, inducing significant T cell activation and substantial transcriptomic changes. [000378] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference. [000379] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims. 107 Include Draft Include Date Include Time

Claims

VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 What is claimed is: 1. An IL-2 variant, which differs from wild type IL-2 (SEQ ID NO: 102) in terms of from three to eight amino acid substitutions selected from amino acid residues H16, D20, R38, F42, Y45, E61, E62, L72, and V91 of SEQ ID NO: 1. 2. The IL-2 variant of claim 1, wherein a first amino acid substitution is selected from H16A, H16R, H16S, D20A, or D20Q, a second amino acid substitution is selected from R38D, F42A, and V91H, and a third amino acid substitution is selected from Y45A and E62N. 3. The IL-2 variant of claim 2, wherein the first amino acid substitution is H16A, H16R, or H16S. 4. The IL-2 variant of claim 2, wherein the first amino acid substitution is D20A or D20Q. 5. The IL-2 variant of any one of claims 2-4, wherein the third amino acid substitution is Y45A. 6. The IL-2 variant of any one of claims 2-4, wherein the third amino acid substitution is E62N. 7. The IL-2 variant of any one of claims 2-6, wherein the second amino acid substitution is F42A. 8. The IL-2 variant of any one of claims 3-7, further comprising a fourth amino acid substitution is selected from R38D, E61A, L72G, and V91H. 9. The IL-2 variant of any one of claims 3-7, further comprising a fourth amino acid substitution is selected from D20A, D20Q, R38D, Y45A, E61A, L72G, and V91H; and wherein the first amino acid substitution is H16A, H16R, or H16S. 10. The IL-2 variant of claim 9, further comprising a fifth amino acid substitution comprising R38D; and wherein the fourth amino acid substitution is selected from D20A, D20Q, Y45A, E61A, L72G, and V91H. 108 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 11. The IL-2 variant of claim 10, further comprising a sixth amino acid substitution comprising L72G; and wherein the fourth amino acid substitution is selected from D20A, D20Q, Y45A, E61A, and V91H. 12. The IL-2 variant of claim 11, further comprising a seventh amino acid substitution comprising E62N; wherein the third amino acid substitution is Y45A; and wherein the fourth amino acid substitution is selected from D20A, D20Q, E61A, and V91H. 13. The IL-2 variant of any one of claims 1-12, which differs from wild-type IL-2 having the amino acid sequence of SEQ ID NO: 102, in terms of having amino acid substitutions at H16A, and F42A; or amino acid substitutions at D20Q, F42A, and E62N; or amino acid substitutions at D20Q, F42A, and Y45A; or amino acid substitutions at H16A, F42A, and E62N; or amino acid substitutions at H16A, F42A, and Y45A; or amino acid substitutions at H16R, F42A, and E62N; or amino acid substitutions at H16S, F42A, and Y45A; or amino acid substitutions at D20Q, V91H, F42A, and E62N; or amino acid substitutions at H16A, F42A, Y45A, and L72G; or amino acid substitutions at H16R, D20Q, F42A, and E62N; or amino acid substitutions at H16S, D20Q, F42A, and E62N; or amino acid substitutions at H16S, F42A, Y45A, and L72G; or amino acid substitutions at H16A, D20A, R38D, F42A, and E62N; or amino acid substitutions at H16R, D20Q, R38D, F42A, and E62N. 14. A chimeric antigen receptor (CAR)-engager, comprising: a first moiety that binds an epitope on an extracellular domain of a CAR, connected to a second moiety comprising a first immune cell effector domain comprising the IL-2 variant of any one of claims 1-13. 15. The CAR-engager of claim 14, wherein the first moiety of the CAR-engager comprises an ectodomain of the antigen present on the cancer cell. 16. The CAR-engager of claim 15, wherein the ectodomain is derived from AFP, AXL, B4GALNT1, B-cell maturation antigen (BCMA), CA9, CD5, CD7, CD19, CD20, CD22, CD23, CD33, CD34, CD38, CD44, CD52, CD70, CD80, CD86, CD123, CD133, CD174, CD274, CD276, CDS, Cancer/Testis Antigen 1B (CTAG1B), carcinoembryonic antigen (CEA), 109 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 CLEC12A, claudin 18.2 (CLDN 18.2), CSPG4, DLL3, EGFR, EPCAM, EPHA2, ERBB2, FAP, FOLH1, FOLR1, GD2, GPC3, GPRC5D, GPNMB, HER2, HPV E7, IL1RAP, IL3RA, 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. 17. The CAR-engager of claim 16, wherein the ectodomain comprises the amino acid sequence MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA (SEQ ID NO: 1). 18. The CAR-engager of claim 17, wherein the ectodomain comprises the amino acid sequence MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAGGG SGGGSPRGSGGGSMLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNAS VTNSVKGTN (SEQ ID NO: 2). 19. The CAR-engager of claim 15, wherein the ectodomain is derived from CD19. 