Engineered polypeptides for immune cell activation

Abstract

Compositions and methods for immunotherapy are provided. In some embodiments, a system for immunotherapy includes: a first composition including an engineered polypeptide conjugated to a small molecule activator, where the engineered polypeptide has a molecular weight less than or equal to 10 kDa, and where the engineered polypeptide binds to a marker on a target cell; and a second composition including an immune cell expressing an engineered receptor that binds to the small molecule activator.

Description

ENGINEERED POLYPEPTIDES FOR IMMUNE CELL ACTIVATIONCROSS-REFERENCE TO RELATED APPLICATION(S)|0001| The present application claims the benefit of priority to U.S. Provisional Application No. 63 / 656,501, filed June 5, 2024, and U.S. Provisional Application No. 63 / 691,884, filed September 6, 2024, each of which is incorporated by reference herein in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application contains an electronic Sequence Listing in XML file format named “DCT_009WO_SL,” created on May 15, 2025, and having a size of 43.9 kilobytes, the contents of which are incorporated by reference herein in their entirety.TECHNICAL FIELD100031 The present technology relates to immunotherapy and, in particular, to engineered polypeptides for immune cell activation.BACKGROUND

[0004] Since its development, chimeric antigen receptor (CAR) T-cell therapy has shown promise for treating cancers, particularly blood cancers, that may not be effectively treated using more conventional cancer therapies, such as chemoradiation therapy. However, cancer relapse following CAR T-cell therapy continues to be a concern. One mechanism of relapse following CAR T-cell therapy is due to poor persistence of T-cells in the patient over time. Additional limitations of conventional CAR T-cell therapy include toxicity from off-tumor targeting, reduced efficacy over time due to innate and acquired tumor heterogeneity, and time and high cost of CAR T-cell manufacturing.BRIEF DESCRIPTION OF THE DRAWINGS100051 Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale. Instead, emphasis is placed on illustrating clearly the principles of the present disclosure.

[0006] FIGS. 1A-1E schematically illustrate representative examples of immune- activating conjugates.

[0007] FIG. 2A is a schematic illustration of an immune-activating conjugate together within an immune cell and a target cell.

[0008] FIG. 2B is a schematic illustration of an immune-activating conjugate together within an immune cell and a target cell.

[0009] FIG. 3 illustrates the structure of an engineered polypeptide targeting CD 123 (“MP”).

[0010] FIG. 4 illustrates a conjugation scheme for coupling of an activator to an engineered polypeptide via acylation.

[0011] FIG. 5A illustrates a conjugation scheme for coupling a fluorescein (FL) or DOTA activator to an engineered polypeptide via lysine or N-terminal acylation.

[0012] FIG. 5B illustrates four different species that may result from the conjugation scheme of FIG. 5A: an engineered polypeptide with no activator (DAR = 0), an engineered polypeptide with an activator conjugated to the lysine residue only (DAR = 1), an engineered polypeptide with an activator conjugated to the N-terminus only (DAR = 1), and engineered polypeptide with activators conjugated to both the lysine residue and the N-terminus (DAR = 2).

[0013] FIG. 6 is a flow diagram of a process for preparing an immune-activating conjugate.

[0014] FIG. 7 illustrates the chemical structures of NHS-functionalized fluorescein activators with varying linkers: no linker (NHS-5-FL), short alkyl linker (NHS-5-EX-FL), PEG linker with 6 repeating ethylene oxide units (fluorescein-PEG6-NHS ester), and PEG linker with 12 repeating ethylene oxide units (carboxyfluorescein-PEG12-NHS).

[0015] FIGS. 8A-8D illustrate conjugation schemes for coupling an engineered polypeptide to NHS-5-FL (FIG. 8A), NHS-5-EX-FL (FIG. 8B), fluorescein-PEG6-NHS ester (FIG. 8C), and carboxyfluorescein-PEG12-NHS (FIG. 8D).

[0016] FIG. 9 is a representative HPLC trace of an MP-43K-5-FL conjugate at 215 nm (top), 280 nm (middle), and 493 nm (bottom).

[0017] FIGS. 10A-10D illustrate anti-FL CAR T-cell killing mediated by different MP lysine variant-FL conjugates. FIG. 10A is a comparison of anti-FL CAR T-cell killing of MV-4- 11 cells mediated by different MP lysine variant-FL conjugates with no linker. FIG. 10B is a comparison of anti-FL CAR T-cell killing of MV-4-11 cells mediated by different MP lysine variant-FL conjugates with a short alkyl linker. FIG. 10C is a comparison of anti-FL CAR T-cell killing of MV-4-11 cells mediated by different MP lysine variant-FL conjugates with a PEG6linker. FIG. 10D is a comparison of anti-FL CAR T-cell killing of MV-4-11 cells mediated by different MP lysine variant-FL conjugates with a PEG12 linker. The MP polypeptides in FIGS. 10A-10D were synthesized by biological expression and coupled to the FL activator by lysine conjugation.

[0018] FIG. 10E is a comparison of anti-FL CAR T-cell killing of MV-4-11 cells mediated by a talacotuzumab 5-maleimidyl-fluorescein conjugated antibody (“TALA-FL HI,” a DAR 5.5 IgGl stochastic cysteine conjugate) relative to a fluoresceinated MP-43K conjugate with a short alkyl linker (MP-43K(PS) / 5-EX-FL) and a MP-43K conjugate with no linker (MP- 43K(PS) / FITC). “PS” signifies that the MP polypeptide was synthesized by chemical peptide synthesis. MP-43K(PS) / 5-EX-FL was prepared by NHS-5-EX-FL conjugation of chemically synthesized MP-43K. MP-43K(PS) / FITC was prepared by chemical peptide synthesis using a FITC-modified amino acid during peptide synthesis.

[0019] FIG. 11 is a graph showing graphs illustrating MV-4-11 tumor killing efficacy of anti-FL CAR T-cells mediated by different MP -FL lysine conjugates and talacotuzumab (TALA)- FL stochastic cysteine conjugates.[0020[ FIG. 12A shows tumor killing mediated by the MP-43K-5-EX-FL conjugate against second generation aF7 anti-FL CAR T-cells.

[0021] FIG. 12B shows tumor killing mediated by the TALA-FL HI antibody against second generation aF7 anti-FL CAR T-cells.

[0022] FIG. 12C shows tumor killing mediated by the MP-43K-5-EX-FL versus the TALA-FL HI antibody against aF6 anti-FL CAR T-cells.

[0023] FIG. 13 schematically illustrates an immune-activating conjugate.

[0024] FIGS. 14A and 14B illustrate reaction schemes for conjugating an engineered polypeptide to an activator via isothiocyanate / lysine coupling.

[0025] FIGS. 15A-15D illustrate reaction schemes for conjugating an engineered polypeptide to an activator via strain-promoted azide-alkyne cycloaddition (SPAAC), metal-free click chemistry.

[0026] FIGS. 16A-16D illustrate reaction schemes for preparation of clickable immune- activating conjugates via one pot sequential lysine and N-terminal conjugations.

[0027] FIGS. 17A and 17B illustrate reaction schemes for preparation of dimeric immune- activating conjugates using Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), click chemistry.

[0028] FIGS. 18A and 18B illustrate reaction schemes for preparation of dimeric immune- activating conjugates using a bifunctional crosslinker and CuAAC.

[0029] FIG. 19A is a graph illustrating anti-FL CAR T-cell killing of tumor cells mediated by MP-43K-5-EX-FL at doses of 1 nM and 0.1 nM. Specific tumor cell killing was mediated by two concentrations of MP-43K-5-EX-FL with two different batches of anti-FL CAR T-cells that had been stored for 5 months.

[0030] FIGS. 19B and 19C are graphs illustrating anti-FL CAR T-cell killing of tumor cells mediated by various clickable and dimeric MP / FL conjugates at a dosage of 1 nM (FIG. 19B) and 0.1 nM (FIG. 19C).

[0031] FIGS. 20 and 21 illustrate reaction schemes for preparing a divalent MP conjugate with a bridging activator.

[0032] FIG. 22A is a schematic illustration of an immune-activating conjugate including a single engineered polypeptide coupled to a single activator (“DAR = 1”).

[0033] FIG. 22B is a schematic illustration of an immune-activating conjugate including a single engineered polypeptide coupled to two activators via a bivalent linker (“DAR = 2”).

[0034] FIG. 22C is a schematic illustration of an immune-activating conjugate including a single engineered polypeptide coupled to three activators via a trivalent linker (“DAR = 3”).

[0035] FIG. 22D is a schematic illustration of an immune-activating conjugate including a single engineered polypeptide coupled to two different activators (“Engager 1” and “Engager 2” via linker.

[0036] FIG. 23 A illustrates a reaction scheme for preparing a DAR = 1 immune-activating conjugate via lysine acylation and SPAAC.

[0037] FIG. 23B illustrates a reaction scheme for preparing a DAR = 2 immune-activating conjugate via lysine acylation and SPAAC.

[0038] FIGS. 23C and 23D illustrate a reaction scheme for preparing a DAR = 3 immune- activating conjugate via lysine acylation and SPAAC.

[0039] FIG. 24A illustrates a reaction scheme for preparing a DAR = 1 immune-activating conjugate via lysine acylation and CuAAC.

[0040] FIG. 24B illustrates a reaction scheme for preparing a DAR = 2 immune-activating conjugate via lysine acylation and CuAAC.

[0041] FIGS. 24C and 24D illustrate a reaction scheme for preparing a DAR = 3 immune- activating conjugate via lysine acylation and CuAAC.

[0042] FIG. 25 is a graph illustrating anti-FL CAR T-cell killing of MV-4-11 cells mediated by MP conjugates with different DARs and 5-isomers or 6-isomers of fluorescein.

[0043] FIG. 26A schematically illustrates an assay for indirect measurement of internalization of antibodies / conjugates.

[0044] FIG. 26B schematically illustrates an assay for direct measurement of internalization of antibodies / conjugates.

[0045] FIG. 27 illustrates a reaction scheme for conjugation of MP-43K to a pHAb pH- sensitive dye.

[0046] FIG. 28 is a graph illustrating anti-FL CAR T-cell killing of MV-4-11 cells mediated by MP-43K-5-FL (no linker) and MP-43K-5-EX-FL conjugates relative to the TALA- FL stochastic cysteine conjugate control.

[0047] FIG. 29 provides graphs illustrating the gMFI of cells incubated with the TALA- FL antibody (left) compared to the MP conjugates (right).

[0048] FIG. 30A provides flow cytometry plots illustrating fluorescence of MV-4-11 cells incubated with TALA-FL antibodies versus MP-43K-5-FL conjugates.

[0049] FIG. 30B provides graphs illustrating the mean fluorescence of MV-4-11 cells incubated with TALA-FL antibodies versus MP-43K-5-FL conjugates.

[0050] FIG. 31 A provides flow cytometry plots illustrating fluorescence of MV-4-11 cells incubated with TALA-FL antibodies versus MP-43K-5-FL conjugates.]0051 ] FIG. 3 IB provides graphs illustrating the mean fluorescence of MV-4-11 cells incubated with TALA-FL antibodies versus MP-43K-5-FL conjugates.

[0052] FIG. 32A provides flow cytometry plots illustrating fluorescence of TALA-pHAb versus MP-pHAb conjugates in the presence of varying pH buffers.

[0053] FIG. 32B provides flow cytometry plots illustrating fluorescence of MV-4-11 cells incubated with TALA-pHAb antibodies versus MP-pHAb conjugates.

[0054] FIG. 32C provides graphs illustrating the mean fluorescence of MV-4-11 cells incubated with TALA-pHAb antibodies versus MP-pHAb conjugates.

[0055] FIGS. 33A and 33B are graphs showing calibration curves for MP-43K-5-EX-FL fluorescence versus concentration for the FLISA assay (excitation at 493 nm and emission at 530 nm).

[0056] FIGS. 34A and 34B are graphs of the mean plasma concentration of the MP conjugate in naive mice over time for 1 mg / kg versus 10 mg / kg dosing (FIG. 34A shows concentration in pg / mL and FIG. 34B shows concentration in nM).

[0057] FIG. 35 is a graph of the mean plasma concentration of the MP conjugate in naive versus tumor-bearing mice over time at 10 mg / kg dosing.

[0058] FIG. 36 is a graph illustrating tumor burden based on mean bioluminescence intensity (BLI) across mice receiving various treatments.

[0059] FIGS. 37A-37F illustrate tumor burden based on mean BLI for individual mice in the vehicle only group (FIG. 37A), BAT-CAR cells with 1 mg / kg Q2D TALA-FL antibody (FIG. 37B), BAT-CAR cells with 1 mg / kg QW TALA-FL antibody (FIG. 37C), BAT-CAR cells with non-binding polypeptide conjugate (FIG. 37D), BAT-CAR cells with MP conjugate (FIG. 37E), and direct CAR T-cells (FIG. 37F).

[0060] FIG. 38 is a graph showing the percent body weight change across mice receiving various treatments.

[0061] FIGS. 39A and 39B are graphs illustrating CAR+ cell counts (FIG. 39A) and CAR+ cell percentages (FIG. 39B) in mice treated with BAT-CAR cells and MP conjugates, BAT-CAR cells and TALA-FL antibodies, or direct CAR T-cells.[0062J FIG. 40 provides charts showing the number of CAR T-cell exhaustion markers (PD-1, TIM3, LAG3) in mice treated with BAT-CAR cells and MP conjugates, BAT-CAR cells and TALA-FL antibodies, or direct CAR T-cells.

[0063] FIG. 41 provides a series of graphs showing the phenotypes of CAR+ cells (CD45RA versus CCR7 and CD45RA versus CD62L) in mice treated with BAT-CAR cells (“5m”), BAT-CAR cells and MP conjugates (“5m+MP”), BAT-CAR cells and TALA-FL antibodies (“5m+TALA”), or direct CAR T-cells.

[0064] FIG. 42 provides flow cytometry plots showing fluorescence in bone marrow cells (left) and graphs showing the percentages of bound receptors and gMFI in bone marrow cells (right) for mice treated with vehicle only, TALA-FL antibodies, or MP conjugates.[00651 FIG. 43 A is a graph illustrating tumor burden based on mean BLI across treatment groups (top) and for individual mice (bottom) for mice treated with BAT-CAR cells and MP-43K- 5 -EX-FL conjugates at a dosage of 10 mg / kg daily up to Day 18.

[0066] FIG. 43B is a graph illustrating tumor burden based on BLI across treatment groups (top) and for individual mice (bottom) for mice treated with BAT-CAR cells and MP-43K-5-EX- FL conjugates at a dosage of 10 mg / kg daily up to Day 28.

[0067] FIG. 44A is a graph illustrating tumor burden based on mean BLI across treatment groups (top) and for individual mice (bottom) for mice treated with IxlO6BAT-CAR cells and 5 mg / kg MP-43K-5-EX-FL conjugates at different dosing frequencies.(0068[ FIG. 44B is a graph illustrating tumor burden based on mean BLI across treatment groups (top) and for individual mice (bottom) for mice treated with 5xl06BAT-CAR cells and 5 mg / kg MP-43K-5-EX-FL conjugates at different dosing frequencies.

[0069] FIG. 45A is a graph illustrating tumor burden based on mean BLI across treatment groups (top) and for individual mice (bottom) for mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL conjugates at different dosing frequencies.

[0070] FIG. 45B is a graph illustrating tumor burden based on mean BLI across treatment groups (top) and for individual mice (bottom) for mice treated with 5xl06BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL conjugates at different dosing frequencies.

[0071] FIG. 46 is a graph illustrating the percent change in body weight across treatment groups for the dose response study.

[0072] FIG. 47 is a series of bioluminescent images of mice treated with IxlO6or 5xl06BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL conjugates at various dosing frequencies.

[0073] FIG. 48A is a graph illustrating tumor burden based on mean BLI (top) and for individual mice (bottom) for mice treated with direct anti-CD123 CAR T-cells.

[0074] FIG. 48B is a graph illustrating tumor burden based on mean BLI (top) and for individual mice (bottom) for mice treated with BAT-CAR cells and MP-43K-5-EX-FL conjugates.

[0075] FIG. 49 is a graph showing the percent survival of mice treated with varying dosages of BAT-CAR cells and MP-43K-5-EX-FL conjugates.

[0076] FIG. 50A is a graph illustrating tumor burden based on mean BLI across various MP-43K-5-EX-FL dosages for mice treated with IxlO6BAT-CAR cells.

[0077] FIG. 50B is a graph illustrating tumor burden in individual mice treated with IxlO6BAT-CAR cells and a MP-43K-5-EX-FL dosage of 0.01 mg / kg.

[0078] FIG. 50C is a graph illustrating tumor burden in individual mice treated with IxlO6BAT-CAR cells and a MP-43K-5-EX-FL dosage of 0.1 mg / kg.

[0079] FIG. 50D is a graph illustrating tumor burden in individual mice treated with IxlO6BAT-CAR cells and a MP-43K-5-EX-FL dosage of 1 mg / kg.

[0080] FIG. 51 A is a graph illustrating tumor burden based on mean BLI across various MP-43K-5-EX-FL dosages for mice treated with 0.5xl06BAT-CAR cells.

[0081] FIG. 5 IB is a graph illustrating tumor burden in individual mice treated with 0.5xl06BAT-CAR cells and a MP-43K-5-EX-FL dosage of 0.1 mg / kg.

[0082] FIG. 51C is a graph illustrating tumor burden in individual mice treated with 0.5xl06BAT-CAR cells and a MP-43K-5-EX-FL dosage of 1 mg / kg.

[0083] FIG. 52 is a series of bioluminescent images of mice treated with varying dosages of BAT-CAR cells and MP-43K-5-EX-FL.

[0084] FIG. 53A is a graph illustrating tumor burden based on mean BLI between mice treated with a vehicle control and mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K- 5-EX-FL.

[0085] FIG. 53B is a graph illustrating CAR+ cell expansion in blood samples from mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL.

[0086] FIG. 53C is a graph illustrating CAR+ cell expansion in bone marrow samples from mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL.

[0087] FIG. 54A shows graphs representing counts and percentages of CD8+ CAR+ cells in blood samples at various time points from mice treated with IxlO6BAT-CAR cells and 1 mg / kgMP-43K-5-EX-FL.

[0088] FIG. 54B shows graphs representing counts and percentages of CD4+ CAR+ cells in blood samples at various time points from mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL.

[0089] FIG. 54C shows graphs representing counts and percentage of CD8+ CAR+ cells in bone marrow samples at various time points from mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL.

[0090] FIG. 54D shows graphs representing the number and percentage of CD4+ CAR+ cells in bone marrow samples at various time points from mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL.[00911 FIGS. 55A-55C are graphs showing the frequency of CAR+ cells in blood and bone marrow samples at various time points from mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL.

[0092] FIGS. 56A and 56B are graphs showing the percentage of CAR+ cells expressing exhaustion markers in blood and bone marrow samples from mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL.

[0093] FIGS. 57A-57E are graphs showing the percentage of Tern, Tcm, Temra, and Tscm phenotypes expressed in CAR+ cell populations in blood samples (FIGS. 57A-57D) and bone marrow samples (FIG. 57E) from mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K- 5-EX-FL.

[0094] FIG. 58A is a schematic showing the detection of surface MP-43K-5-EX-FL on BAT-CAR cells.

[0095] FIG. 58B is a series of flow cytometry plots showing the detection of surface MP- 43K-5-EX-FL in blood samples from individual mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL.

[0096] FIG. 58C is a series of flow cytometry plots showing the detection of surface MP- 43K-5-EX-FL in bone marrow samples from individual mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL.

[0097] FIG. 58D is a pair of graphs showing the surface residency of MP-43K-EX-FL on BAT-CAR cells in blood and bone marrow samples at various time points from mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL.DETAILED DESCRIPTION

[0098] The present technology provides compositions for use in immunotherapy, such as chimeric antigen receptor (CAR) T-cell therapy, and associated methods. In some embodiments, for example, a system for immunotherapy is provided, the system including a first composition including an engineered polypeptide conjugated to a small molecule activator, and a second composition including an immune cell expressing an engineered receptor that binds to the small molecule activator. The engineered polypeptide can be a low molecular weight protein (e.g., a miniprotein) having a molecular weight less than or equal to 10 kDa. The engineered polypeptide can bind to a marker on a target cell, thereby directing the immune cell to the target cell. In some embodiments, the immune cell is a CAR T-cell, and the engineered receptor is a CAR of the CAR T-cell that initiates a cytolytic response to kill the target cell upon binding of the engineered receptor to the small molecule activator and binding of the engineered polypeptide to the marker on the target cell.

[0099] The compositions and methods described herein can provide many advantages compared to conventional immunotherapy approaches. For instance, many conventional immunotherapies use antibody-based therapeutics to target diseased cells. However, antibodybased therapeutics generally require complex and costly manufacturing processes and have stringent storage requirements to maintain stability. In contrast, the engineered polypeptides described herein exhibit high binding affinity, binding specificity, and stability, and can be manufactured at an industrial scale via simple and cost-effective processes. The small size of the engineered polypeptides can also provide various therapeutic advantages, such as rapid clearance for tighter control over dosing and avoiding prolonged immune cell activation that may lead to immune cell exhaustion, as well as reducing the size of the immunological synapse for more efficient T-cell activation. Moreover, the engineered polypeptides herein can be designed with conjugation sites at specific locations, thus allowing for precise control over the architecture and valency of the resulting conjugate. In some embodiments, the engineered polypeptides exhibit some or all of the following advantages compared to larger biologies such as antibodies and antibody fragments: monovalent binding; enhanced stability toward pH, temperature, organic solvents, and chemical modification; reduced immunogenicity; and ability to be synthesized by either biological expression or chemical synthesis.

[0100] Embodiments of the present disclosure will be described more fully hereinafter with reference to the accompanying drawings in which example embodiments are shown. Embodiments of the claims may, however, be embodied in many different forms and should notbe construed as limited to the embodiments set forth herein. The examples set forth herein are nonlimiting examples and are merely examples among other possible examples.

[0101] The headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed present technology. Embodiments under any one heading may be used in conjunction with embodiments under any other heading.I. Immune- Activating Conjugates

[0102] The present technology provides compositions for activating immune cells, such as CAR T-cells. In some embodiments, a composition includes an engineered polypeptide that binds to a target cell and an activator that binds to a receptor on an immune cell. The composition can be administered to a subject together with a population of immune cells having the receptor that recognizes the activator. The composition can direct the immune cells to the target cell via binding of the engineered polypeptide to the target cell and binding of the receptor to the activator. In some embodiments, binding of the activator to the receptor causes activation of immune cell activity, such as cytolytic activity, cytokine signaling activity, conversion to a desired immune cell phenotype, homing to lymphoid organs, etc.

[0103] FIGS. 1A-1E schematically illustrate representative examples of immune- activating conjugates, in accordance with embodiments of the present technology. Referring first to FIG. 1A, an immune-activating conjugate 100a can include an engineered polypeptide 102 coupled to an activator 104. The engineered polypeptide 102 can be a low molecular weight protein (e.g., a miniprotein) that has high binding affinity for a marker on a target cell. Additional details of the engineered polypeptide 102 are provided in Section I. A. below. The activator 104 can be an exogenous small molecule that is recognized by an engineered receptor on an immune cell, such as a CAR or a chimeric cytokine receptor (CCR). Additional details of the activator 104 are provided in Section I.B below, and additional details of immune cells with engineered receptors are provided in Section II below. In the illustrated embodiment, the engineered polypeptide 102 is covalently conjugated to the activator 104 via a linker 106. Alternatively, the engineered polypeptide 102 may be conjugated directly to the activator 104 without a linker. The conjugation sites on the engineered polypeptide 102 and the activator 104, as well as the characteristics of the linker 106 (when present) can be selected to avoid interfering with binding of the engineered polypeptide 102 to the target and recognition of the activator 104 by the immune cell receptor. Additional details of the linker 106 are provided in Section I.C below, and additional details of conjugation techniques are provided in Section I D. below.

[0104] Although FIG. 1A illustrates the immune-activating conjugate 100a as being composed of a single engineered polypeptide 102 conjugated to a single activator 104, in other embodiments, an immune-activating conjugate may include a plurality of engineered polypeptides 102 (e.g., two, three, four, five, or more engineered polypeptides 102) and / or a plurality of activators 104 (e.g., two, three, four, five, or more activators 104). Moreover, an immune- activating conjugate may include any suitable number of linkers 106 (e.g., two, three, four, five, or more linkers 106) to couple to the engineered polypeptide(s) 102 and / or activator(s) 104 to each other in the desired architecture.

[0105] For example, FIG. IB illustrates an immune-activating conjugate 100b including a single engineered polypeptide 102 coupled to two activators 104. The activators 104 can be the same molecule or can be different molecules. The activators 104 can be conjugated to different sites on the engineered polypeptide 102 via respective linkers 106. The immune-activating conjugate 100b with two activators 104 may be advantageous, for example, if receptor dimerization is beneficial for eliciting immune cell activity.

[0106] FIG. 1C illustrates an immune-activating conjugate 100c including a single engineered polypeptide 102 coupled to two activators 104. The activators 104 can be the same molecule or can be different molecules. The activators 104 can be conjugated to the engineered polypeptide 102 via a multivalent linker 106. The immune-activating conjugate 100c with two activators 104 may be advantageous, for example, if receptor dimerization is beneficial for eliciting immune cell activity.{0107] FIG. ID illustrates an immune-activating conjugate lOOd including two engineered polypeptides 102 coupled to a single activator 104. The engineered polypeptides 102 can be the same polypeptide or can be different polypeptides. The engineered polypeptides 102 can be conjugated to the activator 104 via a multivalent linker 106. The immune-activating conjugate lOOd with two engineered polypeptides 102 may be advantageous, for example, to increase binding avidity to the target cell.

[0108] FIG. IE illustrates an immune-activating conjugate lOOe including two engineered polypeptides 102 and two activators 104. The engineered polypeptides 102 can be the same polypeptide or can be different polypeptides, and the activators 104 can be the same molecule or can be different molecules. As shown in FIG. IE, a first engineered polypeptide 102 can be conjugated to a first activator 104 via a first linker 106, a second engineered polypeptide 102 can be conjugated to a second activator 104 via a second linker 106, and the first engineeredpolypeptide 106 can be conjugated to the second engineered polypeptide 106 via a third linker 106. The immune-activating conjugate lOOe with two engineered polypeptides 102 and two activators 104 may be advantageous, for example, to increase binding avidity to the target cell and / or to promote receptor dimerization.

[0109] FIG. 2A is a schematic illustration of the immune-activating conjugate 100a together within an immune cell 200 and a target cell 202, in accordance with embodiments of the present technology. The engineered polypeptide 102 of the immune-activating conjugate 100a can bind to a surface marker 204 expressed by the target cell 202. For instance, the surface marker 204 can be a tumor antigen and the target cell 202 can be a cancer cell. The immune cell 200 can be a CAR T-cell that expresses a CAR 206 that binds to the activator 104 of the immune-activating conjugate 100a, rather than binding the surface marker 204 directly. The binding interactions shown in FIG. 2 A can activate a cytolytic response in the CAR T-cell, resulting in killing of the target cell 202.[one] FIG. 2B is a schematic illustration of the immune-activating conjugate 100b together within an immune cell 200 and a target cell 202, in accordance with embodiments of the present technology. The engineered polypeptide 102 of the immune-activating conjugate 100b can bind to a surface marker 204 expressed by the target cell 202. For instance, the surface marker 204 can be a tumor antigen and the target cell 202 can be a cancer cell. The immune cell 200 can be a CAR T-cell that expresses a CCR 208 that binds to the activators 104 of the immune-activating conjugate 100b. In the illustrated embodiment, the CCR 208 is composed of two receptor chains that each bind to a respective activator 104. Binding of the receptor chains to the activators 104 can cause dimerization and initiation of cytokine signaling by the CCR 208, leading to immune cell activity such as phenotypic conversion of the immune cell 200, homing to lymphoid organs, etc.

[0111] The embodiments of FIGS. 2A and 2B may be combined with each other, such that the immune cell 200 expresses both a CAR 206 and a CCR 208 that bind to the immune-activating conjugates 100a, 100b, respectively. In such embodiments, the CAR 206 and CCR 208 can be engineered to recognize the same activator 104 or different activators 104. The engineered polypeptide 102 that is conjugated to the activator 104 recognized by the CAR 206 may be the same as the engineered polypeptide 102 that is conjugated to the activator 104 recognized by the CCR 208. Accordingly, the immune-activating conjugate 100a for the CAR 206 and the immune- activating conjugate 100b for the CCR 208 may recognize the same surface marker 204 on the target cell 202 or may recognize different surface markers 204 on the target cell 202.

[0112] Although FIGS. 2A and 2B are illustrated and described in connection with the immune-activating conjugates 100a, 100b of FIGS. 1A and IB, respectively, in other embodiments, any of the other immune-activating conjugates described herein may be used to activate an immune cell, such as the embodiments of FIGS. 1C-1E. The architecture of the conjugate (e.g., the number and type of engineered polypeptides, activators, and linkers) may be varied as appropriate based on the immune cell receptor, surface marker, desired immune cell activity, etc.A. Engineered Polypeptides101131 The compositions and methods described herein incorporate engineered polypeptides that have high binding affinity to a marker on a target cell. In some embodiments, the engineered polypeptides described herein have a small size compared to conventional proteinbased targeting molecules, such as antibodies and antibody fragments. Without wishing to be bound by theory, it is hypothesized that the small size of the engineered polypeptides herein can provide various advantages for immunotherapy, such as formation of a smaller immunological synapse for more effective CAR T-cell activation (e.g., in embodiments where the engineered polypeptide is part of an immune-activating conjugate that binds to a CAR), rapid clearance of unbound polypeptide conjugates for better control of immune cell activation and / or reduced immune cell exhaustion, improved stability, and simpler and more cost-effective manufacturing by biological expression or chemical peptide synthesis. Other benefits of the engineered polypeptides disclosed herein may include monovalent binding instead of bivalent binding exhibited by conventional antibodies, which may lead to slower target cell internalization relative to conventional antibodies. In some embodiments of the present technology, it is advantageous for the engineered polypeptide to remain on the surface of the cell with minimal internalization, e.g., to facilitate immune cell recognition of the activator conjugated to the polypeptide. In embodiments where the engineered polypeptide is conjugated to a small molecule activator, the relatively small size of the polypeptide-activator conjugates described herein may allow for formation of a highly stable complex with CAR T-cells leading to improved and more durable tumor cell killing.

[0114] In some embodiments, an engineered polypeptide of the present technology has a molecular weight less than or equal to 40 kDa, 30 kDa, 20 kDa,10 kDa, 9 kDa, 8 kDa, 7 kDa, 6 kDa, 5 kDa, 4 kDa, 3 kDa, 2 kDa, or 1 kDa; and / or the molecular weight can be within a range from 1 kDa to 40 kDa, 1 kDa to 10 kDa, 1 kDa to 5 kDa, 1 kDa to 2 kDa, 2 kDa to 8 kDa, 2 kDa to 5 kDa, 3 kDa to 7 kDa, 4 kDa to 6 kDa, 5 kDa to 8 kDa, 5 kDa to 10 kDa, 8 kDa to 10 kDa, 10kDa to 20 kDa, or 20 kDa to 40 kDa. In some embodiments, an engineered polypeptide of the present technology is composed of no more than 500 amino acid residues, 400 amino acid residues, 300 amino acid residues, 200 amino acid residues, 100 amino acid residues, 90 amino acid residues, 80 amino acid residues, 70 amino acid residues, 60 amino acid residues, 50 amino acid residues, 40 amino acid residues, 30 amino acid residues, 20 amino acid residues, or 10 amino acid residues; and / or the engineered polypeptide has from 10 amino acid residues to 500 amino acid residues, 10 amino acid residues to 100 amino acid residues, 10 amino acid residues to 50 amino acid residues, 20 amino acid residues to 80 amino acid residues, 30 amino acid residues to 70 amino acid residues, 40 amino acid residues to 60 amino acid residues, 100 amino acid residues to 200 amino acid residues, or 200 amino acid residues to 500 amino acid residues.

[0115] In some embodiments, the engineered polypeptides of the present technology have a highly stable 3D structure compared to conventional protein-based targeting molecules, such as antibodies and antibody fragments. For example, the engineered polypeptides described herein can be stable under one or more of the following conditions: exposure to a temperature of at least 40 °C, 50 °C, 60 °C, 70°C, 80 °C, 90°C, or 100 °C; exposure to a temperature less than 30 °C, 20 °C, or 10 °C; exposure to a pH less than 7, 6, 5, 4, 3, 2, or 1; exposure to a pH greater than 8, 9, 10, 11, 12, or 13; placement in a solution containing a concentration (% w / v) of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% organic solvent (e.g., dimethylsulfoxide (DMSO)); exposure to proteases (e.g., pepsin, trypsin, serum proteases); and / or exposure to a pressure greater than 1 atm. An engineered polypeptide may be considered stable if the polypeptide maintains its 3D structure and / or functional properties when exposed to non-physiological and / or extreme conditions (e.g., high temperatures, acidic pH), or if the polypeptide recovers its 3D structure and / or functional properties after temporary exposure to non-physiological and / or extreme conditions (e.g., exposure for at least 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 12 hours, or 24 hours). In some embodiments, an engineered polypeptide may partially unfold under elevated temperatures and may refold when returned to room temperature (e.g., 20-25 °C) or physiological temperature (e.g., 37 °C). This may be advantageous in embodiments where partial unfolding of the engineered polypeptide improves accessibility of a conjugation site to another molecule (e.g., an activator or linker) during a conjugation reaction.10116] The stability of the engineered polypeptides may be attributable to features such as formation of a hydrophobic core and / or presence of intramolecular linkages (e.g., disulfide bridges between cysteine resides), for example. In some embodiments, the engineered polypeptides hereininclude a hydrophobic core and one or more intramolecular linkages (e.g., one or more disulfide bridges). In some embodiments, the engineered polypeptides herein include a hydrophobic core without any intramolecular linkages (e.g., no disulfide bridges). In some embodiments, the engineered polypeptides herein one or more intramolecular linkages (e.g., one or more disulfide bridges) without a hydrophobic core.

[0117] In some embodiments, an engineered polypeptide of the present technology includes 2, 3, 4, 5, 6, or more secondary structure domains; and / or has from 2 to 6, 2 to 5, 2 to 4, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, or 5 to 6 secondary structure domains. A secondary structure domain can be an a-helix or a P-sheet, for example. The secondary structure domain for an a-helix may be at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues in length; and / or may have a length within a range from 4 amino acid residues to 9 amino acid residues, or 4 amino acid residues to 20 amino acid residues. The secondary structure domain for an P-sheet may be at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 acid residues in length; and / or may have a length within a range from 4 amino acid residues to 15 amino acid residues, or 4 amino acid residues to 20 amino acid residues. The secondary structure domains may be connected to each other by one or more loops, with each loop independently having a length within a range from 2 amino acid residues to 5 amino acid residues, 5 amino acid residues to 10 amino acid residues, 10 amino acid residues to 20 amino acid residues, or 20 amino acid residues to 25 amino acid residues, or more than 25 amino acid residues.

[0118] In some embodiments, an engineered polypeptide of the present technology includes one pair of cysteine residues that form one disulfide bridge, 2 pairs of cysteine residues that form 2 disulfide bridges, 3 pairs of cysteine residues that form 3 disulfide bridges, 4 pairs of cysteine residues that form 4 disulfide bridges, 5 pairs of cysteine residues that form 5 disulfide bridges, etc. Each member of a pair of cysteine residues may be present in the same secondary structure domain or may be present on different secondary structure domains. In some embodiments, the engineered polypeptide contains a cysteine knot motif formed by 6 cysteine residues that are linked via 3 disulfide bridges between Cys(I) to Cys(IV), Cys(II) to Cys(V), and Cys(III) to Cys(VI), where the Cys(III)-Cys(VI) bridge crosses the loop formed by the other two disulfide bridges. In other embodiments, however, the engineered polypeptide may not include any cysteine residues that form disulfide bridges.

[0119] At physiological pH (e.g., pH 7.4), the engineered polypeptides described herein can have a net negative charge, a net positive charge, or can have no net charge. In some embodiments, an engineered polypeptide has a net charge less than or equal to 0, -0.5, -1, -1.5, -2,-2.5, -3, -3.5, -4, -4.5, -5, -5.5, -6, -6.5, -7, -7.5, -8, -8.5, -9, -9.5, or -10; and / or the net charge is within a range from -1 to -5, -2 to -4, -2 to -6, -3 to -7, -3 to -5, -4 to -8, -4 to -6, -5 to -10, -5 to - 7, -6 to -8, or -8 to -10. The engineered polypeptide can have at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 negatively charged amino acid residues (e.g., aspartic acid or glutamic acid). In some embodiments, an engineered polypeptide has a net charge greater than or equal to 0, +0.5, +1, +1.5, +2, +2.5, +3, +3.5, +4, +4.5, +5, +5.5, +6, +6.5, +7, +7.5, +8, +8.5, +9, +9.5, or +10; and / or the net charge is within a range from +1 to +5, +2 to +4, +2 to +6, +3 to +7, +3 to +5, +4 to +8, +4 to +6, +5 to +10, +5 to +7, +6 to +8, or +8 to +10. The engineered polypeptide can have at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 positively charged amino acid residues (e.g., lysine, arginine, histidine). The net charge of the engineered polypeptides may be assessed in a suitable solvent, e.g., phosphate-buffered saline at pH 7.4.

[0120] In some embodiments, the engineered polypeptide is a miniprotein. Miniproteins are small size proteins (typically less than 10 kDa) having a stable 3D structure. Examples of miniproteins include hydrophobic core miniproteins, cysteine-reinforced miniproteins, and avimers. Hydrophobic core miniproteins include rigid secondary structures (e.g., a-helices, P- sheets) arranged around a hydrophobic core, with folding driven by solvent exclusion. The hydrophobic core can be composed of aromatic and / or aliphatic side chains of hydrophobic amino acid residues (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, cysteine) that are sequestered from solvent when the miniprotein is folded. In some embodiments, a hydrophobic core miniprotein includes at least one, two, three, four, five, six, seven, eight, nine, ten, or more hydrophobic amino acid residues. Cysteine-reinforced miniproteins (also known as cysteine-rich miniproteins) utilize disulfide bridges between cysteine residues to drive folding and provide stability. Avimers are based on loop-rich A domains of cell surface receptors and involve coordination of a calcium ion.

[0121] An engineered polypeptide of the present technology can exhibit specific binding to a target of interest (e.g., on a marker on a target cell). The target that is recognized by the engineered polypeptide can be any target relevant to immunotherapy. In some embodiments, for example, the engineered polypeptide binds to a tumor antigen expressed by a cancer cell. Examples of tumor antigens include CD19, CD20, CD22, CD123, CD33, CD3, CD4, CD8, CD38, SLAMF7, BCMA, GD2, GPRC5D, MUC16, HER2, EGFR, EGFRvIII, CLL-1, CD44v6, folate receptor-a, mesothelin, CD20, CD37, R0R1, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, surviving, telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, IL13Ra2, B7-H3 (CD276), EPHA2, GRP78, NKG2D, and CD70.[0122| As another example, the engineered polypeptide can bind to a lymphoid marker of a lymphoid organ, such as a lymph node (e.g., a tumor-draining lymph node), spleen, thymus, or bone marrow. The lymphoid marker can be a surface marker (e.g., a cell surface protein) expressed in one or more lymphoid organs, such as the lymph nodes (e.g., tumor-draining lymph nodes, non- tumor-draining lymph nodes), spleen, thymus, bone marrow, or combinations thereof. For example, the lymphoid marker can be a surface-exposed epitope of a cell surface or transmembrane protein. The lymphoid marker can be expressed by a cell that is resident in or otherwise associated with a lymphoid organ, such as an antigen presenting cell (e.g., a dendritic cell), a T-cell, a B-cell, a lymphocyte, a lymphatic endothelial cell, a B cell, a macrophage, or a lymphoid organ stromal cell. The lymphoid marker can be a surface marker expressed by lymphocytes that reside in lymphoid tissue, such as CD3, CD45, CD4, CD2, CD5, CD8, yb-T-cell receptor, T19, CD45, CD205, cell-surface immunoglobulin (sig), and L-selectin. The lymphoid marker can be a marker of a stromal cell of a lymphoid organ (e.g., lymph node stromal cells), such as PNAd, VEGFR-3, LYVE-1, Prox-1, podoplanin, CD31, MadCAMl, CXCL13, RANKL, CXCL12, APRIL, BAFF, IL-7, CCL19, CCL21, and Spns2.

[0123] The engineered polypeptide can bind to the target with an equilibrium dissociation constant (KD) that is less than or equal to 5xlO'7M, 1X10'7M, 5xl0'8M, 1X10'8M, 5xlO'9M, 1x10'9M, 5xlO'10M, IxlO'10M, 5xl0'nM, IxlO'11M, 5xl0'12M, or IxlO'12M; and / or is within a range from IxlO’7M to IxlO’8M, IxlO’8M to IxlO’9M, IxlO’9M to IxlO’10M, IxlO’10M to IxlO’11M, or IxlO'11M to IxlO'12M. Binding experiments to determine the KD can be performed, for example, in phosphate-buffered saline at pH 7.4 and room temperature (e.g., 20-25 °C) or physiological temperature (e.g., 37 °C).

[0124] In some embodiments, an engineered polypeptide includes one or more sites for conjugation to another molecule (e.g., an activator or a linker). The conjugation site can typically be a lysine residue, a cysteine residue, a histidine residue, a non-natural amino acid residue, the N- terminus of the polypeptide, or the C-terminus of the polypeptide. In some embodiments, the conjugation site is a natural or non-natural amino acid residue bearing a polar side chain. The location of the conjugation site on the engineered polypeptide can be selected based on a variety of factors, such as accessibility for conjugation (e.g., the site is not sequestered within the core ofthe polypeptide), avoiding interference with binding of the engineered polypeptide to the target (e.g., the site is located away from residues that are involved in target recognition), and / or ensuring that an activator coupled to the engineered polypeptide via the conjugation site is accessible to an immune cell receptor.

[0125] In some embodiments, the conjugation site is the N-terminus of the engineered polypeptide. In some embodiments, the conjugation site is located no more than 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from the N-terminus of the engineered polypeptide. In some embodiments, the conjugation site is located at least 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from the N-terminus of the engineered polypeptide.

[0126] In some embodiments, the conjugation site is the C-terminus of the engineered polypeptide. In some embodiments, the conjugation site is located no more than 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from the C-terminus of the engineered polypeptide. In some embodiments, the conjugation site is located at least 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from the C-terminus of the engineered polypeptide.

[0127] In some embodiments, the conjugation site is located within a secondary structure domain (e.g., an a-helix or a P-sheet) of the engineered polypeptide. In some embodiments, the conjugation site is located outside of the secondary structure domains of the engineered polypeptide, e.g., within a loop that connects two secondary structure domains.

[0128] In some embodiments, the engineered polypeptide includes a single conjugation site so that the engineered polypeptide is conjugated to a single linker or activator only. Alternatively, the engineered polypeptide can include a plurality of conjugation sites (e.g., two, three, four, five, or more conjugation sites) so that the engineered polypeptide can be coupled to aplurality of linkers and / or activators. In such embodiments, the location of each conjugation site may be independently selected from any of the types and locations described herein, e.g., a first conjugation site may be the N-terminus of the engineered polypeptide and a second conjugation site may be a lysine residue of the engineered polypeptide.

[0129] Representative examples of engineered polypeptides are provided in Table 1 below.

[0130] Table 1 : Engineered Polypeptides

[0131] In some embodiments, an engineered polypeptide of the present technology has a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% sequence identity to any one of SEQ ID NO: 1 - SEQ ID NO: 5. In some embodiments, the engineered polypeptide has a sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 5.

[0132] Various expression vector / host systems can be utilized for the production of the recombinant expression of the engineered polypeptides described herein. Non-limiting examples of such systems include microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a nucleic acid sequence encoding engineered polypeptides described herein, yeast transformed with recombinant yeast expression vectors containing the aforementioned nucleic acid sequence, insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the aforementioned nucleic acid sequence, plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the aforementioned nucleic acid sequence, or animal cell systems infected with recombinant virus expression vectors (e.g., adenovirus, vaccinia virus) including cell lines engineered to contain multiple copies of the aforementioned nucleic acid sequence, either stably amplified (e.g.,CHO / dhfr, CHO / glutamine synthetase) or unstably amplified in double-minute chromosomes (e.g., murine cell lines). Disulfide bond formation and / or folding of the peptide may occur during expression, after expression, or both.[01331 A host cell can be adapted to express one or more engineered polypeptides described herein. The host cells can be prokaryotic, eukaryotic, or insect cells. In some embodiments, host cells are capable of modulating the expression of the inserted sequences, or modifying and processing the gene or protein product in the specific fashion desired. For example, expression from certain promoters can be elevated in the presence of certain inducers (e.g., zinc and cadmium ions for metallothionine promoters). In some embodiments, modifications (e.g., phosphorylation) and processing (e.g., cleavage) of peptide products can be important for the function of the engineered polypeptide. Host cells can have characteristic and specific mechanisms for the post-translational processing and modification of a polypeptide. In some embodiments, the host cells used to express the engineered polypeptides secrete minimal amounts of proteolytic enzymes.

[0134] In the case of cell- or viral-based samples, organisms can be treated prior to purification to preserve and / or release a target polypeptide. In some embodiments, the cells are fixed using a fixing agent. In some embodiments, the cells are lysed. The cellular material can be treated in a manner that does not disrupt a significant proportion of cells, but which removes proteins from the surface of the cellular material, and / or from the interstices between cells. For example, cellular material can be soaked in a liquid buffer or, in the case of plant material, can be subjected to a vacuum, in order to remove proteins located in the intercellular spaces and / or in the plant cell wall. If the cellular material is a microorganism, proteins can be extracted from the microorganism culture medium. Alternatively, the peptides can be packed in inclusion bodies. The inclusion bodies can further be separated from the cellular components in the medium. In some embodiments, the cells are not disrupted. A cellular or viral peptide that is presented by a cell or virus can be used for the attachment and / or purification of intact cells or viral particles. In addition to recombinant systems, polypeptides can also be synthesized in a cell-free system using a variety of known techniques employed in protein and peptide synthesis.

[0135] In some embodiments, the engineered polypeptides of the present technology are prepared by conventional solid phase chemical synthesis techniques, for example, according to the Fmoc solid phase peptide synthesis method.B. Activators

[0136] An activator may activate an immune cell of the present technology by binding to an engineered receptor of the immune cell (e.g., a CAR or CCR). The activator may be a small molecule, a peptide, an oligonucleotide, or a protein. In some embodiments, an activator may be selected to have low toxicity, low immunogenicity, low cross-reactivity, or combinations thereof to reduce unfavorable side effects when administered to a subject (e.g., a human subject). For instance, the activator can be a molecule that is non-toxic to humans, included in an Inactive Ingredients Database, or both. In some embodiments, the activator is an exogenous activator (e.g., an exogenous small molecule, an exogenous peptide, an exogenous oligonucleotide, or an exogenous protein) that is not naturally present in a target environment (e.g., a human subject) to prevent activation of the engineered receptor in the absence of an external stimulus (e.g., administration of the activator), prevent cross-reactivity of the activator with other biological components, and to enable dynamic control of receptor signaling.

[0137] A small molecule activator can have a molecular weight less than or equal to 5 kDa, 4 kDa, 3 kDa, 2 kDa, 1 kDa, 500 Da, 200 Da, or 100 Da; and / or can have a molecular weight within a range from 50 Da to 500 Da, 100 Da to 500 Da, 100 Da to 1 kDa, 200 Da to 500 Da, 500 Da to 1 kDa, 1 kDa to 2 kDa, 1 kDa to 5 kDa, or 2 kDa to 5 kDa. Examples of small molecule activators (e.g., haptens) that may be used to activate an engineered receptor include fluorophores (e.g., fluorescein, fluorescein derivatives, indocyanines, indocyanine derivatives, cyanines, cyanine derivatives), chelators (e.g., DOTA), or other small molecules. For example, the fluorescein derivative may be fluorescein isothiocyanate (FITC), fluorescein 5-maleimide, fluorescein-5-carboxamide, fluorescein-6-carboxamide, or 6-FAM phosphoramidite.

[0138] In some embodiments, the activator is selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), fluorescein 5-maleimide, fluorescein-5- carboxamide, fluorescein-6-carboxamide, 6-FAM phosphoramidite, topiramate hemi succinate, creatine, acetaminophen, ketamine, propofol, lidocaine, ractopamine, salicylate, salicylic acid, sulfasalazine, dapsone, albendazole, ivermectin, levamisole, permethrin, pyrantel, thiabendazole, procainamide, sulfamethazine, amikacin, amoxicillin, ampicillin, cefazolin, cefuroxime, cephalexin, chloramphenicol, chloramphenicol, ciprofloxacin, clenbuterol, cioxacillin, colistin A, dicloxacillin, enrofloxacin, furaltadone, gentamicin, gentamicin, kanamycin, kanamycin, kincomycin, lincomycin, metronidazole, nafcillin, nalidixic acid, neomycin, neomycin, nitrofurazone, norfloxacin, ofloxacin, oxacillin, spectinomycin, streptomycin, streptomycin, sulfabenzamide, sulfacetamide, sulfadiazine, sulfadimidine, sulfametoxydiazine, sulfanilamide,trimethoprim, carbamazepine, ethosuximide, lamotrigine, primidone, cetirizine, chlorpheniramine, diphenhydramine, doxylamine, promethazine, sulfadimethoxine, benzothiazinone, butylated hydroxytoluene, tripelennamine, chlorpromazine, clozapine, haloperidol, olanzapine, paliperidone, quetiapine, ribavirin, meprobamate, acebutolol, atenolol, penbutolol, warfarin, salmeterol, aflatoxin Bl, tetraxetan (DOTA), 4-[(6-methylpyrazin-2-yl) oxy]benzoate (MPOB), biotin, melamine, methotrexate, amphetamine, diethylpropion, dextromethorphan, pseudoephedrine, dihydrochlorothiazide, hydrochlorothiazide, clonazepam, diazepam, nitrazepam, rhodamine B, fluorescent brightener Ksn, zearalenone, Sudan Redl, acetaminophen, acrylamide, benzoic acid, benzophenone, benzothiazine, mercaptobenzothiazole, erythrosine, Sudan, tartrazine, erythromycin, sirolimus, atropine, ethyl glucuronide, aflatoxin Ml, methocarbamol, fentanyl, hydromorphone, morphine, remifentanil, tapentadol, tramadol, pregabalin, gabapentin, amitriptyline, desipramine, imipramine, nortriptyline, venlafaxine, dinitrophenyl, His-tag, PEG methoxy group, etodolac, ibuprofen, ketoprofen, meclofenamic acid, phenylbutazone, acetyl salicylic acid, acetamiprid, acetochlor, carbadazim, carbaryl, chlorothalonil, chlorpyrifos, fenpropathrin, imazalil, imidacloprid, parathion, abscisic acid, dibutyl phthalate, clonazepam, lorazepam, oxazepam, phenobarbital, secobarbital, zaleplon, zolpidem, trazodone, fluoxetine, fluvoxamine, cortisone, dexamethasone, dihydrotestosterone, fluocinolone, methylprednisolone, prednisolone, stanozolol, triamcinolone, mazindol, methamphetamine, methylphenidate, modafinil, chrysoidine, deoxynivalenol, fumonisin, microcystin Lr, ochratoxin, sterigmatocystin, T-2 toxin, sildenafil, tadalafil, scopolamine, florfenicol, pirlimycin, and sulfaquinoxaline.|0139| An activator can include or can be modified to include a functional group for attachment to another molecule (e.g., to a linker or an engineered polypeptide). Non-limiting examples of the functional groups for attachment can include functional groups capable of forming an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond, or a thioether bond. Non-limiting examples of functional groups capable of forming such bonds can include amino groups; carboxyl groups; hydroxyl groups; aldehyde groups; azide groups; alkyne and alkene groups; ketones; hydrazides; acid halides such as acid fluorides, chlorides, bromides, and iodides; acid anhydrides, including symmetrical, mixed, and cyclic anhydrides; carbonates; carbonyl functionalities bonded to leaving groups such as cyano, succinimidyl, and N- hydroxysuccinimidyl (NHS) groups; hydroxyl groups; sulfhydryl groups; and molecules possessing, for example, alkyl, alkenyl, alkynyl, allylic, or benzylic leaving groups, such as halides, mesylates, tosylates, tritiates, epoxides, phosphate esters, sulfate esters, and besylates.

[0140] Alternatively or in combination, an activator can include or can be modified to include a linker, such as any of the linkers discussed in Section I.C below. The linker can include a functional group for attachment to another molecule (e.g., to an engineered polypeptide), which may be any of the functional groups described herein. Optionally, an activator can be coupled to an amino acid residue that is incorporated into the engineered polypeptide during synthesis of the engineered polypeptide, such that the engineered polypeptide can be coupled to the activator without requiring a separate conjugation reaction.10141] Representative examples of activators including functional groups for conjugation and / or linkers are provided in Tables 2A and 2B below.