20. The CAR-engager of claim 19, wherein the ectodomain comprises an amino acid sequence having at least about 90% sequence identity with PEEPLVVKVEEGDEAWLPCLKGTSDGPTQQLTWSRESPLKPFLKVSFGVPGLGVHVRP NAVSLVISNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLG CGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSRDMTVA PGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVTGTRLFL PRATAQDAGKYYCHRGNLTMSFHLEVKARPVSAHTKLRTGGWK (SEQ ID NO: 3). 21. The CAR-engager of claim 20, wherein the ectodomain comprises the amino acid sequence of SEQ ID NO: 3. 22. The CAR-engager of claim 20, wherein the ectodomain comprises the amino acid sequence of PEEPLVVKVEEGDEAWLPCLKGTSDGPTQQLTWSRESPLKPFLKVSFGVPGLGVHVRP NAVSLVISQVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWQVSDLGGLG CGLKQRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLQQSLSRDMTVA PGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVTGTRLFL 110 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 PRATAQDAGKYYCHRGQLTMSFHLEVKARPVSAHTKLRTGGWK (SEQ ID NO: 4) or PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRP LAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLG CGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMA PGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLL PRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWK (SEQ ID NO: 5). 23. The CAR-engager of claim 15, wherein the ectodomain is derived from CD20. 24. The CAR-engager of claim 23, wherein the ectodomain comprises the amino acid sequence KISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQS (SEQ ID NO: 6). 25. The CAR-engager of claim 15, wherein the ectodomain is derived from SLAMF7. 26. The CAR-engager of claim 25, wherein the ectodomain comprises the amino acid sequence SGPVKELVGSVGGAVTFPLKSKVKQVDSIVWTFNTTPLVTIQPEGGTIIVTQNRNRERV DFPDGGYSLKLSKLKKNDSGIYYVGIYSSSLQQPSTQEYVLHVYEHLSKPKVTMGLQS NKNGTCVTNLTCCMEHGEEDVIYTWKALGQAANESHNGSILPISWRWGESDMTFICV ARNPVSRNFSSPILARKLCEGAADDPDSSM (SEQ ID NO: 6). 27. The CAR-engager of claim 15, wherein the ectodomain is derived from PD-1. 28. The CAR-engager of claim 27, wherein the ectodomain comprises the amino acid sequence FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR AELRVTERRAEVPTAHPSPSPRPAGQFQTLV (SEQ ID NO: 7). 29. The CAR-engager of claim 15, wherein the ectodomain is derived from KIT. 30. The CAR-engager of claim 29, wherein the ectodomain comprises the amino acid sequence QPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITE KAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPE VTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSEKFI LKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEKY NSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFP 111 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 MINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYV SELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAA GFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVE CKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTP (SEQ ID NO: 8). 31. The CAR-engager of claim 15, wherein the ectodomain is derived from CD38. 32. The CAR-engager of claim 31, wherein the ectodomain comprises the amino acid sequence VPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPC NITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADD LTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSR SKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQ FSCKNIYRPDKFLQCVKNPEDSSCTSEI (SEQ ID NO: 9). 33. The CAR-engager of claim 15, wherein the ectodomain is derived from CD22. 34. The CAR-engager of claim 33, wherein the ectodomain comprises the amino acid sequence DSSKWVFEHPETLYAWEGACVWIPCTYRALDGDLESFILFHNPEYNKNTSKFDGTRL YESTKDGKVPSEQKRVQFLGDKNKNCTLSIHPVHLNDSGQLGLRMESKTEKWMERIH LNVSERPFPPHIQLPPEIQESQEVTLTCLLNFSCYGYPIQLQWLLEGVPMRQAAVTSTSL TIKSVFTRSELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKHTPKLEIKVTPS DAIVREGDSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVTKDQSGKYC CQVSNDVGPGRSEEVFLQVQYAPEPSTVQILHSPAVEGSQVEFLCMSLANPLPTNYTW YHNGKEMQGRTEEKVHIPKILPWHAGTYSCVAENILGTGQRGPGAELDVQYPPKKVT TVIQNPMPIREGDTVTLSCNYNSSNPSVTRYEWKPHGAWEEPSLGVLKIQNVGWDNT TIACAACNSWCSWASPVALNVQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKE VQFFWEKNGRLLGKESQLNFDSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRR LRVSMSPGDQVMEGKSATLTCESDANPPVSHYTWFDWNNQSLPYHSQKLRLEPVKV QHSGAYWCQGTNSVGKGRSPLSTLTVYYSPETIGRR (SEQ ID NO: 7). 35. The CAR-engager of claim 33, wherein the ectodomain comprises the amino acid sequence PHIQLPPEIQESQEVTLTCLLNFSCYGYPIQLQWLLEGVPMRQAAVTSTSLTIKSVFTRS ELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQPKLEIKVTPSDAIVREGDSVTMTCE 112 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 VSSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVTKDQSGKYCCQVSNDVGPGRSEEV FLQ (SEQ ID NO: 103). 36. The CAR-engager of any one of claims 14-35, wherein the first moiety is an antibody that binds an epitope on the extracellular domain (ED) of the CAR, or derivative thereof. 