[0142] Table 2 A: Fluorescein Activators

[0143] Table 2B: DOTA Activators

[0144] In some embodiments, an immune-activating conjugate of the present technology is prepared using an activator of any one of Compounds 1-13. For example, an activator of anyone of Compounds 1-13 can be conjugated to any of the engineered polypeptides described herein, e.g., directly or via a linker.C. Linkers

[0145] In some embodiments, an engineered polypeptide of the present technology is coupled to an activator of the present technology via at least one linker. The linker can be configured to spatially separate the engineered polypeptide from the activator. For instance, the linker can be sufficiently long so that the activator is accessible for recognition by the engineered receptor, but also sufficiently short so that the activated immune cell can exert a desired effect on the target cell that is recognized by the engineered polypeptide (e.g., cytolytic activity). The characteristics of the linker (e.g., composition, structure, length, rigidity, hydrophobicity / hydrophilicity) can be optimized to produce the desired immune cell activity. For example, the linker length and / or rigidity may control accessibility of the activator for binding to an engineered receptor on an immune cell (e.g., to a CAR or a CCR). In some embodiments, a short rigid linker may be desirable to hold an activator in a particular orientation that is beneficial or necessary to activate signaling. A shorter linker may also be desirable for providing a shorter immunological synapse for efficient T-cell signaling. However, in some instances, excessively short linkers may not provide good recognition by the immune cell, while excessively long linkers may provide suboptimal linkers.

[0146] The linker can have at least two functional groups for attachment to other molecules, and a spacer portion between the two functional groups. Non-limiting examples of the functional groups for attachment can include functional groups capable of forming an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond, or a thioether bond. Non-limiting examples of functional groups capable of forming such bonds can include amino groups; carboxyl groups; hydroxyl groups; aldehyde groups; azide groups; alkyne and alkene groups; ketones; hydrazides; acid halides such as acid fluorides, chlorides, bromides, and iodides; acid anhydrides, including symmetrical, mixed, and cyclic anhydrides; carbonates; carbonyl functionalities bonded to leaving groups such as cyano, succinimidyl, and N-hydroxysuccinimidyl (NHS) groups; hydroxyl groups; sulfhydryl groups; and molecules possessing, for example, alkyl, alkenyl, alkynyl, allylic, or benzylic leaving groups, such as halides, mesylates, tosylates, tritiates, epoxides, phosphate esters, sulfate esters, and besylates.(0147] The linker can be a linear or branched structure. A linear linker can include a functional group at both ends to conjugate two molecules to each other, e.g., a single activator toa single engineered polypeptide, a first engineered polypeptide to a second engineered polypeptide, or a first activator to a second activator. A branched linker can include three or more ends, with each end including a respective functional group to conjugate three or more molecules to each other, e.g., a single activator to two or more engineered polypeptides, a single engineered polypeptide to two or more activators, two or more activators to two or more polypeptides, etc.

[0148] The linker can be a polymer linker, an alkyl linker, a peptide linker, or suitable combinations thereof. Examples of polymers that may be used in a linker include polyethylene glycol (PEG), polypropylene glycol (PPG), polyethylene, polyacrylamide, polyacrylate, polyurea, polycarbonate, polycarbamate, polyester, polyvinyl alcohol, polyvinylchloride, polypeptides (e.g., polyalanine), an oligomeric portion thereof, and combinations thereof. The polymer can be water soluble, for example, PEG or polyvinyl alcohol. The polymer can be rigid (e.g., such as polyphenylene) or flexible (e.g., such as PEG).|0149[ The linker can have any suitable length. For instance, the linker can have a length (e.g., along the backbone of the linker and excluding the functional groups for attachment) of at least 2 atoms, 3 atoms, 4 atoms, 5 atoms, 6 atoms, 7 atoms, 8 atoms, 9 atoms, 10 atoms, 15 atoms, 20 atoms, 25 atoms, 30 atoms, 35 atoms, 40 atoms, 45 atoms, 50 atoms, 60 atoms, 70 atoms, 80 atoms, 90 atoms, 100 atoms, 125 atoms, 150 atoms, 175 atoms, or 200 atoms; and / or the linker can be no more than 200 atoms, 175 atoms, 150 atoms, 125 atoms, 100 atoms, 90 atoms, 80 atoms, 70 atoms, 60 atoms, 50 atoms, 45 atoms, 40 atoms, 35 atoms, 30 atoms, 25 atoms, 20 atoms, 15 atoms, 10 atoms, 9 atoms, 8 atoms, 7 atoms, 6 atoms, 5 atoms, 4 atoms, 3 atoms, or 2 atoms in length. In embodiments where the linker includes a plurality of repeating units (e.g., ethylene oxide groups in a PEG linker), the linker can include at least 2 repeating units, 5 repeating units, 10 repeating units, 12 repeating units, 15 repeating units, 20 repeating units, or 25 repeating units; and / or can include no more than 25 repeating units, 20 repeating units, 15 repeating units, 12 repeating units, 10 repeating units, 5 repeating units, or 2 repeating units. In embodiments where the linker is a peptide linker, the linker can include at least 2 amino acid residues, 5 amino acid residues, 10 amino acid residues, 12 amino acid residues, 15 amino acid residues, 20 amino acid residues, or 25 amino acid residues; and / or can include no more than 25 amino acid residues, 20 amino acid residues, 15 amino acid residues, 12 amino acid residues, 10 amino acid residues, 5 amino acid residues, or 2 amino acid residues.

[0150] In some embodiments, the linker is a rigid linker. The rigidity of the linker can also be a tunable parameter to control activation. Linker rigidity may help to hold the activator in an orientation that is beneficial or necessary for activation. A rigid linker may be or include aromaticrings, highly substituted carbon centers, peptides, and / or other groups that restrict linker rotation. In other embodiments, however, the linker can be a flexible linker. The linker may be cleavable or non-cleavable (e.g., under physiological conditions), and may be hydrophobic or hydrophilic.D. Conjugation

[0151] The components of the immune-activating conjugates described herein (e.g., activator, engineered polypeptide, linker) can include functional groups for covalent coupling to each other. Non-limiting examples of the functional groups for attachment can include functional groups capable of forming an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond, or a thioether bond. Non-limiting examples of functional groups capable of forming such bonds can include amino groups; carboxyl groups; hydroxyl groups; aldehyde groups; azide groups; alkyne and alkene groups; ketones; hydrazides; acid halides such as acid fluorides, chlorides, bromides, and iodides; acid anhydrides, including symmetrical, mixed, and cyclic anhydrides; carbonates; carbonyl functionalities bonded to leaving groups such as cyano, succinimidyl, and N-hydroxysuccinimidyl (NHS) groups; hydroxyl groups; sulfhydryl groups; and molecules possessing, for example, alkyl, alkenyl, alkynyl, allylic, or benzylic leaving groups, such as halides, mesylates, tosylates, tritiates, epoxides, phosphate esters, sulfate esters, and besylates. For example, an activator or linker can include an NHS ester or an isothiocyanate configured to selectively couple to a lysine side chain or an N-terminus of an engineered polypeptide, a maleimide configured to selectively couple to a cysteine side chain of an engineered polypeptide, or an epoxide configured to selectively couple to a histidine side chain of an engineered polypeptide. In some embodiments, click chemistry is used for activator conjugation, following incorporation of a click handle in the engineered polypeptide by amine acylation.101521 In some embodiments, an immune-activating conjugate of the present technology includes an engineered polypeptide that is expressed as a fusion protein with another molecule. For example, the other molecule can be another engineered polypeptide, which may be the same as or different than the engineered polypeptide. As another example, the other molecule can be a peptide or protein activator. The engineered polypeptide may be connected directly to the other molecule or may be coupled to the other molecule via a peptide linker. In such embodiments, the immune-activating conjugate may be synthesized (e.g., via biological expression or chemical synthesis) without requiring a separate conjugation reaction to couple the engineered polypeptide to the other molecule.

[0153] In some embodiments, an immune-activating conjugate of the present technology includes an activator coupled to an engineered polypeptide (e.g., directly or via a linker) at a desired degree of functionalization. The degree of functionalization may be expressed as an activator-to-polypeptide ratio that indicates the average number of activator molecules that are coupled to each polypeptide. In some embodiments, the activator-to-polypeptide ratio is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 18, 20, 24, or 32. The activator-to-polypeptide ratio can be within a range from 1 to 32, 1 to 20, 1 to 10, 1 to 5, 1 to 2, 2 to 10, 2 to 5, 5 to 10, 10 to 20, or 20 to 32. The activator-to-polypeptide ratio may influence the efficacy with which the immune-activating conjugate activates the immune cell of the present technology. For instance, a higher activator-to- polypeptide ratio may be advantageous in some instances to facilitate dimerization of an engineered receptor having two or more receptor chains. A higher activator-to-polypeptide ratio may lead to an increased binding avidity that increases activation.E. Compositions of Immune- Activating Conjugates

[0154] Further provided herein are compositions of immune-activating conjugates suitable for administration to a subject in need thereof. A composition can include a therapeutically effective amount of an immune activating conjugate and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the composition includes a viscosity-enhancing agent (e.g., sodium carboxymethylcellulose, dextran, or glycerol). In some embodiments, the composition includes an isotonicity imparting agent, such as sodium chloride, potassium chloride, or monosodium phosphate. In some embodiments, the composition includes a stabilizing agent, such as carboxymethyl cellulose, alginate, polyethylene glycol, or a polyol. In some embodiments, the composition includes a preservative, such as thimerosal, m- or o-cresol, formalin or benzyl alcohol. In some embodiments, the composition includes an adjuvant, such as aluminum hydroxide. In some embodiments, the composition includes a buffer, such as bicarbonate, TRIS, HEPES, MOPS, CHES, CHAPS, or phosphate buffered saline.II. Engineered Immune Cells

[0155] An engineered immune cell, as described herein, may include an immune cell (e.g., a lymphocyte or macrophage) that expresses one or more engineered receptors that are activated by the immune-activating conjugates of Section I. The immune cell can be a T-cell, a regulatory T-cell, a B-cell, a natural killer (NK) cell, a FcsRIy deficient NK cell (g-NK cell), a neutrophil, an eosinophil, a macrophage, a y5 T-cell, or other immune cell type. In some embodiments, the immune cell is a CAR T-cell. For example, the immune cell can express a CAR that initiatescytolytic activity upon recognition of an activator. As another example, the immune cell can express a CCR that initiates cytokine signaling activity upon recognition of an activator. In a further example, the immune cell can express a CCR that initiates cytokine signaling activity upon recognition of an activator, and a CAR that initiates cytolytic activity upon recognition of an activator or an endogenous antigen.A. Chimeric Antigen Receptors (CARs)

[0156] In some embodiments, an engineered immune cell of the present technology includes a CAR. A CAR may include an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain of the CAR may include an antigen binding domain that binds specifically to an antigen. The antigen binding domain can be any protein, protein fragment, or peptide capable of selectively binding the antigen. In some embodiments, for example, the antigen binding domain may be or include an antibody (e.g., a monoclonal antibody), an antibody fragment, an scFv, a nanobody, or a peptide. In some embodiments, an antigen binding domain may be or include a fragment of an antibody (e.g., a variable fragment) that binds to a selected antigen. Antibodies, antibody fragments, scFvs, and nanobodies may be produced using various methods known in the art to target a specific antigen. In some embodiments, the antigen binding domain may be a VHH antibody, an scFv, a VH, a VL, or a ligand specific for a target antigen. The antigen binding domain of the CAR may bind to a specific epitope of the target antigen.

[0157] In some embodiments, the CAR is a “direct CAR” having an antigen binding domain that recognizes an antigen expressed by a target cell (e.g., a tumor antigen expressed by a cancer cell). A direct CAR may be targeted to a target cell via direct binding of the antigen binding domain to the antigen expressed by the target cell. In some embodiments, the antigen recognized by a direct CAR is a tumor antigen, such as a tumor cell surface marker, a tumor-specific antigen, or a tumor-associated antigen. For example, the antigen may be CD 19, CD20, CD22, CD 123, CD33, CD3, CD4, CD8, CD38, SLAMF7, BCMA, GD2, GPRC5D, MUC16, HER2, EGFR, EGFRvIII, CLL-1, CD44v6, folate receptor-a, mesothelin, CD20, CD37, ROR1, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, surviving, telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, IL13Ra2, B7-H3 (CD276),EPHA2, GRP78, NKG2D, or CD70. In some embodiments, the CAR may be selected to target a tumor cell antigen associated with a cancer of interest.

[0158] In some embodiments, the antigen binding domain of a direct CAR is a VHH antibody, an scFv, a VH, or a VL of an antibody, or a ligand that recognizes any of the tumor antigens described herein. For example, the antigen binding domain can be a VHH antibody, an scFv, a VH, or a VL of an anti-BCMA antibody (e.g., as described in U.S. Patent Publication Nos. 2020 / 0261501 and 2022 / 0127371, the disclosures of which are incorporated herein by reference in their entirety). As another example, the antigen binding domain can be a VHH antibody, an scFv, a VH, or a VL of an anti-CD123 antibody (e.g., as described in U.S. Patent Publication No. 2020 / 0254023, the disclosure of which is incorporated by reference herein in its entirety). In a further example, the antigen binding domain can be a VHH antibody, an scFv, a VH, or a VL of an anti-GD2 or anti-B7-H3 antibody.|0159| In some embodiments, the CAR is an “indirect CAR” having an antigen binding domain that recognizes an antigen that is not expressed by the target cell of the CAR. For instance, the antigen may be a “synthetic antigen” that is not expressed by normal cells or cancer cells of the subject. An indirect CAR may be targeted to a target cell via an immune-activating conjugate including (1) an activator that serves as the synthetic antigen and (2) an engineered polypeptide that binds to an antigen expressed by the target cell (e.g., a tumor antigen expressed by a tumor cell). The indirect CAR may bind indirectly to the target cell by binding of the antigen binding domain to the activator of the immune-activating conjugate, and by binding of the engineered polypeptide of the immune-activating conjugate to the antigen expressed by the target cell. The activator and engineered polypeptide can be any of the embodiments described herein, e.g., in Section I above.

[0160] In some embodiments, the antigen binding domain of an indirect CAR is a VHH antibody, an scFv, a VH, or a VL of an antibody, or a ligand that recognizes any of the activators described herein. For example, the antigen binding domain can be a VHH antibody, an scFv, a VH, or a VL of an anti-FITC antibody (e.g., a 4M5.3 anti-FITC antibody). As another example, the antigen binding domain can be a VHH antibody, an scFv, a VH, or a VL of an anti-DOTA antibody (e.g., a C8.2.5 anti-DOTA antibody). In a further example, the antigen binding domain can be a VHH antibody, an scFv, a VH, or a VL of an anti-MPOB antibody.

[0161] In some embodiments, the antigen binding domain of an indirect CAR may be synthetic (e.g., engineered de novo to bind an activator). In some embodiments, the antigen bindingdomain may be humanized to reduce immunogenicity and prevent an immune reaction to the indirect CAR when administered to a subject (e.g., a human subject). Commercially available small molecule binding domains may be suitable for use as an antigen binding domain in an indirect CAR.

[0162] The transmembrane domain of the CAR (e.g., a direct CAR or an indirect CAR) may link the extracellular domain to the intracellular domain. In some embodiments, the transmembrane domain may be a transmembrane domain derived from any transmembrane protein. The transmembrane domain may include a transmembrane region of a T-cell receptor (TCR) a chain, TCR 0 chain, TCR chain, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. For example, the transmembrane domain may include a CD8 transmembrane domain, including a CD8a hinge domain and / or a CD8a transmembrane domain. In some embodiments, the transmembrane domain may be synthetic. For example, a synthetic transmembrane domain may include mostly hydrophobic residues (e.g., glycine, leucine, isoleucine, alanine, valine, proline, methionine, phenylalanine, and tryptophan). In some embodiments, a first peptide linker (e.g., including glycine, serine, or combinations thereof) may connect the transmembrane domain to the extracellular domain. In some embodiments, a second peptide linker (e.g., including glycine, serine, or combinations thereof) may connect the transmembrane domain to the intracellular domain.

[0163] The intracellular domain of the CAR (e.g., a direct CAR or an indirect CAR), also referred to as the cytoplasmic domain, may be capable of activating a specialized immune cell function (e.g., an immune response). For example, the specialized immune cell function of a T- cell may include cytolytic activity, cytokine secretion, or both. In some embodiments, the intracellular domain of the CAR may include the intracellular domain of TCR C, chain, FcRy, FcR0, CD3< CD3y, CD35, CD3s, CD5, CD22, CD79a, CD79b, or CD66d. In some embodiments, the intracellular domain of the CAR may include a portion of the intracellular domain of TCR C, chain, FcRy, FcR0, CD3< CD3y, CD35, CD3s, CD5, CD22, CD79a, CD79b, or CD66d sufficient to activate the specialized immune cell function. In some embodiments, the intracellular domain of the CAR includes one or more co- stimulatory domains, such as CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, HVEM (LIGHTR), lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3. In some embodiments, the intracellular domain of the CAR may include a 4- IBB signaling domain, a CD28 signaling domain, and / or a CD3(^ signaling domain.

[0164] In some embodiments, the CAR (e.g., a direct CAR or an indirect CAR) includes an antigen binding domain, a transmembrane domain, a CD3(^ intracellular domain, and a 4-1BB intracellular domain. In some embodiments, the CAR (e.g., a direct CAR or an indirect CAR) includes an antigen binding domain, a transmembrane domain, a CD3(^ intracellular domain, a 4- 1BB intracellular domain, and a CD28 intracellular domain.

[0015] Binding of the antigen to the antigen binding domain of the CAR may initiate signal transduction through the transmembrane domain to the cytoplasmic domain to activate the specialized immune cell function. For example, binding of the antigen binding domain to a tumor antigen may activate the intracellular domain of the CAR to trigger cytokine release. In some embodiments, the CAR may facilitate antigen-specific cancer cell killing by binding directly or indirectly to a tumor cell surface antigen present on the cancer cell via the antigen binding domain, transducing a signal through the transmembrane domain to the intracellular domain, and activating the specialized immune cell function (e.g., cytokine release) via activation of the intracellular domain. The specialized immune cell function may kill the cancer cell or may facilitate killing of the cancer cell.

[0166] Examples of polynucleotide sequences for CARs are provided in Table 3.

[0167] Table 3: CARs[0168| In some embodiments, a CAR is encoded by a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% sequence identity to any one of SEQ ID NO: 6 - SEQ ID NO: 8. In some embodiments, the CAR is encoded by a sequence of any one of SEQ ID NO: 6 - SEQ ID NO: 8.

[0169] Additional details and examples of CARs that are applicable to the present technology are provided in International Application No. PCT / US2023 / 082603, the disclosure of which is incorporated by reference herein in its entirety.B. Chimeric Cytokine Receptors (CCRs)

[0170] In some embodiments, an engineered immune cell of the present technology includes a CCR. As described herein, a CCR (which may also be referred to as a small molecule activated receptor (SMAR)) may be engineered to activate an intracellular response (e.g., a cytokine signaling pathway) upon binding of an activator to the CCR. A CCR of the presenttechnology may be expressed on its own or in conjunction with a CAR (e.g., a direct CAR or an indirect CAR) to produce an immunotherapeutic effect (e.g., an anti-cancer effect). For example, a CAR T-cell of the present technology may be engineered to co-express a CCR and a CAR that binds to a tumor antigen or to a synthetic antigen.[01711 In some embodiments, a CCR includes an activator binding domain, a transmembrane domain, and an intracellular signaling domain. The activator binding domain may bind an activator to activate the intracellular signaling domain. In some embodiments, the activator binding domain is a small molecule binding domain that binds a small molecule (e.g., fluorescein or a fluorescein derivative (e.g., FITC, tetraxetan (DOTA), biotin or linker-specific biotin, or MPOB). The activation signal may be communicated through the transmembrane domain to convert an extracellular stimulus (e.g., binding of the activator) to an intracellular effect (e.g., activation of a cytokine signaling pathway).[0.172] In some embodiments, the CCR may further include a hinge connecting the activator binding domain to the transmembrane domain. A hinge may increase flexibility of the CCR, which may reduce spatial constraints between the activator binding domain and the activator. The CCR may further include a signal peptide to direct expression of the CCR to the endoplasmic reticulum (ER). In some embodiments, a signal peptide present at the N-terminus of the protein may direct the protein to be synthesized in the ER membrane and subsequently trafficked to the plasma membrane as a transmembrane protein.

[0173] A CCR of the present technology may include a domain (e.g., an intracellular signaling domain, a transmembrane domain, a hinge, a signal peptide, or combinations thereof) derived from an endogenous cytokine receptor. In some embodiments, a CCR may include a domain derived from an interleukin 2 receptor subunit a (IL2Ra), an interleukin 2 receptor subunit P (IL2RP), an interleukin 2 receptor subunit y (IL2Ry), an interleukin 4 receptor subunit a (IL4Ra), an interleukin 7 receptor subunit a (IL7Ra), an interleukin 15 receptor subunit a (IL15Ra), an interleukin 21 receptor subunit a (IL21Ra), an interleukin 1 receptor (IL1R), a CD123, a CD124, an interleukin 5 receptor subunit a (IL5Ra), an interleukin 5 receptor subunit P (IL5RP), a CD 126, a CD132, a CD129, an interleukin 11 receptor subunit a (ILl lRa), an interleukin 12 receptor subunit pi (IL12RP1), an interleukin 12 receptor subunit P2 (IL12RP2), interleukin 13 receptor subunit al (IL13Ral), a CD122, an interleukin 18 receptor (IL18R), an interleukin 23 receptor (IL23R), an interleukin 27 receptor subunit a (IL27Ra), a CD 130, a CD8, a CD3, a CD4, a CD28, a 4- IBB, a CD28, an 0X40, an inducible T cell costimulatory (ICOS), a CD27, or combinations thereof.

[0174] Examples of CCRs and their associated polynucleotide sequences are provided inTable 4.10175] Table 4: CCRs

[0176] In some embodiments, a CCR may include a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% sequence identity to any one of SEQ ID NO: 9 - SEQ ID NO: 15. In some embodiments, a CCR is encoded by a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% sequence identity to any oneof SEQ ID NO: 16 - SEQ ID NO: 22. In some embodiments, a CCR may include a sequence of any one of SEQ ID NO: 9 - SEQ ID NO: 15. In some embodiments, a CCR is encoded by a sequence of any one of SEQ ID NO: 16 - SEQ ID NO: 22.|0177| In some embodiments, a CCR may be a single-chain CCR. Examples of singlechain CCRs include any one of SEQ ID NO: 9 - SEQ ID NO: 14 (based on IL2Ra, IL2RP, IL2Ry, IL7Ra, IL15Ra, and IL21Ra, respectively). A single-chain CCR can be derived from a single cytokine receptor chain (e.g., an a, P, or y chain). The cytokine receptor chain may be a wild-type cytokine receptor chain, or may be a chimeric or mutant cytokine receptor chain. In some embodiments, the single cytokine receptor chain is capable of initiating signaling via dimerization with an endogenous cytokine receptor chain. For example, IL7 signaling occurs through the IL7R receptor, which is composed of the IL7Ra and IL2Ry chains. The IL2Ry chain (also known as the common gamma chain (yc)) is shared by other members of the in the common gamma chain receptor family. Accordingly, a single-chain CCR (e.g., derived from IL7Ra) may heterodimerize with an endogenous cytokine receptor (e.g., IL2Ry) and bind to an activator to initiate intracellular signaling. Some cytokine receptor chains are capable of initiating signaling via homodimerization (e.g., IL7Ra can form homodimers and initiate IL7 signaling without IL2Ry). Thus, in some embodiments, a pair of single-chain CCRs (e.g., derived from IL7Ra) may bind to respective activators and homodimerize with each other to initiate intracellular signaling. Single-chain CCRs may be used to initiate novel signaling pathways by dimerization with endogenous cytokine receptor chains, depending on how the dimerization occurs and which receptor chains are dimerized. Additionally, in some embodiments, production of viral vectors and engineered immune cells may be easier for single-chain CCRs.101781 In some embodiments, a CCR may be a dual-chain CCR. For example, a dual-chainCCR may include a first CCR of SEQ ID NO: 10 (based on IL2RP) and a second CCR of SEQ ID NO: 11 (based on IL2Ry). As another example, a dual-chain CCR may include a first CCR of SEQ ID NO: 12 (based on IL7Ra) and a second CCR of SEQ ID NO: 11 (based on IL2Ry). In a further example, a dual-chain CCR may include a first CCR of SEQ ID NO: 14 (based on IL21Ra) and a second CCR of SEQ ID NO: 11 (based on IL2Ry). A dual-chain CCR can be derived from two cytokine receptor chains (e.g., a combination of a, P, or y chains), each of which can be independently selected from any of the cytokine receptor chains described herein. For example, a dual-chain CCR including a first cytokine receptor chain derived from IL2RP and a second cytokine receptor chain derived from IL2Ry can mimic the IL2-IL2R signaling pathway. In some embodiments, each chain of a dual-chain CCR may bind to a respective activator andheterodimerize with each other to activate intracellular signaling. Optionally, a dual-chain CCR can be expressed as a single protein including both cytokine receptor chains. The single protein can be subsequently cleaved (e.g., via the inclusion of a 2A peptide or other cleavage sequence) to produce the two separate cytokine receptor chains. SEQ ID NO: 15 provides an example of a dual-chain CCR that is initially expressed as a single protein.

[0179] In some embodiments, a CCR includes one or more cytokine receptor chains with one or more chimeric, tandem, and / or mutant intracellular domains. For example, a CCR can include a chimeric cytokine receptor chain including a first intracellular domain derived from IL2RP and a second intracellular domain derived from IL2RY. AS another example, a CCR can include a chimeric cytokine receptor chain including a first intracellular domain derived from IL7Ra and a second intracellular domain derived from IL2Ry. In another example, a CCR can include a chimeric cytokine receptor chain including a first intracellular domain derived from IL21Ra and a second intracellular domain derived from IL2RY. In a further example, a CCR can include a tandem cytokine receptor chain including first and second intracellular domains derived from IL2Rp. As yet another example, a CCR can include a tandem cytokine receptor chain including first and second intracellular domains derived from IL7Ra. As another example, a CCR can include a mutant cytokine receptor chain including a mutant intracellular domain derived from IL2Rp. In another example, a CCR can include a mutant cytokine receptor chain including a mutant intracellular domain derived from IL7Ra. A mutant intracellular domain can include one or more mutations relative to the wild-type intracellular domain, such as point mutations, truncations, etc.|0180| A CCR of the present technology may include an activator binding domain. The activator binding domain may be positioned in an extracellular region of the CCR and may be designed to bind an activator (e.g., a small molecule, a peptide, an oligonucleotide, a protein) to activate intracellular signaling through the intracellular signaling domain. The activator can be any of the embodiments described herein, e.g., in Section I above. The activator binding domain can be any protein, protein fragment, or peptide capable of selectively binding the activator. In some embodiments, for example, the activator binding domain may include an antibody (e.g., a monoclonal antibody), an antibody fragment, a single chain variable fragment (scFv), a nanobody, or a peptide. In some embodiments, an activator binding domain may include a fragment of an antibody (e.g., a variable fragment) that binds to a selected activator. Antibodies, antibody fragments, scFvs, and nanobodies may be produced using various methods known in the art to target a specific activator.

[0181] In some embodiments, the activator binding domain is an scFv, a heavy chain variable domain (VH), or a light chain variable domain (VL) of an antibody, or a VHH antibody that recognizes any of the activators described herein. For example, the activator binding domain can be an scFv, a VH, or a VL of an anti-FITC antibody (e.g., a 4M5.3 anti-FITC antibody). As another example, the activator binding domain can be an scFv, a VH, or a VL of an anti-DOTA antibody (e.g., a C8.2.5 anti-DOTA antibody). In a further example, the activator binding domain can be an scFv, a VH, or a VL of an anti-MPOB antibody.

[0182] In some embodiments, the activator binding domain may be synthetic (e.g., engineered de novo to bind an activator). In some embodiments, an activator binding domain may be humanized to reduce immunogenicity and prevent an immune reaction to the CCR when administered to a subject (e.g., a human subject). Commercially available small molecule binding domains may be suitable for use as an activator binding domain in a CCR.|0183[ The activator binding domain may have a molecular weight of from about 1 kDa to about 150 kDa, from about 1 kDa to about 100 kDa, from about 1 kDa to about 90 kDa, from about 1 kDa to about 80 kDa, from about 1 kDa to about 70 kDa, from about 1 kDa to about 60 kDa, from about 1 kDa to about 50 kDa, from about 1 kDa to about 40 kDa, from about 1 kDa to about 35 kDa, from about 1 kDa to about 30 kDa, from about 1 kDa to about 25 kDa, from about 1 kDa to about 10 kDa, from about 5 kDa to about 150 kDa, from about 5 kDa to about 100 kDa, from about 5 kDa to about 90 kDa, from about 5 kDa to about 80 kDa, from about 5 kDa to about 70 kDa, from about 5 kDa to about 60 kDa, from about 5 kDa to about 50 kDa, from about 5 kDa to about 40 kDa, from about 5 kDa to about 35 kDa, from about 5 kDa to about 30 kDa, from about 5 kDa to about 25 kDa, from about 5 kDa to about 10 kDa, from about 10 kDa to about 150 kDa, from about 10 kDa to about 100 kDa, from about 10 kDa to about 90 kDa, from about 10 kDa to about 80 kDa, from about 10 kDa to about 70 kDa, from about 10 kDa to about 60 kDa, from about 10 kDa to about 50 kDa, from about 10 kDa to about 40 kDa, from about 10 kDa to about 35 kDa, from about 10 kDa to about 30 kDa, from about 10 kDa to about 25 kDa, from about 20 kDa to about 150 kDa, from about 20 kDa to about 100 kDa, from about 20 kDa to about 90 kDa, from about 20 kDa to about 80 kDa, from about 20 kDa to about 70 kDa, from about 20 kDa to about 60 kDa, from about 20 kDa to about 50 kDa, from about 20 kDa to about 40 kDa, from about 20 kDa to about 35 kDa, or from about 20 kDa to about 30 kDa. For example, the activator binding domain may include an scFv having a molecular weight of about 20 kDa to about 35 kDa. The activator binding domain may include a peptide having a molecular weight of about 1 kDa to about 10 kDa.

[0184] In embodiments where the CCR is expressed in an immune cell that also expresses an indirect CAR, the activator recognized by the CCR may be the same as the activator recognized by the indirect CAR (e.g., the antigen binding domain of the indirect CAR can be the same as the activator binding domain of the CCR). In such embodiments, the indirect CAR may recognize the same epitope on the activator as the CCR, or may recognize a different epitope on the activator than the CCR. Alternatively, the activator recognized by the indirect CAR can be different from the activator recognized by the CCR (e.g., the antigen binding domain of the CAR can be different than the activator binding domain of the CCR). In some embodiments, the antigen binding domain of the indirect CAR binds to a first small molecule, and the activator binding domain of the CCR binds to a second small molecule, where the first small molecule may be the same as or different than the second small molecule. In some embodiments, the antigen binding domain of the indirect CAR binds to a first epitope on a small molecule, and the activator binding domain of the CCR binds to a second epitope on the small molecule, where the first epitope may be the same as or different than the second epitope.

[0185] A CCR of the present technology may include an intracellular domain (also referred to herein as an “intracellular signaling domain”). The intracellular signaling domain may be positioned in an intracellular region of the CCR and may be designed to activate intracellular signaling upon binding of an activator to the activator binding domain. The intracellular signaling domain may activate a cytokine signaling pathway, such as a Jak-STAT pathway. In some embodiments, activation of the cytokine signaling pathway may promote conversion of an immune cell expressing the CCR to a desired phenotype, such as a memory phenotype (e.g., a central memory phenotype, a stem cell memory phenotype, an effector memory phenotype, or an effector memory re-expressing CD45RA phenotype). Alternatively or in combination, activation of the cytokine signaling pathway may upregulate expression of cell surface markers that enable homing of the immune cell to lymphoid organs (e.g., CD62L, CCR7), such as homing to the lymph nodes, spleen, thymus, and / or bone marrow.10186] An intracellular signaling domain may be derived from an endogenous cytokine receptor. For example, an intracellular signaling domain may be derived from an interleukin 2 receptor subunit a (IL2Ra), an interleukin 2 receptor subunit P (IL2RP), an interleukin 2 receptor subunit y (IL2Ry), an interleukin 4 receptor subunit a (IL4Ra), an interleukin 7 receptor subunit a (IL7Ra), an interleukin 15 receptor subunit a (IL15Ra), an interleukin 21 receptor subunit a (IL21Ra), an interleukin 1 receptor (IL1R), a CD123, a CD124, an interleukin 5 receptor subunit a (IL5Ra), an interleukin 5 receptor subunit P (IL5RP), a CD 126, a CD 132, a CD 129, aninterleukin 11 receptor subunit a (ILl lRa), an interleukin 12 receptor subunit pi (IL12RP1), an interleukin 12 receptor subunit P2 (IL12RP2), interleukin 13 receptor subunit al (IL13Ral), a CD 122, an interleukin 18 receptor (IL18R), an interleukin 23 receptor (IL23R), an interleukin 27 receptor subunit a (IL27Ra), a CD 130, or a GM-CSF.

[0187] In some embodiments, the intracellular signaling domain may include an intracellular domain, a fragment of an intracellular domain, or a variant of an intracellular domain of an endogenous cytokine receptor. For example, the intracellular signaling domain may include an intracellular domain, a fragment of an intracellular domain, or a variant of an intracellular domain of an interleukin 2 receptor subunit a (IL2Ra), an interleukin 2 receptor subunit P (IL2RP), an interleukin 2 receptor subunit y (IL2Ry), an interleukin 4 receptor subunit a (IL4Ra), an interleukin 7 receptor subunit a (IL7Ra), an interleukin 15 receptor subunit a (IL15Ra), an interleukin 21 receptor subunit a (IL21Ra), an interleukin 1 receptor (IL1R), a CD123, a CD124, an interleukin 5 receptor subunit a (IL5Ra), an interleukin 5 receptor subunit P (IL5RP), a CD 126, a CD132, a CD129, an interleukin 11 receptor subunit a (ILl lRa), an interleukin 12 receptor subunit pi (IL12RP1), an interleukin 12 receptor subunit P2 (IL12RP2), interleukin 13 receptor subunit al (IL13Ral), a CD122, an interleukin 18 receptor (IL18R), an interleukin 23 receptor (IL23R), an interleukin 27 receptor subunit a (IL27Ra), a CD130, or a GM-CSF. The intracellular domain, fragment of the intracellular domain, or variant of the intracellular domain may be capable of activating the cytokine signaling pathway activated by the endogenous cytokine receptor from which it was derived.

[0188] In some embodiments, a CCR includes a single intracellular signaling domain. Alternatively, a CCR can include a plurality of intracellular signaling domains in tandem (e.g., two, three, four, five, or more intracellular domains in tandem). In such embodiments, some or all of the intracellular signaling domains may be the same intracellular signaling domain, or some or all of the intracellular signaling domains may be different intracellular signaling domains.

[0189] In some embodiments, an intracellular signaling domain may include a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% sequence identity to an intracellular domain of an endogenous cytokine receptor (e.g., an interleukin 2 receptor subunit a (IL2Ra), an interleukin 2 receptor subunit P (IL2RP), an interleukin 2 receptor subunit y (IL2Ry), an interleukin 4 receptor subunit a (IL4Ra), an interleukin 7 receptor subunit a (IL7Ra), an interleukin 15 receptor subunit a (IL15Ra), an interleukin 21 receptor subunit a (IL21Ra), aninterleukin 1 receptor (IL1R), a CD123, a CD124, an interleukin 5 receptor subunit a (IL5Ra), an interleukin 5 receptor subunit P (IL5RP), a CD126, a CD132, a CD129, an interleukin 11 receptor subunit a (ILl lRa), an interleukin 12 receptor subunit pi (IL12RP1), an interleukin 12 receptor subunit P2 (IL12RP2), interleukin 13 receptor subunit al (IL13Ral), a CD122, an interleukin 18 receptor (IL18R), an interleukin 23 receptor (IL23R), an interleukin 27 receptor subunit a (IL27Ra), a CD130, or a GM-CSF).

[0190] A CCR of the present technology may include a transmembrane domain. The transmembrane domain may connect an intracellular portion and an extracellular portion of the CCR and may be designed to span a cell membrane and transduce a signal from an activator binding domain to an intracellular signaling domain upon binding of an activator to the activator binding domain.

[0191] A transmembrane domain may be derived from an endogenous cytokine receptor or from another type of receptor. For example, a transmembrane domain may be derived from an interleukin 2 receptor subunit a (IL2Ra), an interleukin 2 receptor subunit P (IL2RP), an interleukin 2 receptor subunit y (IL2Ry), an interleukin 4 receptor subunit a (IL4Ra), an interleukin 7 receptor subunit a (IL7Ra), an interleukin 15 receptor subunit a (IL15Ra), an interleukin 21 receptor subunit a (IL21Ra), an interleukin 1 receptor (IL1R), a CD123, a CD124, an interleukin 5 receptor subunit a (IL5Ra), an interleukin 5 receptor subunit P (IL5RP), a CD126, a CD132, a CD129, an interleukin 11 receptor subunit a (ILl lRa), an interleukin 12 receptor subunit pi (IL12RP1), an interleukin 12 receptor subunit P2 (IL12RP2), interleukin 13 receptor subunit al (IL13Ral), a CD122, an interleukin 18 receptor (IL18R), an interleukin 23 receptor (IL23R), an interleukin 27 receptor subunit a (IL27Ra), a CD130, an immunoglobulin (e.g., an IgGl, an IgG2, an IgG3, an IgG4, an IgM, an IgA, an IgD, an IgE), a CD8, a CD28, a GM-CSF, or an erythropoietin receptor (EpoR).

[0192] In some embodiments, the transmembrane domain may include a transmembrane domain or a variant of a transmembrane domain of an endogenous cytokine receptor or another type of receptor. For example, the transmembrane domain may include a transmembrane domain or a variant of a transmembrane domain of an interleukin 2 receptor subunit a (IL2Ra), an interleukin 2 receptor subunit P (IL2RP), an interleukin 2 receptor subunit y (IL2Ry), an interleukin 4 receptor subunit a (IL4Ra), an interleukin 7 receptor subunit a (IL7Ra), an interleukin 15 receptor subunit a (IL15Ra), an interleukin 21 receptor subunit a (IL21Ra), an interleukin 1 receptor (IL1R), a CD123, a CD124, an interleukin 5 receptor subunit a (IL5Ra), an interleukin 5 receptor subunit P (IL5RP), a CD126, a CD132, a CD129, an interleukin 11 receptor subunit a(ILl lRa), an interleukin 12 receptor subunit pi (IL12RP1), an interleukin 12 receptor subunit P2 (IL12RP2), interleukin 13 receptor subunit al (IL13Ral), a CD122, an interleukin 18 receptor (IL18R), an interleukin 23 receptor (IL23R), an interleukin 27 receptor subunit a (IL27Ra), a CD130, an immunoglobulin, a CD8, a CD28, a GM-CSF, or an EpoR. The transmembrane domain, fragment of the transmembrane domain, or variant of the transmembrane domain may be capable of activating the cytokine signaling pathway activated by the endogenous cytokine receptor from which it was derived.10193] In some embodiments, the transmembrane domain may include a transmembrane domain derived from any transmembrane protein. In some embodiments, the transmembrane domain may be a synthetic transmembrane domain. For example, the transmembrane domain may include a synthetic transmembrane a-helix, helical bundle, or P-barrel.

[0194] In some embodiments, the transmembrane domain may include a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% sequence identity to a transmembrane domain of an endogenous cytokine receptor or another receptor (e.g., an interleukin 2 receptor subunit a (IL2Ra), an interleukin 2 receptor subunit P (IL2RP), an interleukin 2 receptor subunit y (IL2Ry), an interleukin 4 receptor subunit a (IL4Ra), an interleukin 7 receptor subunit a (IL7Ra), an interleukin 15 receptor subunit a (IL15Ra), an interleukin 21 receptor subunit a (IL21Ra), an interleukin 1 receptor (IL1R), a CD123, a CD124, an interleukin 5 receptor subunit a (IL5Ra), an interleukin 5 receptor subunit P (IL5RP), a CD126, a CD132, a CD129, an interleukin 11 receptor subunit a (ILl lRa), an interleukin 12 receptor subunit pi (IL12RP1), an interleukin 12 receptor subunit P2 (IL12RP2), interleukin 13 receptor subunit al (IL13Ral), a CD122, an interleukin 18 receptor (IL18R), an interleukin 23 receptor (IL23R), an interleukin 27 receptor subunit a (IL27Ra), a CD 130, an immunoglobulin, a CD8, a CD28, a GM- CSF, or an EpoR).|0.195] A CCR of the present technology may include a signal peptide. The signal peptide may be positioned at the N-terminus of the CCR and may be designed to direct expression of the CCR to the ER. The CCR may be synthesized in the ER membrane and may be trafficked to the plasma membrane as a transmembrane protein.(0196] A signal peptide may be derived from an endogenous cytokine receptor or another type of receptor. For example, signal peptide may be derived from an interleukin 2 receptor subunita (IL2Ra), an interleukin 2 receptor subunit P (IL2RP), an interleukin 2 receptor subunit y (IL2Ry), an interleukin 4 receptor subunit a (IL4Ra), an interleukin 7 receptor subunit a (IL7Ra), an interleukin 15 receptor subunit a (IL15Ra), an interleukin 21 receptor subunit a (IL21Ra), an interleukin 1 receptor (IL1R), a CD123, a CD124, an interleukin 5 receptor subunit a (IL5Ra), an interleukin 5 receptor subunit P (IL5RP), a CD126, a CD132, a CD129, an interleukin 11 receptor subunit a (ILl lRa), an interleukin 12 receptor subunit pi (IL12RP1), an interleukin 12 receptor subunit P2 (IL12RP2), interleukin 13 receptor subunit al (IL13Ral), a CD122, an interleukin 18 receptor (IL18R), an interleukin 23 receptor (IL23R), an interleukin 27 receptor subunit a (IL27Ra), a CD 130, an immunoglobulin (e.g., an IgGl, an IgG2, an IgG3, an IgG4, an IgM, an IgA, an IgD, an IgE), a CD8, a CD28, or a GM-CSF.

[0197] In some embodiments, a signal peptide may include the signal peptide portion of an endogenous cytokine receptor or another receptor. For example, the signal peptide may include the signal peptide portion of an interleukin 2 receptor subunit a (IL2Ra), an interleukin 2 receptor subunit P (IL2RP), an interleukin 2 receptor subunit y (IL2Ry), an interleukin 4 receptor subunit a (IL4Ra), an interleukin 7 receptor subunit a (IL7Ra), an interleukin 15 receptor subunit a (IL15Ra), an interleukin 21 receptor subunit a (IL21Ra), an interleukin 1 receptor (IL1R), a CD 123, a CD 124, an interleukin 5 receptor subunit a (IL5Ra), an interleukin 5 receptor subunit P (IL5RP), a CD126, a CD132, a CD129, an interleukin 11 receptor subunit a (ILl lRa), an interleukin 12 receptor subunit pi (IL12RP1), an interleukin 12 receptor subunit P2 (IL12RP2), interleukin 13 receptor subunit al (IL13Ral), a CD122, an interleukin 18 receptor (IL18R), an interleukin 23 receptor (IL23R), an interleukin 27 receptor subunit a (IL27Ra), a CD 130, an immunoglobulin, a CD8, a CD28, or a GM-CSF.

[0198] In some embodiments, the signal peptide may be a signal peptide from any transmembrane or membrane-bound protein. The signal peptide may be sufficient to direct expression of the CCR to the ER.

[0199] In some embodiments, the signal peptide may include a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% sequence identity to a signal peptide of an endogenous cytokine receptor or another receptor (e.g., an interleukin 2 receptor subunit a (IL2Ra), an interleukin 2 receptor subunit P (IL2RP), an interleukin 2 receptor subunit y (IL2Ry), an interleukin 4 receptor subunit a (IL4Ra), an interleukin 7 receptor subunit a (IL7Ra), an interleukin 15 receptor subunit a (IL15Ra), an interleukin 21 receptor subunit a (IL21Ra), aninterleukin 1 receptor (IL1R), a CD123, a CD124, an interleukin 5 receptor subunit a (IL5Ra), an interleukin 5 receptor subunit P (IL5RP), a CD126, a CD132, a CD129, an interleukin 11 receptor subunit a (ILl lRa), an interleukin 12 receptor subunit pi (IL12RP1), an interleukin 12 receptor subunit P2 (IL12RP2), interleukin 13 receptor subunit al (IL13Ral), a CD122, an interleukin 18 receptor (IL18R), an interleukin 23 receptor (IL23R), an interleukin 27 receptor subunit a (IL27Ra), an immunoglobulin, a CD8, a CD28, or a CD 130).

[0200] A CCR of the present technology may include a hinge (also referred to herein as a “hinge domain”). The hinge may be positioned between the activator binding domain and the transmembrane domain and may be designed to increase the flexibility of the CCR. Increased flexibility may reduce spatial constraints between the activator binding domain and the activator (e.g., a small molecule activator adhered to a surface), facilitating access to the activator. In some embodiments, the hinge may be engineered to provide a desired distance between the plasma membrane of a cell expressing the CCR and an activator bound to the CCR.

[0201] In some embodiments, the hinge may be a synthetic peptide designed to provide a desired length, flexibility, or both. In some embodiments, the hinge may be derived from an endogenous transmembrane protein. For example, the hinge may be derived from a CD8 (e.g., a CD8a), a CD3, a CD4, a CD28, a 4-1BB, a CD28, an 0X40, an inducible T cell costimulatory (ICOS), a CD27, an immunoglobulin (e.g., an IgGl, an IgG2, an IgG3, an IgG4, an IgM, an IgA, an IgD, an IgE), or an EpoR. In some embodiments, the hinge may include a hinge of an endogenous transmembrane protein. For example, the hinge may be derived from a hinge of a CD8 (e.g., a CD8a), a CD3, a CD4, a CD28, a 4-1BB, a CD28, an 0X40, an ICOS, a CD27, an immunoglobulin, or an EpoR.

[0202] In some embodiments, the signal peptide may include a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% sequence identity to a hinge of an endogenous transmembrane protein (e.g., a CD8 (e.g., a CD8a), a CD3, a CD4, a CD28, a 4- IBB, a CD28, an 0X40, an ICOS, a CD27, an immunoglobulin, or an EpoR).

[0203] In some embodiments, a CCR may include or be co-expressed with a marker domain. The marker domain may be co-expressed with the cytokine receptor chain(s) of the CCR for purposes of identifying immune cells that are expressing the CCR (“positive cells”), enriching and purifying positive cells, acting as a conditional suicide switch for positive cells, and / or otherrelevant functions. The marker domain can be truncated (e.g., in the intracellular domain) such that the expressed truncated marker does not have the biological function of the native marker. The marker domain can be any cell surface molecule that is not present on natural T cells. For example, a CCR may include a CD19 domain (e.g., a truncated CD19 domain), a CD20 domain (e.g., a truncated CD20 domain), a CD22 domain (e.g., a truncated CD22 domain), a CD34 domain (e.g., a truncated CD34 domain), or an EGFR domain (e.g., a truncated EGFR domain).

[0204] Alternatively or in combination, the marker domain can be a detection marker, such as a fluorescent protein. For example, a CCR may include EBFP, Sapphire, T-Sapphire, ECFP, mCFP, Cerulean, CyPet, AmCyanl, Midori -Ishi Cyan, mTFPl, GFP, EGFP, AcGFP, TurboGFP, Emerald, Azami Green, ZsGreen, EYFP, Topaz, Venus, mCitrine, yPet, PhiYFP, ZsYellowl, mBanana, Kusabira Orange, mOrange, dTomato, tdTomato, DsRed, DsRed2, DsRed-Express, DsRed-Monomer, mTangerine, mStrawberry, AsRed2, mRFPl, JREd, mCherry, HcRedl, mRaspberry, HcRed-Tandem, mPlum, or AQ143.

[0205] In some embodiments, a construct for expression of a CCR may include a cleavage sequence, such as a 2A self-cleaving peptide sequence (e.g., a P2A peptide, a T2A peptide, a E2A peptide, or a F2A peptide). A 2A self-cleaving peptide sequence (also known as a “2A peptide”) may be included to link a CCR to one or more additional CCRs for co-expression. In some embodiments, a 2 A peptide may link a first CCR to a CCR. For example, a 2 A peptide may link a first cytokine receptor chain (e.g., an IL2RP cytokine receptor chain or an IL2Ry cytokine receptor chain) to a second cytokine receptor chain (e.g., an IL2RP cytokine receptor chain or an IL2RY cytokine receptor chain) to form a single protein that encompasses both chains of an engineered dual-chain CCR (e.g., a dual-chain CCR of SEQ ID NO: 15). After expression, the protein can be cleaved at the cleavage sequence to produce the separate two cytokine receptor chains of the dualchain CCR. As another example, a 2A peptide may be included to link a cytokine receptor chain to a marker domain to form a single protein that encompasses the cytokine receptor chain and the marker domain. After expression, the protein can be cleaved at the cleavage sequence to separate the cytokine receptor chain from the marker domain.

[0206] Alternatively or in combination, a construct for expression of a CCR may include an internal ribosome entry site (IRES) to allow for co-expression of an CCR with an additional CCR and / or to a marker domain. In some embodiments, an IRES may link a first CCR to a second CCR in an expression construct, such that the first CCR and the second CCR are translated as separate proteins. In some embodiments, an IRES may link a CCR to a marker domain, such that the CCR and the marker domain are translated as separate proteins.

[0207] Additional details and examples of CCRs that are applicable to the present technology are provided in International Application No. PCT / US2023 / 082603, the disclosure of which is incorporated by reference herein in its entirety.C. Vectors

[0208] The present technology provides polynucleotides encoding one or more engineered receptors described herein (e.g., a CAR, a CCR, or both). A polynucleotide may include an RNA sequence or a DNA sequence encoding an engineered receptor, or an RNA sequence or a DNA sequence reverse complementary to a sequence encoding an engineered receptor. In some embodiments, the polynucleotide encoding the engineered receptor may be part of a polynucleotide construct (also referred to herein as a polynucleotide expression cassette) capable of expressing the engineered receptor in a cell (e.g., an immune cell). The polynucleotide expression cassette can be a plasmid, a cosmid, a viral vector, or a combination thereof.

[0209] The polynucleotide expression cassette may include a promoter, an open reading frame (e.g., encoding the engineered receptor), a 3’ untranslated region, or combinations thereof. In some embodiments, the polynucleotide expression cassette may include two or more open reading frames. For example, the polynucleotide expression cassette may include a first open reading frame encoding a CAR and a second open reading frame encoding a CCR. The expression cassette may further include an origin of replication, a restriction endonuclease site, a selectable marker, or combinations thereof. The expression cassette may be capable of expressing a CAR, a CCR, or both in a cell (e.g., an immune cell). In some embodiments, the cell may be a mammalian cell (e.g., a human cell). For example, the cell may be a human T-cell. A polynucleotide or polynucleotide expression cassette may be obtained using recombinant methods known in the art. Alternatively or in addition, the polynucleotide or polynucleotide expression cassette may be generated synthetically.

[0210] The polynucleotide expression cassette can also encode elements for gene editing, nucleases, reverse transcriptases, integrases, recombinases, and combinations thereof. As nonlimiting examples, a nuclease encoded by the polynucleotide expression cassette can include a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), a Cas3 nuclease, a Cas9 nuclease, a CRISPR / Casl2 nuclease, a CRISPR / Casl4 nuclease, a Fokl nuclease, or a combination thereof. The polynucleotide expression cassette can also encode additional transcripts and proteins which further affect cell phenotype, such as small interfering RNA(siRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, transcription factors, or immunomodulatory elements.

[0211] In some embodiments, a polynucleotide expression cassette for expressing two or more proteins may include a cleavage sequence, such as a 2 A self-cleaving peptide sequence (e.g., a P2A peptide, a T2A peptide, a E2A peptide, or a F2A peptide). For instance, a 2 A peptide may be included to link a CAR to a CCR for co-expression. After expression, the protein can be cleaved at the cleavage sequence to separate the CAR from the CCR as separate proteins, at the cleavage sequence to separate the cytokine receptor chain from the marker domain. Alternatively or in combination, a polynucleotide expression cassette for expressing two or more proteins may include an IRES. For instance, an IRES may be included to link a CAR to a CCR for co-expression, such that the CAR and the CCR are translated as separate proteins.