37. The CAR-engager of claim 36, wherein the CAR present on the CAR immune cell binds AFP, AXL, B4GALNT1, BCMA, CA9, CD5, CD7, CD19, CD20, CD22, CD23, CD33, CD34, CD38, CD44, CD52, CD70, CD80, CD86, CD123, CD133, CD174, CD274, CD276, CDS, CTAG1B, CEA, CLEC12A, CLDN 18.2, CSPG4, DLL3, EGFR, EPCAM, EPHA2, ERBB2, FAP, FOLH1, FOLR1, GD2, GPC3, GPRC5D, GPNMB, HER2, HPV 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, TNFRSF8, TNFRSF10B, TNFRSF13C, TNFRSF17, ULBP1, or ULBP2. 38. The CAR-engager of claim 14, wherein the ED of the CAR further comprises a linker and wherein the first moiety binds an epitope on the linker. 39. The CAR-engager of claim 14, wherein the CAR binds CD19 and wherein the first moiety has the amino acid sequence of SEQ ID NO: 124. 40. The CAR-engager of any one of claims 14-39, further comprising a first linker that connects the first and second moieties. 41. The CAR-engager of claim 40, further comprising a dimerization domain disposed between the first linker and the second moiety comprising the first immune cell effector domain. 42. The CAR-engager of claim 41, further comprising a second linker that connects the dimerization domain and the second moiety, wherein the first and second linkers may be the same or different. 43. The CAR-engager of claim 42, wherein the first linker and the second linker are flexible. 44. The CAR-engager of claim 44, wherein the first linker and/or the second linker are derived from the hinge region of CD3ζ, CD4, CD8α, CD28, IgG1, IgG2, or IgG4. 113 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 45. The CAR-engager of claim 44, wherein the first linker and/or the second linker comprises the amino acid sequence GGGX, GGGGX (SEQ ID NO: 55), or GSSGSX (SEQ ID NO: 56), or GSPRG (SEQ ID NO: 58), wherein X is either C or S. 46. The CAR-engager of claim 45, wherein the first linker has the amino acid sequence of GGGGS (SEQ ID NO: 57), or GSPRG (SEQ ID NO: 32) and the second linker has the amino acid sequence of GSPRGGGGSGGGGSGGGGS (SEQ ID NO: 62). 47. The CAR-engager of any one of claim 41-46, wherein the dimerization domain is derived from IgA, IgD, IgG, IgM, or IgE. 48. The CAR-engager of claim 47, wherein the dimerization domain comprises an IgG1 constant heavy (CH) 3 domain. 49. The CAR-engager of claim 47, wherein the dimerization domain further comprises an IgG CH2 domain and an IgG CH3 domain. 50. The CAR-engager of any one of claims 14-49, wherein the second moiety further comprises a second immune cell effector domain comprising a cytokine or immune cell-activating moiety. 51. The CAR-engager of claim 50, wherein the second immune cell effector domain is different from the IL-2 variant. 52. The CAR-engager of claim 50, wherein the second immune cell effector domain comprises a second IL-2 variant of any one of claims 1-13, wherein the IL-2 variant and the second immune cell effector domain are the same or different. 53. The CAR-engager of claim 52, wherein the IL-12 variant and the immune cell effector domain are different. 54. The CAR-engager of any one of claims 14-53, wherein the CAR-engager is in the form of a fusion protein and the first and the second moieties are connected by peptide bonds. 55. The CAR-engager of any one of claims 14-53, wherein the first moiety is connected to the second moiety or the first linker by an azide-alkyne connection, an oxime or hydrazine 114 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 connection, a tetrazine-transcyclooctene connection, an azide-nitrone connection, a thiol-alkene connection, an alkene-tetrazole connection, an alkene-tetrazine connection, an alkene-azide connection, a conjugated diene-alkene connection, or an isonitrile-tetrazine connection. 56. The CAR-engager of any one of claims 40-55, which is in the form of a homodimer comprising two of the CAR-engagers. 57. A heterodimeric CAR-engager comprising: a first moiety that binds an epitope on an extracellular domain of the CAR connected to first dimerization domain; and a second moiety comprising the IL-2 variant of any one of claims 1-13 connected to a second dimerization domain; wherein the first and the second dimerization domains dimerize to form a heterodimer. 58. The CAR-engager of claim 57, wherein the first dimerization domain comprises a protuberance, the second dimerization domain comprises a cavity sterically compensatory to the protuberance, and wherein the protuberance is positionable within the cavity. 59. A nucleic acid encoding the IL-2 variant of any one of claims 1-13 or the CAR-engager of claim 54. 60. A nucleic acid encoding a first moiety that binds an extracellular domain of the CAR fused to a first dimerization domain. 61. A nucleic acid encoding the IL-2 variant of any one of claims 1-13 fused to a second dimerization domain. 62. The nucleic acid of any one of claims 59-61, further comprising a sequence encoding a signal peptide. 63. A vector comprising the nucleic acid of any one of claims 59-62. 64. A cell comprising the vector of claim 63. 65. The cell of claim 64, which is a mammalian cell. 115 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 66. The cell of claim 65, which is a bacterial cell. 67. A pharmaceutical composition comprising the CAR-engager of any one of claims 14-58 and a pharmaceutically acceptable carrier. 68. The pharmaceutical composition of claim 67, further comprising an effective number of immune cells comprising a CAR that comprises an extracellular domain that binds the ectodomain of the CAR-engager, a transmembrane domain and an intracellular domain comprising a stimulatory domain. 69. A method of making an IL-2 variant or a CAR-engager comprising: culturing the cell of any one of claims 64-66 in medium under conditions wherein the nucleic acid encoding the IL-2 variant or the CAR-engager is expressed; and isolating the IL-2 variant or the CAR-engager from the cell and/or medium. 70. A method of treating cancer, comprising: administering to the subject a first course of an effective amount of the CAR-engager therapy of any one of claims 14-58, wherein prior to, substantially contemporaneous with, or subsequent to the administering of the CAR-engager, the subject is administered an effective number of immune cells comprising a chimeric antigen receptor (CAR) that comprises an extracellular domain that binds the ectodomain of the CAR-engager, a transmembrane domain, and an intracellular domain comprising a stimulatory domain (CAR immune cells). 71. The method of any one of claim 70, wherein the subject is administered the CAR immune cells prior to the administration of the CAR-engager. 72. The method of claim 71, wherein the CAR immune cells are administered at least about 6 months, at least about 9 months, or at least about a year prior to administering the CAR-engager. 73. The method of claim 70, wherein the CAR-engager is administered substantially contemporaneously with the CAR immune cells. 74. The method of claim 73, wherein the co-administering comprises administering the CAR immune cells and the CAR-engager substantially simultaneously. 116 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 75. The method of claim 73, further comprising contacting the CAR-engager with the CAR immune cells in vitro prior to administering the CAR-engager and the CAR immune cells. 76. The method of claim 73, further comprising: measuring a concentration of CAR immune cells present in a sample obtained from the subject after administering the immune cells; and calculating a difference between the concentration of the CAR immune cells administered to the subject and the measured CAR immune cell concentration. 77. The method of claim 76, wherein the co-administering comprises administering the CAR- engager when the measured CAR immune cell concentration is less than the administered CAR immune cell concentration. 78. The method of any one of claims 70-77, wherein the CAR immune cells are T cells or NK cells. 79. The method of claim 78, wherein the T cells are CD8+ T cells. 80. The method of any one of claims 70-79, wherein the cancer is a hematopoietic cancer. 81. The method of claim 80, wherein the hematopoietic cancer is leukemia, lymphoma, or multiple myeloma. 82. The method of claim 81, wherein the hematopoietic cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, or non-Hodgkin lymphoma. 83. The method of any one of claims 70-79, wherein the cancer is characterized by a solid tumor. 84. The method of claim 83, wherein the cancer is malignant mesothelioma, ovarian cancer, breast cancer, pancreatic cancer, lung cancer, liver cancer, glioblastoma, gastric cancer, endometrial cancer, cervical cancer, biliary cancer, uterine serous carcinoma, cholangiocarcinoma, neuroblastoma, sarcoma, lung cancer, or melanoma. 117 Include Draft Include Date Include Time VIA EFS Attorney Docket No.046094-788001WO Date of Deposit: September 11, 2024 85. The method of any one of claims 70-84, further comprising administering to the subject high- dose chemotherapy prior to the administering of the CAR immune cells. 86. The method of claim 85, further comprising administering to the subject bone marrow cells or peripheral blood stem cells. 87. The method of any one of claims 70-86, further comprising administering to the subject an additional active agent comprising one or more of thalidomide, lenalidomide, and bortezomib. 88. The method of any one of claims 70-87, wherein the effective number of CAR immune cells is approximately 1×104 to approximately 6×105 cells per kg of subject body weight. 89. The method of any one of claims 70-88, wherein the subject is in a state of minimal residual disease. 118 Include Draft Include Date Include Time
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