[0212] Also provided herein are vectors including the polynucleotide expression cassette. The vector may be capable of delivering the polynucleotide expression cassette to a target cell (e.g., an immune cell). Upon delivery, a protein encoded by the polynucleotide expression cassette (e.g., a CCR, a CAR, or combinations thereof) may be expressed in the cell. In some embodiments, the polynucleotide expression cassette is encoded within a viral vector. In some embodiments, the viral vector is a lentiviral vector, an adeno-associated viral vector, a vaccinia viral vector, a poxvirus viral vector, a herpes viral vector, an alphavirus viral vector, gamma retrovirus, a polyoma viral vector, or a combination thereof. In some embodiments, the viral vector is a gamma retrovirus, an adeno-associated viral vector or a lentiviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector has a titer of between about 106and about 109virions per ml. In some embodiments, transfection includes delivery of nonviral vectors (e.g., in a lipid or chitosan nanoparticles or with a colloidal dispersion system). A vector encoding an engineered receptor may be generated and delivered to an immune cell using standard cloning and gene delivery protocols, for example as described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), which is herein incorporated by reference. A vector may encode a selectable marker, a reporter gene, or both to facilitate selection of cells (e.g., immune cells) successfully transfected and expressing a protein encoded by the expression cassette.D. Methods for Manufacturing Immune Cells1. Collection

[0213] An immune cell to be engineered (e.g., to express a CAR, a CCR, or both) may be obtained from a subject. Immune cells may be obtained from blood (e.g., peripheral blood mononuclear cells), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, tumors, or combinations thereof collected from the subject. Immune cells may be collected from a subject using any technique known in the art (e.g., Ficoll separation or apheresis). In some embodiments, immune cells collected from a subject may include T-cells, monocytes, granulocytes, B-cells, other nucleated white blood cells, red blood cells, platelets, or combinations thereof.

[0214] In some embodiments, immune cells (e.g., T-cells, regulatory T-cells, B-cells, NK cells, macrophages, y5 T-cells, or combinations thereof) may be collected from a donor. The cells may be obtained from blood (e.g., peripheral blood mononuclear cells), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, tumors, or combinations thereof collected from the subject. Immune cells (e.g., precursors to engineered immune cells of the present technology) may be collected from a subject using any technique known in the art (e.g., Ficoll separation). In some embodiments, the precursor cells are collected from blood, for example through apheresis, leukapheresis, or buffy coat preparation.10215] In some embodiments, precursor cells (e.g., cells to be engineered to express a CAR, a CCR, or both) are immune cells collected from a donor. The immune cells can be of a single type, or can be a heterogeneous collection of cells. The immune cells can include T-cells, B-cells, natural killer cells (NK cells), FcsRIy deficient NK cells (g-NK cells), macrophages, monocytes, basophils, eosinophils, neutrophils, megakaryocytes, thrombocytes, or combinations thereof. In some embodiments, the immune cells are CD4+or CD8+T cells. In some embodiments, the immune cells are naive T and / or naive B cells.

[0216] The immune cells can be enriched for specific cell types. For many of the methods disclosed herein, blood-derived immune cells are separated from other whole blood components, for example through monocyte depletion, centrifugation, filtration, or clotting. The immune cells can also be subjected to positive or negative selection for certain cell types. In many cases, immune cells from a donor are separated from other peripheral blood mononuclear cells (PBMCs) through negative selection for surface markers expressed by non-target cells, such as CD25, CD45, CD103, or FOXP3. In specific cases, the immune cells are depleted of memory (e.g., central memory Tcells, effector memory T cells, virtual memory T cells, memory B cells, etc.) and / or effector cells. In some embodiments, the immune cells are enriched for a particular type of T or B cell, such as a y5 T-cell, a THI cell, a TH2 cell, a TH17 cell, a TH22 cell, a T helper cell, a T regulatory cell, or a combination thereof. As a nonlimiting example, immune cell enrichment can include selectively binding one or more cell type-specific surface markers on a magnetically separatable bead or on a column. In some embodiments, the immune cells are of a single cell type.

[0217] In some embodiments, the immune cells are obtained through leukapheresis, bone marrow biopsy, or a combination thereof. In some embodiments, the immune cells include at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% naive T cells and naive B cells as a percentage of total cell population, e.g., prior to contacting the immune cells with the activator.

[0218] The precursor cells can also include a totipotent, pluripotent, multipotent, or oligopotent cell. In many such cases, the precursor cells include immune precursor cells, such as common myeloid progenitor cells, granulocyte progenitor cells, myeloblasts, monocytes, common lymphoid progenitor cells, lymphoid progenitor cells, progenitor B cells, or combinations thereof. In some embodiments, the precursor cells include a stem cell, such as a tetrapioid reprogrammed cell, an induced pluripotent stem cell, an embryoblast, a lymphoid stem cell, or a myeloid stem cell.2. Transfection

[0219] The immune cells may be isolated and transfected with a polynucleotide expression cassette encoding one or more engineered receptors (e.g., a CAR and / or a CCR), thereby engineering the immune cell to express the engineered receptor(s). The polynucleotide expression cassette can be a plasmid, a cosmid, a viral vector, or a combination thereof. The polynucleotide expression cassette can be naked or delivered via a viral vector or a nonviral vector. A polynucleotide expression cassette may be introduced into a target cell using physical or chemical means. In some embodiments, a polynucleotide expression cassette may be introduced into a target cell using calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. In some embodiments, the polynucleotide expression cassette may be introduced using colloidal dispersion systems (e.g., macromolecule complexes), nanocapsules, microspheres, beads, lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, liposomes), and the like. Additional transfection methods are described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0220] In some embodiments, the immune cells are contacted to a receptor agonist concurrently with and / or prior to transfection. In some embodiments, the receptor agonist is selected from the group consisting of granulocyte macrophage-colony stimulating factor (GM- CSF), stem cell factor (SCF), interleukin-1 (IL1), interleukin-2 (IL2), interleukin-3 (IL3), a CD3 agonist, a CD4 agonist, a CD8 agonist, a CD 16 agonist, a CD23 agonist, a CD28 agonist, a CD47 agonist, a CD 80 agonist, a CD113 agonist, a CD131 agonist, a CD 137 agonist, an HLA-E agonist, a 41BBL agonist, and a combination thereof. In some embodiments, the receptor agonist is selected from the group consisting of a CD3 agonist, a CD23 agonist, a CD28 agonist, an IL2 receptor agonist, and a combination thereof. In some embodiments, the CD3 agonist is an antigen. In some embodiments, the receptor agonist is coupled to a substrate. In some embodiments, the substrate includes a peptide, an antibody, a minibody, a nanobody, a fragment antigen-binding, a nanoparticle, a microparticle, a polymer matrix, a surface, a surface functionalization (e.g., a dextran polymer functionalized with the activator), a carbon nanomaterial, a quantum dot, a surface, or a combination thereof. In some embodiments, the substrate is contacted to the immune cells at a ratio of between about 50: 1 and about 1 : 1 (substrate to immune cells). In some embodiments, the immune cells are contacted with a CD3 agonist and a CD28 agonist prior to transfection. In some embodiments, the immune cells are contacted with IL2 prior to transfection. In some embodiments, the immune cells are contacted with IL2 during transfection. In some embodiments, the immune cells are expanded by about 2-fold, about 5-fold, about 10-fold, about 25-fold, about 50-fold, or about 100-fold prior to transfection and the contacting to the substrate. In some embodiments, the immune cells are expanded by between about 2-fold and about 10-fold, by between about 2-fold and about 25-fold, by between about 5-fold and about 50-fold, or by between about 10-fold and about 100-fold prior to transfection and the contacting to the substrate.

[0221] Following transfection, the immune cells can be fractionated on a substrate. In some embodiments, the immune cells are collected on a substrate that is functionalized with an activator (e.g., a species which binds to an activator binding domain of a CCR), which may also be used to perform ex vivo activation of the immune cells as described herein. Activator functionalized substrates (e.g., test tube surfaces, cell culture flasks, cell culture bags, cell culture plates, G-rex bioreactors, CentriCult chambers, cassettes, column materials, beads such as magnetically separatable beads) can selectively bind cells which express engineered receptors. Alternatively, or in addition thereto, immune cells can be collected on substrates functionalized with agents which bind engineered (e.g., CAR) or native (e.g., phenotype-specific immune cell markers, such as CD45RA or CCR7) receptors. A method can include separating immune cells which express aparticular engineered receptor from immune cells which do not express the receptor by binding the receptor to a substrate. Unbound cells can then be washed or removed, while the receptorexpressing immune cells can be collected from the substrate. Alternatively, the substrate (along with bound immune cells) can be separated from unbound immune cells, for example through gravimetric or magnetic separation.

[0222] In some embodiments, immune cells are fractionated by cell phenotype. During such processes, subsets of the engineered immune cell population which express a particular cell marker (e.g., CCR7 or CD45RA) can be captured and separated from subsets of the engineered immune cell population which do not express the cell marker. For example, T cells can be separated from non-T cells of the immune cell population through CD3 affinity collection. In some embodiments, a method includes selecting memory T cells from an immune cell population, for example by collecting CCR7+ and / or CCR7+ / CD45RA+ cells from the immune cell population.3. Activation102231 Optionally, immune cells expressing a CCR may be activated ex vivo by contacting the cells with an activator. Activating the immune cells may promote conversion to a desired phenotype, such as a memory cell phenotype. As a result, an activated population of engineered immune cells may have a higher proportion of stem-cell memory phenotypes and central memory phenotypes than a population of engineered immune cells that has not been activated. Activating the immune cells may alternatively or additionally promote homing to lymphoid organs (e.g., lymph nodes, spleen, thymus, and / or bone marrow), where the lymphoid environment may facilitate activation, expansion, and / or conversion to memory cell phenotypes.

[0224] In some embodiments, activation is performed on engineered immune cells prior to administration to a subject, e.g., by administration of an activator bound to a substrate, such as a surface (e.g., a plate surface), a bead (e.g., a polystyrene paramagnetic bead), a carrier protein (e.g., an antibody), a carrier polymer (e.g., a synthetic polymer, a biopolymer), a carrier nucleic acid (e.g., an oligonucleotide, a polynucleotide), or combinations thereof. The immune cells can be exposed to the activator for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or 24 hours. Ex vivo activation may be performed at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or 24 hours before administration of the engineered immune cells to the subject. The concentration of the activator for ex vivo activation can be within a range from 1 nM to 1000 nM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 10 nM to 1000 n, 10 nMto 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 50 nM to 1000 nM, 50 nM to 500 nM, 50 nM to 100 nM, 100 nM to 1000 nM, 100 nM to 500 nM, or 500 nM to 1000 nM. Following ex vivo activation, the substrate-bound activator may be removed from the immune cells, e.g., by cleaving the activator from the substrate (e.g., via disulfide reduction, pH-based cleavage, photocleavage, protease cleavage) and / or mechanical disruption.

[0225] Additional details and examples of compositions and methods for immune cell activation that are applicable to the present technology are provided in International Application No. PCT / US2023 / 082604, the disclosure of which is incorporated by reference herein in its entirety.E. Compositions of Engineered Immune Cells

[0226] Further provided herein are compositions including engineered immune cells suitable for administration to a subject in need thereof. The engineered immune cells or precursor cells thereof may be cultured and formulated for delivery to a subject (e.g., a donor of precursors of the engineered immune cells). In some embodiments, precursor cells are cultured prior to transfection with a polynucleotide encoding an engineered receptor. In such cases, the precursor cells can be activated, differentiated, and or expanded. The cells can be further cultured following transfection, for example, to further expand the cells or to affect terminal differentiation. In some embodiments, precursor cells are transfected with a CAR and / or a CCR prior to culturing, as described herein.

[0227] An engineered immune cell composition can be formulated with a solution tolerated by the immune cells. The immune cells are formulated with a solution of biological origin, such as plasma; a synthetic solution, such as saline, Ringer’s solution, dextrose solution, phosphate buffered saline; water; or a combination thereof. The formulation can also include a nonaqueous vehicle, such as ethyl oleate or a fatty acid triglyceride.

[0228] An engineered immune cell composition can include a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the composition includes a viscosityenhancing agent (e.g., sodium carboxymethylcellulose, dextran, or glycerol). In some embodiments, the composition includes an isotonicity imparting agent, such as sodium chloride, potassium chloride, or monosodium phosphate. In some embodiments, the composition includes a stabilizing agent, such as carboxymethyl cellulose, alginate, polyethylene glycol, or a polyol. In some embodiments, the composition includes a preservative, such as thimerosal, m- or o-cresol, formalin or benzyl alcohol. In some embodiments, the composition includes an adjuvant, such asaluminum hydroxide. In some embodiments, the composition includes a buffer, such as bicarbonate, TRIS, HEPES, MOPS, CHES, CHAPS, or phosphate buffered saline.

[0229] Engineered immune cell compositions can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a nonliquid formulation, the excipient can include, for example, dextrose, human serum albumin, and / or preservatives to which sterile water or saline can be added prior to administration.III. Therapeutic Methods

[0230] The immune-activating conjugates and engineered immune cells of the present technology may be administered to a subject to treat a disease or condition. In some embodiments, the disease or condition is a cancer. In some embodiments, the cancer is biliary tract cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, leukemia, liver cancer, lymphoma, lung cancer, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, skin cancer, testicular cancer, or thyroid cancer. In some embodiments, the cancer is a hematological cancer, such as acute myeloid leukemia (AML), multiple myeloma (MM), nonHodgkin lymphoma, acute lymphoblastic leukemia (ALL), mantle cell lymphoma, or follicular lymphoma. In some embodiments, the cancer is gastric cancer, glioma (e.g., diffuse midline glioma), mesothelioma, ovarian cancer, pancreatic cancer, or prostate cancer.

[0231] In some embodiments, a method for treating a disease includes administering to a subject a first composition including an immune-activating conjugate and a second composition including a plurality of engineered immune cells. The immune-activating conjugate can be any of the embodiments described herein, e.g., in Section I above. For example, the immune-activating conjugate can include an engineered polypeptide conjugated to an activator, where the engineered polypeptide binds to a marker on a target cell (e.g., a tumor antigen on a cancer cell). The dosage of the immune-activating conjugate that is administered to the subject can be within a range from 0.01 mg / kg to 10 mg / kg, 0.01 mg / kg to 5 mg / kg, O.Olmg / kg to 2 mg / kg, 0.01 mg / kg to 1 mg / kg, 0.01 mg / kg to 0.5 mg / kg, 0.01 mg / kg to 0.1 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.1 mg / kg to 5 mg / kg, 0.1 mg / kg to 2 mg / kg, 0.1 mg / kg to 1 mg / kg, 0.1 mg / kg to 0.5 mg / kg, 0.5 mg / kg to 10 mg / kg, 0.5 mg / kg to 5 mg / kg, 0.5 mg / kg to 2 mg / kg, 0.5 mg / kg to 1 mg / kg, 1 mg / kg to 10 mg / kg, 1 mg / kg to 5 mg / kg, 1 mg / kg to 2 mg / kg, 2 mg / kg to 10 mg / kg, 2 mg / kg to 5 mg / kg, or 5 mg / kg to 10 mg / kg.

[0232] The engineered immune cells can include at least one engineered receptor that binds to the activator of the immune-activating conjugate, e.g., as described in Section II above. Forexample, the engineered receptor can be an indirect CAR or a CCR that recognizes the activator. Binding of the engineered receptor to the activator can elicit immune cell activity that is beneficial for treatment of the disease, e.g., cytolytic activity, activation of cytokine signaling pathways, differentiation to a desired immune cell phenotype, homing to lymphoid organs, etc. In some embodiments, between about 105and about 5 x 107engineered immune cells are administered to the subject. In some embodiments, between about 5 x 104and about 3 x 107engineered immune cells are administered to the subject. In some embodiments, approximately 0.1 x 106, 0.5 x 106, 1 x 106, 2 x 106, or 5 x 106engineered immune cells are administered to the subject.

[0233] In some embodiments, the engineered immune cells are CAR T-cells, and administration of the CAR T-cells to the subject may treat a cancer in the subject by targeting and killing cancer cells. The CAR of the CAR T-cells may be an indirect CAR that is engineered to recognize the activator. In such embodiments, administration of the activator with an engineered polypeptide that recognizes a tumor antigen on the cancer cells can target the CAR T-cells to the cancer cells to cause activation of the CAR and killing of the cancer cells. The indirect CAR may or may not be expressed with another engineered receptor that recognizes the same activator or a different activator, such as a CCR.

[0234] Alternatively, the CAR of the CAR T-cells may be a direct CAR that recognizes a tumor antigen. In such embodiments, the direct CAR may be co-expressed with an engineered receptor that recognizes the activator, such as a CCR. Administration of the activator with an engineered polypeptide that recognizes a tumor antigen on the cancer cells can facilitate targeting of the CAR T-cells to the cancer cells, whereupon the CAR of the CAR T-cells can recognize the tumor antigen and kill the cancer cells. Moreover, binding of the CCR to the activator may cause activation of the engineered immune cells to promote formation of memory phenotypes (e.g., a stem-cell memory phenotype, a central memory phenotype, or combinations thereof) which increase immune cell persistence in the subject. Increased persistence of the engineered immune cells in the subject may improve patient outcome by reducing the chance of disease recurrence (e.g., cancer recurrence).

[0235] In some embodiments, administration of an immune cell engineered to express an indirect CAR and / or a CCR that recognizes an activator, and an immune-activating conjugate including the activator conjugated to an engineered polypeptide that recognizes CD123, ROR1, or CD33 may be used to treat acute myeloid leukemia. In another example, administration of an immune cell engineered to express an indirect CAR and / or a CCR that recognizes an activator, and an immune-activating conjugate including the activator conjugated to an engineeredpolypeptide that recognizes BCMA, SLAMF7, or GPRC5D may be used to treat multiple myeloma. In another example, administration of an immune cell engineered to express an indirect CAR and / or a CCR that recognizes an activator, and an immune-activating conjugate including the activator conjugated to an engineered polypeptide that recognizes MUC16, HER2, mesothelin antibody, or folate receptor-a may be used to treat ovarian cancer. In another example, administration of an immune cell engineered to express an indirect CAR and / or a CCR that recognizes an activator, and an immune-activating conjugate including the activator conjugated to an engineered polypeptide that recognizes mesothelin may be used to treat mesothelioma. In a further example, administration of an immune cell engineered to express an indirect CAR and / or a CCR that recognizes an activator, and an immune-activating conjugate including the activator conjugated to an engineered polypeptide that recognizes GD2 or B7-H3 may be used to treat glioma.|0236| In some embodiments, the immune-activating conjugate is administered to the subject at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 36 hours, or 48 hours before the subject receives the engineered immune cells. Alternatively or in combination, the immune-activating conjugate is administered to the subject concurrently with the engineered immune cells. Alternatively or in combination, the immune-activating conjugate is administered to the subject at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 36 hours, or 48 hours after the subject receives the engineered immune cells.

[0237] In some embodiments, the immune-activating conjugate is administered to the subject a single time. Alternatively, the immune-activating conjugate may be administered to the subject multiple times, e.g., two, three, four, five, or more times. The administration frequency can be at any suitable time interval, such as daily, weekly, biweekly, monthly, yearly, etc. The immune-activating conjugate may be administered to the subject multiple times to re-activate the engineered immune cells so the engineered immune cells continue to exhibit activity that is beneficial for treatment of the disease, e.g., cytolytic activity, activation of cytokine signaling pathways, differentiation to a desired immune cell phenotype, homing to lymphoid organs, etc.

[0238] In some embodiments, the engineered immune cells may be re-activated about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 16 days, about 18 days, about 20 days, about 22 days, about 24 days, about 26 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, or combinations thereof, afterbeing administered to the subject. In some embodiments, the engineered immune cells may be reactivated about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 21 months, about 24 months, about 27 months, about 30 months, about 33 months, about 36 months, or combinations thereof, after being administered to the subject. In some embodiments, the engineered immune cells may be re-activated about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or combinations thereof, after being administered to the subject.

[0239] In some embodiments, the engineered immune cells may be re-activated one or more times from about 7 days to about 56 days, from about 14 days to about 56 days, from about 21 days to about 56 days, from about 28 days to about 56 days, from about 35 days to about 56 days, from about 42 days to about 56 days, from about 49 days to about 56 days, from about 1 month to about 36 months, from about 2 months to about 36 months, from about 3 months to about 36 months, from about 4 months to about 36 months, from about 5 months to about 36 months, from about 6 months to about 36 months, from about 12 months to about 36 months, from about 18 months to about 36 months, from about 24 months to about 36 months, from about 30 months to about 36 months, from about 1 year to about 10 years, from about 2 years to about 10 years, from about 3 years to about 10 years, from about 4 years to about 10 years, from about 5 years to about 10 years, from about 1 years to about 8 years, from about 2 years to about 8 years, from about 3 years to about 8 years, from about 4 years to about 8 years, from about 5 years to about 8 years, from about 1 years to about 6 years, from about 2 years to about 6 years, from about 3 years to about 6 years, from about 4 years to about 6 years, or from about 5 years to about 6 years after being administered to the subject.

[0240] The dosage of the immune-activating conjugate that is administered to the subject may be the same for some or all of the administrations, or may be different for some or all of the administrations. For instance, a first dosage of an immune-activating conjugate may be provided to the subject at a first time point; and a second, different dosage of the immune-activating conjugate may be provided to the subject at a second, later time point. The second dosage may be higher than, lower than, or the same as the first dosage. The first and second dosages may each be independently selected from any of the following: from 0.1 mg / kg to 10 mg / kg, 0.1 mg / kg to 5 mg / kg, 0.1 mg / kg to 2 mg / kg, 0.1 mg / kg to 1 mg / kg, 0.1 mg / kg to 0.5 mg / kg, 0.5 mg / kg to 10mg / kg, 0.5 mg / kg to 5 mg / kg, 0.5 mg / kg to 2 mg / kg, 0.5 mg / kg to 1 mg / kg, 1 mg / kg to 10 mg / kg, 1 mg / kg to 5 mg / kg, 1 mg / kg to 2 mg / kg, 2 mg / kg to 10 mg / kg, 2 mg / kg to 5 mg / kg, or 5 mg / kg to 10 mg / kg. In some embodiments, administration of different dosages at different times can be used to titrate the extent of immune cell activation, e.g., to improve the efficacy of cancer cell killing, to mitigate side effects from on-target off-tumor targeting, etc.

[0241] In some embodiments, the immune-activating conjugate that is administered to the subject may be the same for some or all of the administrations, or may be different for some or all of the administrations. For instance, a first immune-activating conjugate including an activator and a first engineered polypeptide that recognizes a first tumor antigen may be provided to the subject at a first time point; and a second immune-activating conjugate including the activator and a second engineered polypeptide that recognizes a second, different tumor antigen may be provided to the subject at a second, later time point. This approach can be advantageous for directing the immune cells to different tumor antigens, e.g., to mitigate tumor antigen escape.

[0242] In some embodiments, a plurality of different immune-activating conjugates are administered to the subject at the same time point. For example, a first immune-activating conjugate including an activator and a first engineered polypeptide that recognizes a first tumor antigen may be provided to the subject at a time point; and a second immune-activating conjugate including the activator and a second engineered polypeptide that recognizes a second, different tumor antigen may be provided to the subject at the same time point. This multiplexed approach can facilitate immune cell recognition of multiple tumor antigens expressed on the same cancer cell and / or across multiple cancer cells, which may be advantageous for increasing the speed of cancer cell killing. Moreover, in situations where cancer cells express multiple antigens while normal cells express only a single antigen, multiplexing can mitigate on-target, off-tumor toxicity.

[0243] As another example, a first immune-activating conjugate including a first activator and an engineered polypeptide that recognizes a tumor antigen may be provided to the subject at a time point; and a second immune-activating conjugate including a second different, activator and an engineered polypeptide that recognizes a tumor antigen may be provided to the subject at the same time point. The first activator may be recognized by a first engineered receptor of the immune cell (e.g., an indirect CAR) and the second activator may be recognized by a second engineered receptor of the immune cell (e.g., a CCR), thereby allowing for concurrent activation of both engineered receptors. The engineered polypeptide of the first immune-activating conjugate may be the same as the engineered polypeptide of the second immune-activating conjugate (e.g., both engineered polypeptides recognize the same tumor antigen), or the engineered polypeptide of thefirst immune-activating conjugate may be different than the engineered polypeptide of the second immune-activating conjugate (e.g., the engineered polypeptides recognize different tumor antigens).

[0244] In some embodiments, a method of treating a subject includes administering a first immune-activating conjugate to the subject, and administering a second immune-activating conjugate to the subject. For example, the first immune-activating conjugate can include a first activator for an indirect CAR and a first engineered polypeptide; and the second immune- activating conjugate can include a second activator for a CCR and a second engineered polypeptide. The first activator can be the same as the second activator, or the first activator can be different than the second activator. The first engineered polypeptide can be the same as the second engineered polypeptide, or the first engineered polypeptide can be different than the second engineered polypeptide. The first engineered polypeptide can recognize the same epitope as the second engineered polypeptide, or can recognize a different epitope than the second engineered polypeptide. The dosage of the first immune-activating conjugate can be the same as, greater than, or less than the dosage of the second immune-activating conjugate.

[0245] The first immune-activating conjugate may be administered before, concurrently with, and / or after the second immune-activating conjugate. In some embodiments, the first immune-activating conjugate is administered at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 16 days, at least about 18 days, at least about 20 days, at least about 22 days, at least about 24 days, at least about 26 days, at least about 28 days, at least about 35 days, at least about 42 days, at least about 49 days, at least about 56 days, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months before the second immune-activating conjugate is administered.

[0246] In some embodiments, the second immune-activating conjugate is administered at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 16 days, at least about 18 days, at least about 20 days, at least about 22 days, at least about 24 days, at least about 26 days, at least about 28 days, at least about 35 days, at leastabout 42 days, at least about 49 days, at least about 56 days, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months before the first immune-activating conjugate is administered.IV. Examples

[0247] The present technology is further illustrated by the following non-limiting examples.Example 1 : Engineered Polypeptides for Tumor Recognition

[0248] This example describes the preparation and characterization of engineered polypeptides that recognize tumor antigens.

[0249] Engineered polypeptides of the present technology were designed and screened for binding to tumor antigens of interest. To allow for conjugation of an activator, variants with lysine residues at specified sites were also designed and screened to confirm that binding activity was maintained. Engineered polypeptides were prepared via expression in E. coli or by chemical peptide synthesis (denoted by “PS” in the nomenclature below), and purity (> 90%) was confirmed by high performance liquid chromatography (HPLC). Protein parameters were approximated based on sequence and computational tools, and binding parameters were determined by surface plasmon resonance (SPR) analysis.

[0250] An engineered polypeptide targeting CD 123, an antigen for acute myeloid leukemia (AML), was prepared (“MP” (SEQ ID NO: 1)), along with three lysine variants (“MP- 24K” (SEQ ID NO: 2), “MP-33K” (SEQ ID NO: 3), “MP-43K” (SEQ ID NO: 4)). FIG. 3 illustrates the structure of the MP engineered polypeptide with the three lysine residues indicated. As a control, a non-binding engineered polypeptide based on the MP sequence was also prepared. The characteristics of the MP engineered polypeptides are summarized in Tables 5 and 6 below.

[0251] Table 5: MP Protein Parameters[0252 | Table 6: MP Binding ParametersExample 2: Lysine / N-Terminal Acylation of Engineered Peptides with NHS-Functionalized Activators

[0253] This example describes methods for conjugation of engineered peptides via lysine or N-terminal acylation with NHS-functionalized activators.

[0254] FIG. 4 illustrates a conjugation scheme for coupling of an activator to an engineered polypeptide via acylation. An NHS-functionalized activator with an optional linker was conjugated to the engineered lysine residue or the N-terminus of the engineered polypeptide.

[0255] FIG. 5A illustrates a conjugation scheme for coupling a fluorescein or DOTA activator to an engineered polypeptide via lysine or N-terminal acylation. NHS-functionalized fluorescein (NHS-FL) or NHS-functionalized DOTA (NHS-DOTA) were combined with an engineered polypeptide in phosphate-buffered saline (PBS) at pH 7.4 and DMSO.|0256[ FIG. 5B illustrates four different species that may result from the conjugation scheme of FIG. 5A: an engineered polypeptide with no activator (DAR = 0), an engineered polypeptide with an activator conjugated to the lysine residue only (DAR = 1), an engineered polypeptide with an activator conjugated to the N-terminus only (DAR = 1), and engineered polypeptide with activators conjugated to both the lysine residue and the N-terminus (DAR = 2). In these Examples, the “DAR” of an immune-activating conjugate refers to the number of activators per polypeptide.

[0257] FIG. 6 is a flow diagram of a process for preparing an immune-activating conjugate. Stock solutions of engineered polypeptide (1-1.4 mM (5-8 g / L) polypeptide in 100 mM sodium phosphate buffer, pH 7.4) and NHS-functionalized activator (10 mM in DMSO) were combined at a ratio of 1-1.4 molar equivalents of NHS-functionalized activator to engineered polypeptide. The lysine conjugation reaction was performed for 1 hour at 65 °C in 100 mM sodium phosphatebuffer with 60% DMSO. The resulting product was buffer exchanged into PBS, formulated via dilution to a target dosing concentration, and sterile filtered (0.22 pm) to produce a stock solution of the engineered polypeptide-activator conjugate.

[0258] FIG. 7 illustrates the chemical structures of NHS-functionalized fluorescein activators with varying linkers: no linker (NHS-5-FL), short alkyl linker (NHS-5-EX-FL), PEG linker with 6 repeating units (fluorescein-PEG6-NHS ester), and PEG linker with 12 repeating units (carboxyfluorescein-PEG12-NHS).

[0259] FIGS. 8A-8D illustrate conjugation schemes for coupling an engineered polypeptide to NHS-5-FL (FIG. 8A), NHS-5-EX-FL (FIG. 8B), fluorescein-PEG6-NHS ester (FIG. 8C), and carboxyfluorescein-PEG12-NHS (FIG. 8D).Example 3: Preparation of Immune- Activating Conjugates for Targeting CD123

[0260] This example describes the preparation of immune-activating conjugates from MP engineered polypeptides that recognize CD 123.

[0261] Three lysine variants of the MP engineered polypeptide (MP-24K, MP-33K, and MP-43K of Example 1) were conjugated to NHS-functionalized fluorescein activators according to the protocol described in FIG. 6 above. The activators had varying linker lengths: no linker (NHS-5-FL), short alkyl linker (NHS-5-EX-FL), PEG linker with 6 repeating units (fluorescein- PEG6-NHS ester), and PEG linker with 12 repeating units (carboxyfluorescein-PEG12-NHS). The various conjugates are summarized in Table 7 below. Yield, purity, and DAR of the conjugates were assessed by Qubit fluorometric protein concentration and reversed-phase HPLC (RP-HPLC) at 280 nm and 493 nm.

[0262] Conjugates were purified by size exclusion chromatography (SEC) using Sephadex G15 or G25 resin to remove unbound activator. The purified conjugates were analyzed by liquid chromatography-mass spectroscopy (LC-MS) to verify the molecular weight and purity, and MS / MS peptide mapping was used to determine conjugation site. Antigen binding by SPR was performed to verify that activator conjugation did not significantly impact polypeptide binding. Optionally, anion exchange chromatography (AEX) or preparative RP-HPLC was used for fractionating the DAR = 1 species if higher purity standards were desired.

[0263] Table 7: MP Conjugates

[0264] Binding parameters of the conjugates with no linker or the short alkyl linker are summarized in Table 8 A below. The binding parameters of the conjugated polypeptides did not differ significantly from the binding parameters of the unconjugated polypeptides (see Table 6 above).

[0265] Table 8A: CD123 Binding Parameters of FL and 5-EX-FL MP Conjugates|0266| FIG. 9 is a representative HPLC trace of the MP-43K-5-FL conjugate at 215 nm (top), 280 nm (middle), and 493 nm (bottom). Peaks corresponding to DAR = 0 (“DO”), DAR = 1 (“DI”), and DAR = 2 (“D2”) are labeled. Yield and purity data for small scale initial batches (< 100 pg) of all MP conjugates is summarized in Table 8B below. In Table 8B, “LP eq.” indicates the molar equivalents of the linker-payload (activator) relative to the polypeptide. Conjugation optimization (e.g., switching from SEC purification instead of centrifugal purification) and scale up further improved yield (> 90%) and purity, with the majority of the species being DAR = 1.

[0267] Table 8B: Yield and Purity of MP Conjugates

[0268] These results demonstrate that the MP engineered polypeptide could be successfully conjugated to a fluorescein activator with varying linker lengths, with high yield and purity of the DAR = 1 species. The conjugation reactions were scalable from microgram to milligram level, thus indicating the feasibility of adapting this approach for larger scale manufacturing for the preparation of clinical grade material.Example 4: In Vitro Efficacy of Immune- Activatin Conjugates with Engineered Immune Cells

[0269] This example describes studies to investigate in vitro tumor cell killing using immune cells expressing an indirect CAR and immune-activating conjugates that recognize CD123.[0270 [ Engineered T-cells expressing an indirect CAR that recognizes fluorescein (also referred to herein as “anti-FL CAR T-cells” or “BAT-CAR cells”) were generated from primary T-cells isolated from healthy donor PBMCs. T-cells were activated using Dynabeads CD3 / CD28 (ThermoFisher) and cultured in X-VIVO media (Lonza) containing 5% hAB serum, 1% GlutaMAX, 1% HEPES, and 100 U / mL IL-2. Post activation, cells were transduced with an anti- FL CAR transgene linked to an mCherry detection marker via a 2A peptide. After several days, transduced anti-FL CAR T-cells were debeaded using a magnetic column, and further expanded in a 6M G-Rex culture vessel (Wilson Wolf) until harvest and cryopreservation.(0271 ] Cells were transduced using either: (1) a lentiviral vector encoding an anti-FL scFv(4m5.3), CD8 hinge domain, CD28 transmembrane domain, CD3(^ intracellular domain, and 4- 1BB intracellular domain (SEQ ID NO: 7) (“5M”), (2) a retroviral vector encoding an anti-FL scFv (4m5.3), CD8 hinge domain, CD28 transmembrane domain, CD3(^ intracellular domain, and 4-1BB intracellular domain (SEQ ID NO: 7) (“aF7”), or (3) a retroviral vector encoding an anti-FL scFv (4m5.3), CD8 hinge domain, CD28 transmembrane domain, CD3(^ intracellular domain, 4-1BB intracellular domain, and CD28 intracellular domain (SEQ ID NO: 8) (“aF6”).

[0272] The ability of the immune-activating conjugates to mediate tumor cell killing was assessed in vitro. Anti-FL CAR T-cells were co-cultured with MV-4-l l-luc-GFP cells (human AML cell line with high CD123 expression) at an effector cell to tumor cell ratio of 2: 1. Varying concentrations of the MP conjugates of Example 3 were added and cells were incubated for 20 hours. Cells were collected and stained with a viability dye and assessed through flow cytometry. Specific killing was measured by the percentage decrease of viable CTV+ MV-4-11-luc-GFP cells.

[0273] FIGS. 10A-10E are graphs illustrating MV-4-11 tumor killing efficacy of aF6 anti- FL CAR T-cells mediated by different MP conjugates. The MP polypeptides used in FIGS. 10A- 10D were expressed in E. coh. while the MP polypeptides used in FIGS. 10E were prepared by chemical peptide synthesis. In FIGS. 10A-10E, conjugates that gave > 60% maximum killing had R2values > 0.95.

[0274] FIG. 10A is a comparison of anti-FL CAR T-cell killing of MV-4-11 cells mediated by different MP lysine variant-FL conjugates with no linker. MP-43K-5-FL produced killing activity with a half-maximal effective concentration (ECso) of 417.45 pM. MP-24K-5-FL and MP- 33K-5-FL did not produce any significant killing activity.

[0275] FIG. 10B is a comparison of anti-FL CAR T-cell killing of MV-4-11 cells mediated by different MP lysine variant-FL conjugates with a short alkyl linker. The MP-43K-5-EX-FL and MP-33K-5-EX-FL constructs both produced killing activity, with the MP-43K-5-EX-FL having an ECso of 218.3 pM and the MP-33K-5-EX-FL having an ECso of 254.6 pM. MP-24K-5-EX-FL did not produce any significant killing activity.

[0276] FIG. 10C is a comparison of anti-FL CAR T-cell killing of MV-4-11 cells mediated by different MP lysine variant-FL conjugates with a PEG6 linker. All three MP lysine variants exhibited killing activity, with MP-43K-PEG6-FL having an ECso of 259.8 pM, MP-24K-PEG6- FL having an ECso of 214.9 pM, and MP-33K-PEG6-FL having an ECso of 207.3 pM.[0277J FIG. 10D is a comparison of anti-FL CAR T-cell killing of MV-4-11 cells mediated by different MP lysine variant-FL conjugates with a PEG12 linker. All three MP lysine variants exhibited killing activity, with MP-43K-PEG12-FL having an ECso of 820.25 pM, MP-24K- PEG12-FL having an ECso of 967.55 pM, and MP-33K-PEG12-FL having an ECso of 998 pM.

[0278] FIG. 10E is a comparison of anti-FL CAR T-cell killing of MV-4-11 cells mediated by a 5-maleimidyl-fluorescein conjugated talacotuzumab biosimilar antibody (“TALA-FL HI,” aDAR 5.5 IgGl stochastic cysteine conjugate) relative to a peptide synthesized MP-43K conjugated to fluorescein via short alkyl linker (MP-43K(PS)-5-EX-FL), and MP-43K / FITC with no linker (MP-43K(PS)-FITC). For the latter, FITC was incorporated directly into MP-43K during chemical peptide synthesis through use of a FITC-containing amino acid. These MP conjugates displayed cell killing performance that was similar to MP-43K-5-FL and MP-43K-5-EX-FL described above, except that the MP polypeptides were prepared by chemical peptide synthesis instead of biological expression in E. coli. As shown in FIG. 10E, both MP conjugates exhibited superior killing compared to the TALA-FL control. The TALA-FL control had an ECso of 89.93 pM, the MP-43K(PS)-5-EX-FL had an ECso of 65.495 pM, and the MP-43K(PS)-FITC had an ECso of 290.65 pM.

[0279] FIG. 11 is a graph showing graphs illustrating MV-4-11 tumor killing efficacy of aF6 anti-FL CAR T-cells mediated by different MP / FL lysine conjugates and talacotuzumab FITC-conjugated antibodies (“TALA-FL LO” is DAR=1.0 IgGl low DAR stochastic cysteine conjugate). For conjugates with no linker, at both 5nM and 50nM, MP-43K performed better than MP-24K and MP-33K. For conjugates with the short alkyl linker, at 5 nM and 50 nM, MP-43K and MP-33K performed equally well, while MP-24K performed less well. Extending the linker to PEG6 and PEG12 at 5 nM and 50 nM, all three lysine variants performed equally well. The talacotuzumab conjugates provided less efficient cell killing at 50 nM, relative to 5 nM. Peptide synthesized (PS) MP performed equally well as E. coli expressed MP.

[0280] The results of FIGS. 10A-11 demonstrate that tumor killing efficacy by anti-FL CAR T-cells mediated by MP conjugates was affected by both the conjugation site and linker length. The MP-43K variant produced specific killing for all linker variations. The MP-33K variant did not produce specific killing when no linker was used but produced specific killing for all other linkers. The MP-24K only produced killing when the longer PEG6 and PEG12 linkers were used. Without wishing to be bound by theory, it is hypothesized that the location of the lysine residue affected the accessibility of the conjugated activator, and that longer linkers could improve activator recognition at locations that are otherwise less accessible. The MP-43K conjugates mediated improved killing compared to a talacotuzumab antibody conjugate. The MP-43K-EX- FL conjugate produced the best ECso, potentially attributable to the formation of a smaller immunological synapse, and thus was selected for further studies.

[0281] FIGS. 12A-12C are graphs illustrating tumor killing efficacy of anti-FL CAR T- cells mediated by the MP-43K-5-EX-FL conjugate versus a talacotuzumab-FL antibody conjugate (“TALA-FL HI,” a DAR 6.3 IgGl cysteine conjugate). Specifically, FIG. 12A shows tumor killingmediated by the MP-43K-5-EX-FL conjugate with second generation aF7 anti-FL CAR T-cells, FIG. 12B shows tumor killing mediated by the TALA-FL HI antibody with second generation aF7 anti-FL CAR T-cells, and FIG. 12C shows tumor killing mediated by the MP-43K-5-EX-FL versus the TALA-FL HI antibody with third generation aF6 anti-FL CAR T-cells. The background killing was 54.5% for the initial aF7 assays and 12.58% for the aF6 assays. As can be seen in FIGS. 12A- 12C, the specific killing percentage for the TALA-FL HI antibody declines at higher doses, but this effect is not observed in the MP-43K-5-EX-FL conjugate for doses below 1000 nM. Without wishing to be bound by theory, it is hypothesized that the MP polypeptides may not be internalized or may be internalized at a much slower rate than the antibody, and that the drop-off in killing at high concentrations is caused by saturation of binding sites on the CAR and target antigen. There appears to be a larger concentration range over which the polypeptide-activator conjugate can achieve maximal killing relative to the antibody-activator conjugate.Example 5: Methods for Preparation of Immune- Activating Conjugates

[0282] This example describes additional conjugation schemes for preparing immune- activating conjugates.

[0283] FIG. 13 schematically illustrates an immune-activating conjugate. Various conjugation chemistries can be used to couple a first functional group of an engineered polypeptide to a second functional group of a small molecule activator-linker complex (the linker is optional and may be omitted). Examples of conjugation chemistries that may be used include NHS-ester- lysine acylations (e.g., as described in Example 2 above), isthiocyanate / lysine couplings, and metal-free click chemistry.

[0284] FIGS. 14A and 14B illustrate reaction schemes for conjugating an engineered polypeptide to an activator via isothiocyanate / lysine coupling. Isothiocyanate modified activators (FITC (FIG. 14A) and p-SCN-Bn-DOTA (FIG. 14B)) were combined with an engineered polypeptide (MP-43K) in sodium bicarbonate with 60% DMSO at pH 9.4 and incubated at 65 °C for one hour. The resulting conjugates were MP-43K conjugated to FITC (“MP-43K-FITC,” FIG. 14A) and MP-43K conjugated to DOTA (“MP-43K-Bn-DOTA,” FIG. 14B). Yield and purity data for the conjugates is summarized in Table 9 below.

[0285] Table 9: Yield and Purity of MP Conjugates

[0286] FIGS. 15A-15D illustrate reaction schemes for conjugating an engineered polypeptide to an activator via strain-promoted azide-alkyne cycloaddition (SPAAC), metal-free click chemistry.

[0287] As shown in FIG. 15 A, an azidation reaction was preformed to functionalize an engineered polypeptide (MP-43K) with an NHS-linker-azide (azido-propanoic NHS ester). MP- 43K and azido-propanoic NHS ester were combined at 1.5-1.6 eq of the NHS ester to polypeptide in a 100 mM solution of sodium phosphate at pH 7.4 with 60% DMS). The reaction was then incubated at 65°C for 1 hour to produce an azide-functionalized polypeptide “MP-43k-azide”).

[0288] As shown in FIG. 15B, azide-functionalized polypeptide (“MP-43K-azide”) was conjugated to a bicyclononyne (BCN) functionalized DOTA activator (“BCN-DOTA”) via strain- promoted azide-alkyne cycloaddition (SPAAC). The polypeptide and activator were combined in a PBS solution at pH 7.2 with 60% DMSO. The reaction was then incubated at room temperature for 1 hour to produce a DOTA-functionalized polypeptide (“MP-43K-Tz-BCN-DOTA”).

[0289] As shown in FIG. 15C, azide-functionalized polypeptide (“MP-43K-azide”) was conjugated to a bicyclononyne (BCN) functionalized fluorescein activator (“BCN-PEG3-FL”) via SPAAC. The polypeptide and activator were combined in a PBS solution at pH 7.2 with 60% DMSO. The reaction was then incubated at room temperature for 1 hour to produce a fluorescein- functionalized polypeptide (“MP-43K-Tz-BCN-PEG3-FL”).

[0290] As shown in FIG. 15D, azide-functionalized polypeptide (“MP-43K-azide”) was conjugated to a dibenzocylooctyne (DBCO) functionalized fluorescein activator (“DBCO-FL”) via SPAAC. The polypeptide and activator were combined in a PBS solution at pH 7.2 with 60% DMSO. The reaction was then incubated at room temperature for 1 hour to produce a fluorescein- functionalized polypeptide (“MP-43K-DBCO-FL”).

[0291] Yield and purity data for MP conjugates prepared according to the protocols ofFIGS. 15A-15C is summarized in Table 10 below.

[0292] Table 10: Yield and Purity of MP-43K ConjugatesExample 6: Preparation and Characterization of Multimeric Immune-Activating Conjugates

[0293] This example describes the preparation and characterization of immune-activating conjugates including multiple engineered polypeptides via chemical synthesis.|0294| FIGS. 16A-16D illustrate reaction schemes for preparation of clickable immune- activating conjugates via one pot sequential lysine and N-terminal conjugations.

[0295] FIG. 16 A illustrates a reaction scheme for preparation of an engineered polypeptide conjugated to an activator at an engineered lysine residue and with an azide group at the N- terminus (“MP-43K-5-EX-FL-N-PEG4-Azide”). As shown in FIG. 16A, an engineered polypeptide ( “MP-43K”) was combined with an NHS-functionalized fluorescein activator (NHS- 5-EX-FL) (1.35 eq) in a 100 mM sodium phosphate solution at pH 7.5 with 60% DMSO and incubated at 65°C for 20 minutes. Then, azido-PEG4-NHS (7 eq) was added and incubated at room temperature for 1 hour.

[0296] FIG. 16B illustrates a reaction scheme for preparation of an engineered polypeptide conjugated to an activator at an engineered lysine residue and with an alkyne group at the N- terminus (“MP -43K-5-EX-FL-N-PEG1 -Alkyne”). As shown in FIG. 16B, an engineered polypeptide ( “MP-43K”) was combined with an NHS-functionalized fluorescein activator (NHS- 5-EX-FL) (1.35 eq) in a 100 mM sodium phosphate solution at pH 7.5 with 60% DMSO and incubated at 65°C for 20 minutes. Then, propargyl-PEGl-NHS (7 eq) was added and incubated at room temperature for 1 hour.

[0297] FIG. 16C illustrates a reaction scheme for preparation of an engineered polypeptide conjugated to an activator at the N-terminus and with an azide group at the engineered lysine residue (“MP-43K-PEG4-Azide-N-FITC”). As shown in FIG. 16C, an engineered polypeptide ( “MP-43K”) was combined with azido-PEG4-NHS (1.2 eq) in a 100 mM sodium phosphate solution at pH 7.0 with 58% DMSO and incubated at 65°C for 25 minutes to add an azide group to the engineered lysine residue (“MP-43K-PEG4-Azide”). Then, FITC (30 eq.) in 60% DMSO was added and incubated at room temperature for 2 hours.10298] FIG. 16D illustrates a reaction scheme for preparation of an engineered polypeptide conjugated to an activator at the N-terminus and with an alkyne group at the engineered lysine residue (“MPR-REK-PEGl-Alkyne-N-FITC”). As shown in FIG. 16D, an engineered polypeptide ( “MP-REK”) was combined with propargyl-PEGl-NHS in a 100 mM sodium phosphate solution at pH 7.0 with 58% DMSO and incubated at 65°C for 25 minutes to add an azide group to the engineered lysine residue (“MP-43K-PEG1 -Alkyne”). Then, FITC (30 eq.) in 60% DMSO was added and incubated at room temperature for 2 hours.

[0299] FIGS. 17A and 17B illustrate reaction schemes for preparation of dimeric immune- activating conjugates using Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), click chemistry.

[0300] FIG. 17A illustrates a reaction scheme for preparation of an immune-activating conjugate including two engineered polypeptides with respective activators (“Bis-(MP-43K-N- FITC)-PEG5”). MP-43K-PEG4-azide-N-FITC and MP-43K-PEGl-Alkyne-A-FITC were combined with CuSO4 AH2O, THPTA, and (+)-sodium L-ascorbate in PBS at pH 7.4 and incubated at room temperature for 1 hour. The resulting conjugate was a MP-43K dimer with a short PEG5 linker between the engineered lysine residues of the polypeptides and fluorescein activators at the N-termini of the polypeptides.

[0301] FIG. 17B illustrates a reaction scheme for preparation of an immune-activating conjugate including two engineered polypeptides with respective activators (“Bis-(MP-43K-5- EX-FL)-PEG5”). MP-43K-5-EX-FL-N-PEG4-azide and MP-43K-5-EX-FL-N-PEG1 -Alkyne were combined with CuSCU 5H2O, THPTA, and (+)-sodium L-ascorbate in PBS at pH 7.4 and incubated at room temperature for 1 hour. The resulting conjugate was a MP-43K dimer with a short PEG5 linker between the N-termini of the polypeptides and fluorescein activators at the engineered lysine residues of the polypeptides.

[0302] FIGS. 18A and 18B illustrate reaction schemes for preparation of dimeric immune- activating conjugates using a bifunctional crosslinker and CuAAC.

[0303] FIG. 18A illustrates a reaction scheme for preparation of an immune-activating conjugate including two engineered polypeptides with respective activators (“Bis-(MP-43K-N- FITC)-PEG11”). MP-43K-PEG4-Azide-N-FITC was combined with a bis-propargyl PEG3 bifunctional crosslinker, CuSC>4 5H2O, (+)-sodium L-ascorbate, THPTA, and aminoguanidine in PBS and incubated at room temperature for 1 hour. The resulting conjugate was a MP-43K dimer with a long PEGU linker between the engineered lysine residues of the polypeptides and fluorescein activators at the N-termini of the polypeptides.

[0304] FIG. 18B illustrates a reaction scheme for preparation of an immune-activating conjugate including two engineered polypeptides with respective activators (“Bis-(MP-43K-5- EX-FL)-PEG11”). MP-43K-5-EX-FL-N-PEG4-Azide was combined with a bis-propargyl PEG3 bifunctional crosslinker, CuSC 'SEEO, (+)-sodium L-ascorbate, THPTA, and aminoguanidine in PBS and incubated at room temperature for 1 hour. The resulting conjugate was a MP-43K dimer with a long PEG11 linker between the N-termini of the polypeptides and fluorescein activators at the engineered lysine residues of the polypeptides.

[0305] Yield and purity data for MP conjugates prepared according to the protocols of FIGS. 16A-18B is summarized in Table 11 below.10306 [ Table 11 : Yield and Purity of MP-43K Conjugates[03071 The ability of the MP conjugates to mediate tumor cell killing by anti-FL CAR T- cells was assessed in vitro according to the protocol of Example 4 above. 5M anti-FL CAR T-cells were co-cultured with MV-4-11-luc-GFP cells at an effector cell to tumor cell ratio of 2: 1. Varying concentrations of the MP conjugates were added and cells were incubated for 20 hours before specific killing was measured.

[0308] FIG. 19A is a graph illustrating anti-FL CAR T-cell killing of tumor cells mediated by MP-43K-5-EX-FL at doses of 1 nM and 0.1 nM. Tumor cell killing efficiencies are shown for two batches of anti-FL CAR T-cells after 5 months of storage, and demonstrate that killing efficiencies were maintained across both batches.

[0309] FIGS. 19B and 19C are graphs illustrating anti-FL CAR T-cell killing of tumor cells mediated by various MP conjugates at a dosage of 1 nM (FIG. 19B) and 0.1 nM (FIG. 19C). This data shows that bivalent MP conjugates having the activator conjugated at the engineered lysine and crosslinked via the N-termini provided comparable tumor cell killing as the monomeric MP-43K-5-EX-FL conjugate. Bivalent MP conjugates having the activator conjugated to the N- terminus and crosslinked via the engineered lysines exhibited suboptimal cell killing. For the clickable monomeric MP conjugates, it was advantageous to conjugate the activator at theengineered lysine and have the clickable handle at the N-terminus. The clickable fluorescein conjugated MP performed comparably well as the MP-43K-5-EX-FL conjugate.Example 7: Preparation and Characterization of Bivalent Immune- Activating Conjugates|0310| This example describes the preparation and characterization of immune-activating conjugates with two engineered polypeptides conjugated to a single activator via chemical synthesis.

[0311] FIG. 20 illustrates a reaction scheme for preparing a divalent MP conjugate with a bridging activator (“Bis-(MP-43K-PEG6)-N-PEGl-5-EX-FL”). An engineered polypeptide (“MP-43K”) was combined with azido-PEG4-NHS ester in 100 mM sodium phosphate at pH 7 with 60% DMSO and incubated at 65°C for 30 minutes to produce an azide-functionalized polypeptide (“MP -43K-C3 -azide”) (Step A). Subsequently, a trifunctional linker with an amine group and two azide groups (“Bis-(PEG2-alkyne)-N-PEGl -amine”) was combined with an NHS- functionalized fluorescein activator (NHS-5-EX-FL) in 100 mM sodium phosphate at pH 7 with 60% DMSO and incubated for 30 minutes to produce a fluorescein activator bridge with two alkyne groups (Step B). The fluorescein activator bridge was combined with MP-43K-C3 -azide and CuSO45H2O, (+)-sodium L-ascorbate, and THPTA in PBS and incubated at room temperature for 1 hour (Step C). The resulting conjugate included two engineered polypeptides connected to each other via a single fluorescein activator.[03121 FIG. 21 illustrates a reaction scheme for preparing a divalent MP conjugate with a bridging activator (“Bis-(mp-43K-PEG2)-N-PEGl-FITC”). An engineered polypeptide ( “MP- 43K”) was combined with 3-azido-propanoic acid NHS ester in 100 mM sodium phosphate at pH 7 with 60% DMSO and incubated at 65°C for 20 minutes to produce an azide-functionalized polypeptide (“MP -43K-C3 -azide”) (Step A). Subsequently, a trifunctional linker with an amine group and two azide groups (“Bis-(PEG2-alkyne)-N-PEGl -amine”) was combined with FITC in 100 mM sodium phosphate at pH 7 with 60% DMSO and incubated for 30 minutes to produce a fluorescein activator bridge with two azide groups (“Bis-(PEG2-alkyne)-N-PEGl-FITC”) (StepB). Bis-(PEG2-alkyne)-N-PEGl-FITC was combined with MP-43K-C3 -azide and CUSO45H2O, (+)-sodium L-ascorbate, and THPTA in PBS and incubated at room temperature for 1 hour (StepC). The resulting conjugate included two engineered polypeptides connected to each other via a single fluorescein activator.Example 8: Preparation and Characterization of Immune-Activating Conjugates with Multiple Activators

[0313] This example describes the preparation and characterization of immune-activating conjugates including multiple activators conjugated to a single engineered polypeptide (“high DAR conjugates”).

[0314] FIG. 22A is a schematic illustration of an immune-activating conjugate including a single engineered polypeptide coupled to a single activator (“DAR = 1”). FIG. 22B is a schematic illustration of an immune-activating conjugate including a single engineered polypeptide coupled to two activators via a bivalent linker (“DAR = 2”). FIG. 22C is a schematic illustration of an immune-activating conjugate including a single engineered polypeptide coupled to three activators via a trivalent linker (“DAR = 3”). FIG. 22D is a schematic illustration of an immune-activating conjugate including a single engineered polypeptide coupled to two different activators (“Engager 1” and “Engager 2”) via linker.

[0315] FIG. 23 A illustrates a reaction scheme for preparing a DAR = 1 immune-activating conjugate (“MP-43K-PEG4-FAM”) via SPAAC. An engineered polypeptide ( “MP-43K”) was combined with NHS-PEG4-azide in 100 mM sodium phosphate at pH 7.4 with 60% DMSO and incubated at 65°C for 30 minutes to prepare an azide-functionalized polypeptide (“MP-43K- PEG4-Azide”). DBCO-FAM (6-isomer) in PBS was then added and incubated at room temperature to prepare a fluorescein functionalized polypeptide with DAR = 1.

[0316] FIG. 23B illustrates a reaction scheme for preparing a DAR = 2 immune-activating conjugate (“MP-43K-C5-Bis-PEG3-SPAAC-FAM”) via SPAAC. An engineered polypeptide ( “MP-43K”) was combined with NHS-C5-Bis-PEG3-azide in 100 mM sodium phosphate at pH 7.4 with 60% DMSO and incubated at 65°C for 30 minutes to prepare a polypeptide functionalized with two azide groups (“MP-43K-Bis-PEG3-Azide”). DBCO-FAM (6-isomer) in PBS was then added and incubated at room temperature to prepare a fluorescein functionalized polypeptide with DAR = 2.

[0317] FIGS. 23C and 23D illustrate a reaction scheme for preparing a DAR = 3 immune- activating conjugate (“MP-43K-C5-Tris-PEG3-SPAAC-FAM”) via SPAAC. An engineered polypeptide ( “MP-43K”) was combined with NHS-C5-Tris-PEG3-azide in 100 mM sodium phosphate at pH 7.4 with 60% DMSO and incubated at 65°C for 30 minutes to prepare a polypeptide functionalized with three azide groups (“MP-43K-Bis-PEG3-Azide”) (FIG. 23C).DBCO-FAM (6-isomer) in PBS was then added and incubated at room temperature to prepare a fluorescein functionalized polypeptide with DAR = 3 (FIG. 23D).

[0318] Yield and purity data for the MP conjugates prepared via SPAAC is summarized in Table 12 below.

[0319] Table 12: Yield and Purity of MP Conjugates Prepared via SPAAC

[0320] FIG. 24A illustrates a reaction scheme for preparing a DAR = 1 immune-activating conjugate (“MP-43K-PEG4-CuAAC-FAM”) via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). An engineered polypeptide ( “MP-43K”) was combined with NHS-PEG4-azide in 100 mM sodium phosphate at pH 7.4 with 60% DMSO and incubated at 65°C for 30 minutes to prepare an azide-functionalized polypeptide (“MP-43K-PEG4-Azide”). FAM-alkyne (5-isomer) in CuSC>4, THPTA, sodium ascorbate, and PBS was then added and incubated at room temperature for 30 minutes to prepare a fluorescein functionalized polypeptide with DAR = 1.

[0321] FIG. 24B illustrates a reaction scheme for preparing a DAR = 2 immune-activating conjugate (“MP-43K-Bis-PEG3-CuAAC-FAM”) via CuAAC. An engineered polypeptide ( “MP- 43K”) was combined with NHS-C5-Bis-PEG3-azide in 100 mM sodium phosphate at pH 7.4 with 60% DMSO and incubated at 65°C for 30 minutes to prepare a polypeptide with two azide groups (“MP-43K-Bis-PEG3-Azide”). FAM-alkyne (5-isomer) in CuSO4, THPTA, sodium ascorbate,and PBS was then added and incubated at room temperature for 30 minutes to prepare a fluorescein functionalized polypeptide with DAR = 2.

[0322] FIGS. 24C and 24D illustrate a reaction scheme for preparing a DAR = 3 immune- activating conjugate (“MP-43K-Tris-PEG3-CuAAC-FAM”) via CuAAC. An engineered polypeptide ( “MP-43K”) was combined with NHS-C5-Tris-PEG3-azide in 100 mM sodium phosphate at pH 7.4 with 60% DMSO and incubated at 65°C for 30 minutes to prepare a polypeptide with three azide groups (“MP-43K-Tris-PEG3-Azide”) (FIG. 24C). FAM-alkyne (5- isomer) in CuSC>4, THPTA, sodium ascorbate, and PBS was then added and incubated at room temperature for 30 minutes to prepare a fluorescein functionalized polypeptide with DAR = 3. (FIG.33D)

[0323] Yield and purity data for the MP conjugates prepared via CuAAC is summarized in Table 13 below.

[0324] Table 13: Yield and Purity of MP Conjugates Prepared via CuAAC

[0325] The ability of the immune-activating conjugates to mediate tumor cell killing was assessed in vitro. 5M anti -FL CAR T-cells were prepared according to the protocol of Example 4 above, and co-cultured MV-4-l l-luc-GFP cells at an effector cell to tumor cell ratio of 2: 1. Varying concentrations of conjugates were added and cells were incubated for 24 hours. Cellswere collected and stained with a viability dye and Cell Trace Violet (CTV) and assessed through flow cytometry. Specific killing was measured by the percentage decrease of viable CTV+ MV- 4-11-luc-GFP cells.

[0326] FIG. 25 is a graph illustrating anti-FL CAR T-cell killing of MV-4-11 cells mediated by MP conjugates with different DARs. No significant improvement in cell killing was observed at higher DARs. Immune-activating conjugates containing a fluorescein (5-isomer) provided tumor cell killing in the presence of BAT-CARs, but those containing a fluorescein (6- isomer) did not, thus indicating the significance of the isomeric form for CAR recognition.Example 9: In Vitro Activity of Immune- Activating Conjugates Targeting CD123

[0327] This example describes studies to investigate the in vitro activity of MP immune- activating conjugates.

[0328] The ability of MP conjugates to mediate tumor cell killing by anti-FL CAR T-cells was assessed in vitro according to the protocol of Example 4 above. aF6 anti-FL CAR T-cells were combined with MV-4-11-luc-GFP cells at a CAR+ cell to tumor cell ratio of 5: 1, and a CD8+CAR+ cell to tumor cell ratio of 1 : 1. Varying concentrations of conjugates (MP-43K-5-FL and MP-43K-5-EX-FL) were added and cells were incubated for 20 hours before specific killing was measured. A fluorescein-conjugated talacotuzumab antibody (“TALA-FL,” DAR = 7.1) was used as a control.

[0329] The ability of MP conjugates to bind to CD 123 -expressing AML cell lines in vitro was evaluated using MV-4-11 and MOLM-14 cells. MM. IS cells (a CD 123 -negative multiple myeloma cell line) were used as a negative control. The binding activity of the MP-43K-5-FL conjugate was compared to that of the TALA-FL antibody and the non-binding negative control polypeptide.|0330] Internalization of MP conjugates versus the talacotuzumab antibody was evaluated using two approaches: an indirect measurement via detection of antibodies / conjugates remaining on the cell surface (FIG. 26A), and a direct measurement via detection of internalized antibodies / conjugates within the cell (FIG. 26B).

[0331] For the indirect measurement, TALA-FL antibodies (100 nM) or MP-43K-5-FL conjugates (“MP -FL”) (100 nM or 1000 nM) were incubated with MV-4-11 cells for 15 minutes, followed by a wash out step or no wash out step. Detection was performed using an anti-FITC- biotinylated antibody and streptavidin-PE. Fluorescence measurements were obtained at 0, 2, 6,24, 48, or 72 hours, with cells incubated at 4 °C or 37 °C. The detailed protocol is as follows: MV-4-11 cells were aliquoted at -100,000 cells per well, resuspended in 50 pL of Fcblock (TruStain FcX) and FACS buffer, and incubated at 4 °C for 10 minutes. FL conjugates were prepared at a 2X desired concentration and added to the appropriate wells, and incubated at 4 °C for 15 minutes. Samples were washed by adding 100 pL of staining buffer, the cells were then centrifuged and the supernatant was discarded. A master mix of LIVE / DEAD Fixable Near-IR viability dye stain was prepared and added to each well, and incubated at 4 °C for 30 minutes protected from light. A master mix of anti-FITC-biotin stain (anti-FITC-biotin and FACS buffer) was prepared and added to each well, and incubated at 4 °C for 20 minutes. Samples were then washed with a staining buffer, and a master mix of streptavidin-PE Detection stain (Streptavidin- PE and FACS buffer) was added and incubated at 4 °C for 20 minutes. Samples were washed with a staining buffer. Single color compensation controls for near-IR viability dye using cells and for PE and FITC (and any additional fluorochromes used) using UltraComp eBeads were prepared. Fixation of cells was performed by resuspending thoroughly with 150 pL of FluoroFix, and incubated at room temp for at least 30 minutes or at 4 °C overnight. Samples were analyzed by flow cytometry on a Fortessa, gating on live single cells, and the PE channel was evaluated to detect FL conjugate binding on tumor cells.103321 For the direct measurement, talacotuzumab antibodies conjugated to pHAb, a pH- sensitive dye (TALA-pHAb) (100 nM) or MP-43K polypeptides conjugated to pHab (“MP- pHAb”) (100 nM or 1000 nM) were incubated with MV-4-11 cells for 20 minutes, with or without a subsequent wash out step. Detection was performed based on fluorescence of the pHAb dye in the low pH environment of the lysosome. Fluorescence measurements were obtained at 0, 2, 6, 24, 48, or 72 hours, with cells incubated at 4 °C or 37 °C. The details protocol is as follows: tumor cell culture was split at least once prior to experiment setup. MV-4-11 tumor cell lines were treated with MP-pHAb or TALA-pHAb at the indicated concentrations for 20 minutes at 4 °C. Half of the samples were washed out to remove excess pHAb-labeled MP-43K or Talacotuzumab, and the other half were not washed out. Cells were incubated at 4 °C or 37 °C for 0, 2, 6, 24, 48 or 72 hours, two sets of cells for each timepoint. For cells treated with TALA-pHAb, cells were stained with Live / Dead at 4 °C. For cells treated with MP-pHAb, cells were stained with Live / Dead stain at 4 °C. All cells were then fixed with 150 pL of FluoroFix for 30 minutes at room temperature. Flow cytometry data was acquired using a Fortessa X20 flow cytometer using FACSDiva software. Flow cytometry data analysis was interpreted using FlowJo vlO.7.2. Differences between groups were determined by using t-tests or one-way ANOVA, as appropriate. Allstatistical analyses were performed using Graphpad Prism and statistical significance was accepted at p < 0.05.

[0333] FIG. 27 illustrates a reaction scheme for conjugation of MP-43K to a pHAb pH- sensitive dye. MP-43K polypeptide and a pHAb amine reactive dye were combined in a PBS solution at pH 7.4 with 60% DMSO and incubated at 65°C for 1 hour.(0334] FIG. 28 is a graph illustrating anti-FL CAR T-cell killing of MV-4-11 cells mediated by MP conjugates versus the TALA-FL antibody control. As shown in FIG. 28, the MP conjugates outperformed the TALA-FL antibody control with higher levels of specific cell killing. MP-43K-5-FL had an ECso of 370.2 pM, MP-43K-5-EX-FL had an ECso of 100.3 pM, and TALA- FL had an ECso of 33.54.

[0335] FIG. 29 provides graphs illustrating the gMFI of cells incubated with the TALA- FL antibody (left) compared to the MP conjugates (right). MP-43K-5-FL (right) showed a higher apparent binding affinity to the CD 123 -positive MV-4-11 and MOLM-14 cell lines (KD = 0.45 nM) compared to the TALA-FL antibody (left) (KD = 13 nM). Some non-specific binding was observed with MP-43K-5-FL on the CD 123 -negative MM. IS cell line at high concentrations, but no apparent non-specific binding by the non-binding negative control polypeptide was observed.

[0336] FIGS. 30A and 30B illustrate results of the indirect internalization assay with a wash out step. Specifically, FIG. 30A provides flow cytometry plots illustrating fluorescence of MV-4-11 cells incubated with TALA-FL antibodies versus MP-43K-5-FL conjugates, and FIG. 30B provides graphs illustrating the mean fluorescence of MV-4-11 cells incubated with TALA- FL antibodies versus MP-43K-5-FL conjugates. As shown in FIG. 30A, there was a steady loss of surface-bound TALA-FL antibodies and MP-43K-5-FL conjugates throughout the 72-hour incubation period at 37 °C. As shown in FIG. 30B, fluorescence levels observed for the MP-43K- 5-FL conjugates were lower than those for the TALA-FL antibodies, thus indicating that MP-43K- 5-FL is internalized by the cells at a faster rate.

[0337] FIGS. 31 A and 3 IB illustrate the results of the indirect internalization assay without a wash out step. Specifically, FIG. 31A provides flow cytometry plots illustrating fluorescence of MV-4-11 cells incubated with TALA-FL antibodies versus MP-43K-5-FL conjugates, and FIG. 3 IB provides graphs illustrating the mean fluorescence of MV-4-11 cells incubated with TALA- FL antibodies versus MP-43K-5-FL conjugates. As shown in FIGS. 31 A and 3 IB, both the TALA- FL antibodies and MP-43K-5-FL conjugates were maintained on the cell surface when not subject to a wash out step.

[0338] FIGS. 32A-32C illustrate the results of the direct internalization assay. Specifically, FIG. 32A provides flow cytometry plots illustrating fluorescence of TALA-pHAb antibodies versus MP-pHAb conjugates in the presence of varying pH buffers with unbound dye washed out before incubating at 4 °C or 37 °C. FIG. 32B shows flow cytometry plots illustrating fluorescence of MV-4-11 cells incubated with TALA-pHAb antibodies versus MP-pHAb conjugates with no unbound dye wash out, and FIG. 32C provides graphs of mean fluorescence over time of MV-4-11 cells incubated with TALA-pHAb antibodies versus MP-pHAb conjugates. As shown in FIG. 32A, in the presence of pH 5 buffer, low levels of fluorescence were detected for the TALA-pHAb antibodies, while no fluorescence was detectable for the MP-pHAb conjugates. As shown in FIGS. 32B and 32C, fluorescence levels for both the TALA-pHAb antibodies and MP-pHAb conjugates increased over time, thus providing evidence that both constructs were internalized, albeit at different internalization rates. Comparing the internalization of 100 nM TALA-pHAb versus 100 nM MP-pHAb at 37 °C over time in FIG. 32B, it appears that TALA-pHAb was internalized at a considerably faster rate relative to MP-pHAb. Higher levels of internalization were observed for MP only at a lOx higher polypeptide concentration relative to the antibody.|0339| Overall, the results of the internalization assays indicate that both MP-43K immune-activating conjugates and talacotuzumab antibodies were internalized by the MV-4-11 cell line. The antib ody / activator conjugates were internalized at a faster rate than the MP polypeptide / activator conjugate. Antibody conjugates were not detectable by 24 hours whereas MP conjugates were still detectable on the cell surface at 72 hours.Example 10: In Vivo Pharmacokinetics of Immune-Activating Conjugates Targeting CD123|0340| This example describes studies to investigate the in vivo pharmacokinetics (PK) of MP immune-activating conjugates.

[0341] A first in vivo PK study was performed in female 6-week-old naive NCG mice. 100 mg / kg of human IVIG was administered via intraperitoneal (IP) injection to each mouse. After 24 hours, MP-43K-5 -EX-FL conjugates were administered. One group of n = 18 mice received a dosage of 1 mg / kg and a second group of n = 18 mice received a dosage of 10 mg / kg. Samples were collected via submandibular bleed at 0.08, 0.25, 0.5, 1, 2, 4, and 8 hours following treatment, and by cardiac puncture at 24 hours following treatment. Plasma samples were analyzed by fluorescent-linked immunosorbent assay (FLISA) and the data was then extrapolated in WinNonlin to calculate PK parameters.

[0342] A second in vivo PK study was performed in female 9-week-old MV-4-11 tumor bearing mice. A first group of n = 15 mice received 100 mg / kg of human IVIG administered via IP injection. A second group of n = 15 mice did not receive the human IVIG treatment. After 24 hours, MP-43K-5-EX-FL conjugates were administered to both groups. Each group of mice received a dosage of 10 mg / kg. Samples were collected via submandibular bleed at 0.08, 0.25, 0.5, 1, 2, 4, and 8 hours following treatment, and by cardiac puncture at 24 hours following treatment. Plasma samples were analyzed by FLISA and the data was then extrapolated in WinNonlin to calculate PK parameters.

[0343] FIGS. 33A and 33B are graphs showing calibration curves for MP-43K-5-EX-FL concentration versus fluorescence for the FLISA assay. Calibration standards were diluted > 99% in mouse plasma, and then diluted 2x with assay buffer prior to FLISA.

[0344] FIGS. 34A and 34B are graphs of the mean plasma concentration of the MP conjugate in naive mice over time for 1 mg / kg versus 10 mg / kg dosing (FIG. 34A shows concentration in pg / mL and FIG. 34B shows concentration in nM). The MP conjugate showed 50 times higher exposure with dosing at 10 mg / kg compared to 1 mg / kg, with a half-life of 5 hours. Based on the results of the tumor cell killing assay of FIG. 10B, the plasma concentration of MP conjugate is estimated to be above the EC90 (0.4 nM) out to 24 hours when dosed at 10 mg / kg. Table 14A below summarizes the PK parameters from the naive mouse study.

[0345] Table 14A: PK Parameters of MP Conjugates in Naive Mice

[0346] FIG. 35 is a graph of the mean plasma concentration of the MP conjugate in naive versus tumor-bearing mice overtime at 10 mg / kg dosing. Tumor-bearing mice had lower exposure and longer half-life of the MP conjugate exposure compared to naive mice. Table 14B below summarizes the PK parameters from the tumor-bearing mouse study.

[0347] Table 14B: Pharmacokinetic Parameters of MP-43K

[0348] Overall, these results demonstrate that increased half-life in plasma was observed in tumor-bearing animals. Human IVIG did not show a significant impact on exposure or half-life with MP-43K in tumor-bearing animals.Example 1 1 : / / / Vivo Efficacy of Immune-Activating Conjugates with Indirect CAR T-Cells

[0349] This example describes studies to investigate the in vivo efficacy of MP immune- activating conjugates with indirect CAR T-cells.

[0350] The efficacy of MP conjugates with anti-FL indirect CAR T-cells against MV-4- 11-luc-GFP cells was assessed in vivo. 5M anti-FL indirect CAR T-cells were prepared according to the protocol of Example 4 above. The groups for the study are shown in Table 15 below. 5xl06MV-4-11-luc-GFP tumor cells were administered via intravenous (IV) injection to each female NCG mouse (Charles River Laboratories). 6 days later, 5xl06anti-FL indirect CAR T-cells (“BAT-CAR cells”) or direct anti-CD123 CAR T-cells (“direct CAR T-cells”) were administered via IV injection. Mice received either 10 mg / kg daily (QD) of MP-43K-5-EX-FL conjugate or non-binding polypeptide fluorescein conjugate, or 1 mg / kg weekly (QW) or once every two days (Q2D) of fluorescein-conjugated talacotuzumab antibody (“TALA-FL”). Human IVIG was administered via IP injection at a dosage of 100 mg / kg weekly in the vehicle and TALA-FL groups only. 10 minutes before imaging acquisitions, mice were administered 150 mg / kg of IVISbrite D- Luciferin bioluminescence substrate, subcutaneously. Ventral and dorsal images were acquired on an IVIS Lumina. The bioluminescence signals were calculated by whole body regions of interest (ROI) on each day of imaging using Living Image software. The sum of the ventral and dorsal images was used for the calculations of mean bioluminescence signal. These calculations are reported as total flux (photons / second) from the ROI of each mouse. At the end of the study, data was log transformed and analyzed using an unpaired t-test for statistical analysis.

[0351] Table 15: Treatment Groups for In Vivo Study

[0352] CAR-T cell populations were analyzed post-injection to determine the number and percentage of CAR+ cells (CD3+mCherry+), subtype of CAR+ cells (CD8 versus CD4), phenotype of CAR+ cells (CD45RA versus CCR7), and exhaustion of CAR+ cells (as indicated by number of inhibitory molecules TIM3, PD1, LAG3). 100 pL of blood per mouse were collected and analyzed for various markers.

[0353] To evaluate tumor receptor occupancy of MP conjugates, female NCG mice (Charles River Laboratories, n = 4 per group) were injected with MV-4-11 cells. Approximately 20-25 days later, mice were treated with a single dose of vehicle (PBS), MP-43K-5-EX-FL (10 mg / kg), or TALA-FL antibody (1 mg / kg). At 0.25 hours and 5 hours post-treatment, mice were sacrificed and bone marrow samples were collected. The tumor receptor occupancy in the bone marrow samples was then measured by fluorescence-activated cell sorting (FACS).

[0354] To assess the dose response of BAT-CAR cells and MP immune-activating conjugates in vivo, female NCG mice (Charles River Laboratories) were inoculated with 5xl06MV-4-11-luc-GFP cells via IV injection and randomized into the treatment groups shown in Table 16 below. As shown in Table 16, MP-43K-5-EX-FL was administered via IP injection at dosages of either 1, 5, or 10 mg / kg. 4 hours later, BAT-CAR cells were administered via IV injection at amounts of either IxlO6or 5xl06cells. Subsequent doses of MP-43-5-EX-FL were administered in treatment schedules of either QD, Q2D, or QW. The 10 mg / kg QD mice were treated until Day 18. Four treatment cycles (28 days) were used for QW groups, with the last QW doses given on Day 21. Whole blood was collected for analysis on Day 25 (72 hours after the final QW doses and 24 hours after the final QD doses).

[0355] 10 minutes before imaging acquisitions, mice were administered 150 mg / kg ofIVISbrite D-Luciferin bioluminescence substrate, subcutaneously. Ventral and dorsal images wereacquired on an IVIS Lumina. The bioluminescence signal was calculated by whole body regions of interest (ROI) on each day of imaging using Living Image software. The sum of the ventral and dorsal images was used for the calculations of mean bioluminescence signal. These calculations are reported as total flux (photons / second) from the ROI of each mouse. At the end of study data was log transformed and analyzed using an unpaired t-test for statistical analysis.

[0356] Table 16: Treatment Groups for Dose Response Study|0357| FIG. 36 is a graph illustrating tumor burden based on mean bioluminescence intensity (BLI) across the various treatment groups. As shown in FIG. 36, tumor growth was well controlled in mice treated with the MP conjugate and BAT-CAR cells, with similar efficacy after 28 days and 56 days of daily MP dosing. Efficacy was comparable to treatment with the direct CAR T-cells, and superior to treatment with the BAT-CAR cells with TALA-FL antibodies.

[0358] FIGS. 37A-37F illustrate tumor burden based on mean BLI for individual mice in the vehicle only group (Group 1, FIG. 37A), BAT-CAR cells with 1 mg / kg Q2D TALA-FL antibody (Group 2, FIG. 37B), BAT-CAR cells with 1 mg / kg QW TALA-FL antibody (Group 3, FIG. 37C), BAT-CAR cells with non-binding polypeptide conjugate (Group 4, FIG. 37D), BATCAR cells with MP conjugate (Group 5, FIG. 37E), and direct CAR T-cells (Group 6, FIG. 37F). Between Days 28-35, mice with direct CAR T-cell treatments were found dead or had significant body weight loss (n=4). Between Days 57-59, an additional 2 mice were found dead with direct CAR T-cell treatments. Mice treated with the direct CAR T-cells that were found dead or had significant body weight loss were necropsied. It was found that the mice treated with direct CAR T-cells had significantly larger spleens than the mice treated with BAT-CAR cells and MP conjugates.

[0359] FIG. 38 is a graph showing the percent body weight change across the various treatment groups. The results in FIG. 38 indicate that all treatments were well tolerated.

[0360] FIGS. 39A and 39B are graphs illustrating CAR+ cell counts (FIG. 39 A) and CAR+ cell percentages (FIG. 39B) in mice treated with BAT-CAR cells and MP conjugates, BAT-CAR cells and TALA-FL antibodies, or direct CAR T-cells. As shown in FIGS. 39A and 39B, MP conjugates mediated expansion of BAT-CAR cells similar to the direct CAR T-cells. A significant decrease in CAR positivity was observed in mice treated with TALA-FL antibodies.[03611 FIG. 40 provides charts showing the number of CAR T-cell exhaustion markers (PD-1, TIM3, LAG3) in mice treated with BAT-CAR cells and MP conjugates, BAT-CAR cells and TALA-FL antibodies, or direct CAR T-cells. The charts indicate the percentages of cells expressing 0, 1, 2, and 3 exhaustion markers, respectively. As shown in FIG. 40, mice treated with BAT-CAR cells and MP conjugates demonstrated less CAR-T cell exhaustion than mice treated with direct CAR T-cells, suggesting that the constitutive CAR activation in direct CAR T-cells leads to more exhaustion.

[0362] FIG. 41 provides a series of graphs showing the phenotypes of CAR+ cells (CD45RA versus CCR7 and CD45RA versus CD62L) in mice treated with BAT-CAR cells (“5m”), BAT-CAR cells and MP conjugates (“5m+MP”), BAT-CAR cells and TALA-FL antibodies (“5m+TALA”), or direct CAR T-cells. These graphs depict the memory phenotype percentages from CAR+ cells, acquired through flow cytometry using CD45RA, CCR7, and CD62L markers, which denote stem cell memory (Tscm: CD45RA+CCR7+; CD45RA+CD62L+), central memory (Tcm: CD45RA-CCR7+; CD45RA-CD62L+), effector memory (Tern:CD45RA+CCR7-; CD45RA-CD62L-), and effector memory cells re-expressing CD45RA (Temra: CD45RA-CCR7; CD45RA+CD62L-). Patterns of CD45RA, CCR7 and CD62L expression are used to define the T central memory (Tcm) phenotype in the CAR-T cells Mice treated with MP conjugates had a greater percentage of central memory (Tcm) CAR-T cells than mice treated with TALA-FL antibodies.

[0363] FIG. 42 provides flow cytometry plots showing fluorescence in bone marrow cells (left) and graphs showing the percentages of bound receptors and gMFI in bone marrow cells (right) for mice treated with vehicle only, TALA-FL antibodies, or MP conjugates. As seen in FIG. 42, MP conjugate binding is high at 15 minutes (top row) but more variable after 5 hours (bottom row). These results show that the MP conjugates are trafficked to and bind to tumor cells in the bone marrow more quickly (within 15 minutes) than TALA-FL antibodies, while TALA-FL antibodies show improved and more consistent binding at 5 hours.10364] FIG. 43 A is a graph illustrating tumor burden based on mean BLI across treatment groups (top) and for individual mice (bottom) for mice treated with BAT-CAR cells and MP-43K- 5-EX-FL conjugates at a dosage of 10 mg / kg daily up to Day 18, and FIG. 43B is a graph illustrating tumor burden based on BLI across treatment groups (top) and for individual mice (bottom) for mice treated with BAT-CAR cells and MP-43K-5-EX-FL conjugates at a dosage of 10 mg / kg daily up to Day 28. At 10 mg / kg daily dosage of MP conjugate, no significant difference in tumor control was observed if the conjugate was given for 28 days versus 18 days.

[0365] FIG. 44A is a graph illustrating tumor burden based on mean BLI across treatment groups (top) and for individual mice (bottom) for mice treated with IxlO6BAT-CAR cells and 5 mg / kg MP-43K-5-EX-FL conjugates at different dosing frequencies, and FIG. 44B is a graph illustrating tumor burden based on mean BLI across treatment groups (top) and for individual mice (bottom) for mice treated with 5x106BAT-CAR cells and 5 mg / kg MP-43K-5-EX-FL conjugates at different dosing frequencies. At the 5 mg / kg dosage, no significant difference was observed in tumor control between the QD, Q2D, and QW treatment groups.

[0366] FIG. 45A is a graph illustrating tumor burden based on mean BLI across treatment groups (top) and for individual mice (bottom) for mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL conjugates at different dosing frequencies, and FIG. 45B is a graph illustrating tumor burden based on mean BLI across treatment groups (top) and for individual mice (bottom) for mice treated with 5x106BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL conjugatesat different dosing frequencies. At the 1 mg / kg dosage, superior anti-tumor activity was observed in the QW treatment groups.

[0367] FIG. 46 is a graph illustrating the percent change in body weight across treatment groups for the dose response study, indicating that the MP conjugate treatments were well- tolerated at all dose levels and frequencies.{(1368] FIG. 47 is a series of bioluminescent images of mice treated with IxlO6or 5xl06BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL conjugates at various dosing frequencies. Tumor presence in mice was drastically reduced for all dosage frequencies in comparison to the control.

[0369] Overall, the results of the dose response studies indicate that superior anti-tumor activity could be achieved with weekly MP conjugate treatment at 1 mg / kg (4 doses total) with only IxlO6BAT-CAR cells. Tumor killing efficacy was maintained even when reducing the MP conjugate dosage from 5 mg / kg to 1 mg / kg, when reducing dosing frequency from daily to weekly administration, and when reducing the BAT-CAR dose from 5xl06to IxlO6(5-fold reduction).Example 12: Dose Titration of Immune- Activating Conjugates for use with Indirect CAR T-Cells

[0370] This example describes studies to investigate the in vivo efficacy of different dosages of MP immune-activating conjugates for use with indirect CAR T-cells.

[0371] The efficacy of MP-43K-5-EX-FL conjugates with anti-FL indirect CAR T-cells against MV-4-11-luc-GFP (AML) cells was assessed in vivo according to the protocol of Example 11 above. Briefly, female NCG mice (Charles River Laboratories) were inoculated with 5xl06MV-4-11-luc-GFP cells via IV injection (Day -7) and randomized into the treatment groups shown in Table 17 below. Seven days later (Day 0), each treatment group received a dosage of 0.01, 0.1, or 1 mg / kg MP-43K-5-EX-FL as indicated in Table 17 by IP injection. Four hours later, each treatment group received a dosage of IxlO6, 0.5xl06, or 0. IxlO6BAT-CAR cells indicated in Table 17 by IV injection. MP-43K-5-EX-FL was re-dosed on Days 7, 14, 21, and 28 (QW treatment schedule), and the study was terminated on Day 57. Control groups were dosed with vehicle only (by IV injection on Day 0 and by IP injection on Days 7, 14, 21, and 28) or with O. lxlO6anti- CD123 direct CAR T-cells (by IV injection on Day 0).

[0372] Table 17: Treatment Groups for In Vivo Study

[0373] Bioluminescence imaging was performed according to the protocols described in Example 11 above. CAR T-cell populations were analyzed post-injection to determine the number and percentage of CAR+ cells, subtype of CAR+ cells, phenotype of CAR+ cells, and exhaustion of CAR+ cells according to the protocols described in Example 11 above.

[0374] To evaluate the MP-43K-5-EX-FL receptor occupancy on the surfaces of BATCAR T-cells, blood and bone marrow samples were collected from each mouse at Days 3, 7, and 14. Samples were incubated with phycoerythrin (PE) labeled rCD123 protein (ACRO Biosystems) for 15 minutes at 4 °C, followed by a cell wash using FACS buffer. Cell suspensions were then incubated with an anti-PE-biotin antibody (Biolegend) for 15 minutes at 4 °C, washed again with FACS buffer, then stained with anti-PE biotin / streptavidin-BV-421 (Biolegend) for 15 minutes at 4 °C. Cells were then assessed by FACS using a BD LSRII Fortessa flow cytometer (BD Biosciences).

[0375] FIG. 48A is a graph illustrating tumor burden based on mean BLI (top) and in individual mice (bottom) for mice treated with 0. IxlO6direct anti-CD123 CAR T-cells at different time points, and FIG. 48B is a graph illustrating tumor burden based on mean BLI (top) and in individual mice (bottom) for mice treated with O. lxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL at different time points (arrows in FIG. 48B indicate timing of MP-43K-5-EX-FL treatments). Mice treated with BAT-CAR cells and MP-43K-5-EX-FL showed a significant (p<0.0001) decrease in tumor burden over time in comparison to the vehicle only control, while mice treated with direct CAR T-cells did not show a significant difference in tumor burden in comparison to the vehicle only control. These results indicate that a low dose of BAT-CAR cells in combination with MP-43K-5-EX-FL is more effective in tumor control than a direct anti-CD 123 CAR T-cell treatment at the same dosage.

[0376] FIG. 49 is a graph showing the percent survival of mice across the various treatment groups described in Table 17. Survival endpoints were based on body weight loss from tumor burden and hind limb paralysis. At Day 57, 0% of the mice from Groups 1 and 2 survived, 60% of the mice from Group 5 survived, 80% of the mice from Groups 3 and 6 survived, and 100% of the mice from Groups 4 and 7 survived. These results show that a low dose of BAT-CAR (O. lxlO6cells / mouse) and 1 mg / kg of MP-43K-5-EX-FL (Group 7) were sufficient for achieving survival of tumor-bearing mice.

[0377] FIG. 50A is a graph illustrating tumor burden based on mean BLI across three MP- 43K-5-EX-FL dosages for mice treated with IxlO6BAT-CAR cells at different time points, and FIGS. 50B-50D are graphs illustrating tumor burden for individual mice for mice treated with IxlO6BAT-CAR cells and MP-43K-5-EX-FL dosages of 0.01 mg / kg (FIG. 50B), 0.1 mg / kg (FIG. 50C), and 1 mg / kg (FIG. 50D) at different time points. As shown in FIG. 50A, significant reductions in tumor burden at Day 28 were observed in mice treated with MP-43K-5-EX-FL versus mice treated with vehicle only (p = 0.0186 for vehicle versus 0.01 mg / kg or 0.1 mg / kg MP-43K- 5-EX-FL, p < 0.0001 for vehicle versus 1 mg / kg MP-43K-5-EX-FL). Tumor killing was achieved at Day 35 with a MP-43K-5-EX-FL dosage of 0.1 mg / kg (4 / 5 mice at background BLI levels) and at Day 14 with a MP-43K-5-EX-FL dosage of 1 mg / kg (5 / 5 of mice at background BLI levels). These results indicate that higher dosages of MP-43K-5-EX-FL provide greater tumor killing efficacy, although successfully killing was also achieved with the intermediate 0.1 mg / kg dosage.

[0378] FIG. 51 A is a graph illustrating tumor burden based on mean BLI across two MP- 43K-5-EX-FL dosages for mice treated with 0.5xl06BAT-CAR cells at different time points, and FIGS. 5 IB and 51C are graphs illustrating tumor burden for individual mice for mice treated with 0.5xl06BAT-CAR cells and MP-43K-5-EX-FL dosages of 0.1 mg / kg (FIG. 5 IB) and 1 mg / kg (FIG. 51C) at different time points. As shown in FIG. 51 A, significant reductions in tumor burden at Day 28 were observed in mice treated with MP-43K-5-EX-FL versus mice treated with vehicle only (p = 0.0012 for vehicle versus 0.1 mg / kg MP-43K-5-EX-FL, p = 0.0028 for vehicle versus 1mg / kg MP-43K-5-EX-FL). At a dosage of 1 mg / kg MP-43K-5-EX-FL, BLI signals reached background 4 / 5 mice by Day 49, although tumor killing exhibited higher variability at a dosage of 0.5xl06BAT-CAR cells in comparison to IxlO6BAT-CAR, regardless of MP-43K-5-EX-FL dosage.

[0379] FIG. 52 is a series of bioluminescent images of mice treated with BAT-CAR cells and MP-43K-5-EX-FL according to treatment groups 1-7 described in Table 17. Tumor presence in mice was reduced for most treatment groups in comparison to the vehicle only control, with the most significant reductions in treatment groups that received dosages of 1 mg / kg MP-43K-5-EX- FL.

[0380] FIG. 53A is a graph illustrating tumor burden based on mean BLI between mice treated with the vehicle and mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX- FL (arrows indicate timing of blood and bone marrow collection), and FIGS. 53B and 53C are graphs illustrating expansion of CAR+ cells in the blood (FIG. 53B) and bone marrow (FIG. 53C) at various timepoints. As shown in FIGS. 53B and 53C, CAR T-cell expansion was demonstrated in both the blood and bone marrow.

[0381] FIGS. 54A and 54B are graphs representing counts (left) and percentages (right) of CD8+ CAR+ cells (FIG. 54A) and CD4+ CAR+ cells (FIG. 54B) in blood samples at various time points. FIGS. 54C and 54D are graphs representing counts (left) and percentages (right) of CD8+ CAR+ cells (FIG. 54C) and CD4+ CAR+ cells (FIG. 54D) in bone marrow samples at various time points. The data in FIGS. 54A-54D was obtained from mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL. As shown in FIGS. 54A-54D, the blood favored expansion of CD8+ cells while the bone marrow favored expansion of CD4+ cells.

[0382] FIG. 55A is a graph showing the frequency of CAR+ cells at Day 0, FIG. 55B is a graph showing the frequency of CAR+ cells in blood samples at Days 7 and 14, and FIG. 55C is a graph showing the frequency of CAR+ cells in bone marrow samples at Day 15. The data in FIGS. 55A-55C was obtained from mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP- 4K-5-EX-FL. In both blood and bone marrow samples, the percentage of CAR+ cells remained fairly consistent from the time of injection (Day 0) to Day 14.

[0383] FIG. 56A is a graph showing the percentage of CAR+ cells in blood samples expressing exhaustion markers (TIM3, LAG3, and PD1), and FIG. 56B is a graph showing the percentage of CAR+ cells in bone marrow samples expressing exhaustion markers. The data in FIGS. 56A and 56B was obtained from mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL. Expression of exhaustion markers decreased in the blood between Days 7 and 14, and was low in the bone marrow at Day 14.

[0384] FIGS. 57A-57D are graphs showing the percentages of CAR+ cells in blood samples having the Tern (FIG. 57A), Tcm (FIG. 57B), Temra (FIG. 57C), and Tscm (FIG. 57D) phenotypes. FIG. 57E is a graph showing the percentage of CAR+ cells in bone marrow samples expressing the Tern, Tcm, Temra, and Tscm phenotypes. The data in FIGS. 57A-57E was obtained from mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL. In CAR+ cell populations collected from both blood and bone marrow, the Tern phenotype was favored.

[0385] FIG. 58A is a schematic showing the detection of surface MP-43K-5-EX-FL on BAT-CAR cells. FIGS. 58B and 58C are a series of flow cytometry plots showing the detection of surface MP-43K-5-EX-FL in blood samples (FIG. 58B) and bone marrow samples (FIG. 58C) from individual mice at various time points. FIG. 58D is a pair of graphs showing the percentage of BAT-CAR cells remaining bound to MP-43K-5-EX-FL in blood samples (left) and bone marrow samples (right) at various time points. The data in FIGS. 58A-58D was obtained from mice treated with IxlO6BAT-CAR cells and 1 mg / kg MP-43K-5-EX-FL. In both blood and bone marrow samples, surface residency of MP -43K-5 -EX-FL conjugates on BAT-CAR cells increased throughout the study and remained high at Day 14.

[0386] Overall, the results of these studies indicate that a lower dose of BAT-CAR-T cells (O.lxlO6) administered with MP-43K-5-EX-FL conjugates at 1 mg / kg weekly demonstrated superior tumor killing in comparison to direct CAR T-cell therapy in a mouse model of AML. This could provide an efficacious treatment for AML patients while mitigating the risk of toxicity that has been observed in direct CAR T-cell treatments. The superior efficacy of the BAT-CAR system was evidenced by the improved survival, expansion of CAR+ cell populations in the blood and bone marrow, low expression of exhaustion markers, and shift towards effector memory phenotypes in CAR+ cell populations.V. Additional Examples

[0387] Additional examples of aspects of the present technology are described below as numbered clauses (1, 2, 3, etc.) for convenience. These are provided as examples and do not limit the subject technology.

[0388] Example 1. A system for immunotherapy, the system comprising a first composition comprising an engineered polypeptide conjugated to a small molecule activator, wherein the engineered polypeptide has a molecular weight less than orequal to 10 kDa, and wherein the engineered polypeptide binds to a marker on a target cell; and a second composition comprising an immune cell expressing an engineered receptor that binds to the small molecule activator.

[0389] Example 2. The system of Example 1, wherein the molecular weight of the engineered polypeptide is less than or equal to 9 kDa, 8 kDa, 7 kDa, 5 kDa, 4 kDa, 3 kDa, 2 kDa, or 1 kDa.

[0390] Example 3. The system of Example 1 or 2, wherein the molecular weight of the engineered polypeptide is within a range from 1 kDa to 10 kDa, 1 kDa to 5 kDa, 1 kDa to 2 kDa, 2 kDa to 8 kDa, 2 kDa to 5 kDa, 3 kDa to 7 kDa, 4 kDa to 6 kDa, 5 kDa to 8 kDa, 5 kDa to 10 kDa, or 8 kDa to 10 kDa.

[0391] Example 4. The system of any one of Examples 1 to 3, wherein the engineered polypeptide comprises no more than 100 amino acid residues, 90 amino acid residues, 80 amino acid residues, 70 amino acid residues, 60 amino acid residues, 50 amino acid residues, 40 amino acid residues, 30 amino acid residues, 20 amino acid residues, or 10 amino acid residues.

[0392] Example 5. The system of any one of Examples 1 to 4, wherein the engineered polypeptide comprises from 10 amino acid residues to 100 amino acid residues, 10 amino acid residues to 50 amino acid residues, 20 amino acid residues to 80 amino acid residues, 30 amino acid residues to 70 amino acid residues, or 40 amino acid residues to 60 amino acid residues.

[0393] Example 6. The system of any one of Examples 1 to 5, wherein the engineered polypeptide includes a hydrophobic core.{0394] Example 7. The system of any one of Examples 1 to 6, wherein the engineered polypeptide does not include any disulfide bridges.

[0395] Example 8. The system of any one of Examples 1 to 7, wherein the engineered polypeptide includes 2, 3, 4, 5, 6, or more secondary structure domains.

[0396] Example 9. The system of any one of Examples 1 to 8, wherein the engineered polypeptide has a net charge less than or equal to 0, -0.5, -1, -1.5, -2, -2.5, -3, -3.5, -4, -4.5, -5, - 5.5, -6, -6.5, -7, -7.5, -8, -8.5, -9, -9.5, or -10.

[0397] Example 10. The system of any one of Examples 1 to 9, wherein the engineered polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 negatively charged amino acid residues.

[0398] Example 11. The system of any one of Examples 1 to 10, wherein the engineered polypeptide comprises a conjugation site for conjugation to the small molecule activator or to a linker.

[0399] Example 12. The system of Example 11, wherein the conjugation site is a lysine residue, a cysteine residue, a histidine residue, a non-natural amino acid residue, an N-terminus of the engineered polypeptide, or a C-terminus of the engineered polypeptide.

[0400] Example 13. The system of Example 11 or 12, wherein the conjugation site is located no more than 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from an N-terminus of the engineered polypeptide.

[0401] Example 14. The system of any one of Examples 11 to 13, wherein the conjugation site is located at least 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from an N-terminus of the engineered polypeptide.

[0402] Example 15. The system of any one of Examples 11 to 14, wherein the conjugation site is located no more than 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from a C-terminus of the engineered polypeptide.

[0403] Example 16. The system of any one of Examples 11 to 15, wherein the conjugation site is located at least 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from a C-terminus of the engineered polypeptide.

[0404] Example 17. The system of any one of Examples 1 to 16, wherein the engineered polypeptide comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 1 - SEQ ID NO: 5.

[0405] Example 18. The system of any one of Examples 1 to 17, wherein the engineered polypeptide comprises a sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 5.

[0406] Example 19. The system of any one of Examples 1 to 18, wherein the marker is a tumor antigen and the target cell is a cancer cell.

[0407] Example 20. The system of Example 19, wherein the tumor antigen comprises CD19, CD20, CD22, CD123, CD33, CD3, CD4, CD8, CD38, SLAMF7, BCMA, GD2, GPRC5D, MUC16, HER2, EGFR, EGFRvIII, CLL-1, CD44v6, folate receptor-a, mesothelin, CD20, CD37, R0R1, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, surviving, telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, IL13Ra2, B7-H3 (CD276), EPHA2, GRP78, NKG2D, or CD70.

[0408] Example 21. The system of any one of Examples 1 to 20, wherein the small molecule activator has a molecular weight less than or equal to 5 kDa, 4 kDa, 3 kDa, 2 kDa, 1 kDa, 500 Da, 200 Da, or 100 Da.

[0409] Example 22. The system of any one of Examples 1 to 21, wherein the small molecule activator is an exogenous small molecule.

[0410] Example 23. The system of Example 22, wherein the exogenous small molecule is selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), fluorescein 5-maleimide, fluorescein-5-carboxamide, fluorescein-6-carboxamide, 6-FAM phosphoramidite, topiramate hemi succinate, creatine, acetaminophen, ketamine, propofol, lidocaine, ractopamine, salicylate, salicylic acid, sulfasalazine, dapsone, albendazole, ivermectin, levamisole, permethrin, pyrantel, thiabendazole, procainamide, sulfamethazine, amikacin, amoxicillin, ampicillin, cefazolin, cefuroxime, cephalexin, chloramphenicol, chloramphenicol, ciprofloxacin, clenbuterol, cioxacillin, colistin A, dicloxacillin, enrofloxacin, furaltadone, gentamicin, gentamicin, kanamycin, kanamycin, kincomycin, lincomycin, metronidazole, nafcillin, nalidixic acid, neomycin, neomycin, nitrofurazone, norfloxacin, ofloxacin, oxacillin, spectinomycin, streptomycin, streptomycin, sulfabenzamide, sulfacetamide, sulfadiazine, sulfadimidine, sulfametoxydiazine, sulfanilamide, trimethoprim, carbamazepine, ethosuximide, lamotrigine, primidone, cetirizine, chlorpheniramine, diphenhydramine, doxylamine, promethazine, sulfadimethoxine, benzothiazinone, butylated hydroxytoluene, tripelennamine, chlorpromazine, clozapine, haloperidol, olanzapine, paliperidone, quetiapine, ribavirin, meprobamate, acebutolol,atenolol, penbutolol, warfarin, salmeterol, aflatoxin Bl, tetraxetan (DOTA), 4-[(6-methylpyrazin- 2-yl) oxy]benzoate (MPOB), biotin, melamine, methotrexate, amphetamine, diethylpropion, dextromethorphan, pseudoephedrine, dihydrochlorothiazide, hydrochlorothiazide, clonazepam, diazepam, nitrazepam, rhodamine B, fluorescent brightener Ksn, zearalenone, Sudan Redl, acetominophen, acrylamide, benzoic acid, benzophenone, benzothiazine, mercaptobenzothiazole, erythrosine, Sudan, tartrazine, erythromycin, sirolimus, atropine, ethyl glucuronide, aflatoxin Ml, methocarbamol, fentanyl, hydromorphone, morphine, remifentanil, tapentadol, tramadol, pregabalin, gabapentin, amitriptyline, desipramine, imipramine, nortriptyline, venlafaxine, dinitrophenyl, His-tag, PEG methoxy group, etodolac, ibuprofen, ketoprofen, meclofenamic acid, phenylbutazone, acetyl salicylic acid, acetamiprid, acetochlor, carbadazim, carbaryl, chlorothalonil, chlorpyrifos, fenpropathrin, imazalil, imidacloprid, parathion, abscisic acid, dibutyl phthalate, clonazepam, lorazepam, oxazepam, phenobarbital, secobarbital, zaleplon, zolpidem, trazodone, fluoxetine, fluvoxamine, cortisone, dexamethasone, dihydrotestosterone, fluocinolone, methylprednisolone, prednisolone, stanozolol, triamcinolone, mazindol, methamphetamine, methylphenidate, modafinil, chrysoidine, deoxynivalenol, fumonisin, microcystin Lr, ochratoxin, sterigmatocystin, T-2 toxin, sildenafil, tadalafil, scopolamine, florfenicol, pirlimycin, and sulfaquinoxaline.

[0411] Example 24. The system of any one of Examples 1 to 23, wherein the small molecule activator is fluorescein, a fluorescein derivative, or tetraxetan (DOTA).

[0412] Example 25. The system of Example 24, wherein the small molecule activator is the fluorescein derivative, and the fluorescein derivative is fluorescein isothiocyanate (FITC), fluorescein 5-maleimide, fluorescein-5-carboxamide, fluorescein-6-carboxamide, or 6-FAM phosphoramidite.

[0413] Example 26. The system of any one of Examples 1 to 25, wherein the engineered polypeptide is conjugated to the small molecule activator via a linker.

[0414] Example 27. The system of Example 26, wherein the linker is a linear linker.

[0415] Example 28. The system of Example 26, wherein the linker is a branched linker.

[0416] Example 29. The system of any one of Examples 26 to 28, wherein the linker comprises at least 2 repeating units, 5 repeating units, 10 repeating units, 12 repeating units, 15 repeating units, 20 repeating units, or 25 repeating units.

[0417] Example 30. The system of any one of Examples 26 to 29, wherein the linker comprises no more than 25 repeating units, 20 repeating units, 15 repeating units, 12 repeating units, 10 repeating units, 5 repeating units, or 2 repeating units.[0418 [ Example 31. The system of any one of Examples 26 to 30, wherein the linker has a length of at least 2 atoms, 3 atoms, 4 atoms, 5 atoms, 6 atoms, 7 atoms, 8 atoms, 9 atoms, 10 atoms, 15 atoms, 20 atoms, 25 atoms, 30 atoms, 35 atoms, 40 atoms, 45 atoms, 50 atoms, 60 atoms, 70 atoms, 80 atoms, 90 atoms, or 100 atoms.

[0419] Example 32. The system of any one of Examples 26 to 31, wherein the linker has a length no greater than 100 atoms, 90 atoms, 80 atoms, 70 atoms, 60 atoms, 50 atoms, 45 atoms, 40 atoms, 35 atoms, 30 atoms, 25 atoms, 20 atoms, 15 atoms, 10 atoms, 9 atoms, 8 atoms, 7 atoms, 6 atoms, 5 atoms, 4 atoms, 3 atoms, or 2 atoms.

[0420] Example 33. The system of any one of Examples 1 to 32, wherein the immune cell is a chimeric antigen receptor (CAR) T cell.

[0421] Example 34. The system of Example 33, wherein the engineered receptor comprises a CAR of the CAR T cell.

[0422] Example 35. The system of Example 34, wherein binding of the activator to the CAR causes the CAR T cell to kill the target cell.

[0423] Example 36. The system of Example 34 or 35, wherein the CAR is encoded by a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 6 - SEQ ID NO: 8.

[0424] Example 37. The system of any one of Examples 34 to 36, wherein the CAR is encoded by a sequence of any one of SEQ ID NO: 6 - SEQ ID NO: 8.{0425] Example 38. The system of any one of Examples 1 to 37, wherein the engineered receptor comprises a chimeric cytokine receptor (CCR).

[0426] Example 39. The system of Example 38, wherein binding of the small molecule activator to the CCR causes a change in phenotype of the immune cell.

[0427] Example 40. The system of Example 39, wherein the change in phenotype comprises a change to a memory phenotype.

[0428] Example 41. The system of any one of Examples 38 to 40, wherein the CCR comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 9 - SEQ ID NO: 15.

[0429] Example 42. The system of any one of Examples 38 to 41, wherein the CCR comprises a sequence of any one of SEQ ID NO: 9 - SEQ ID NO: 15.

[0430] Example 43. The system of any one of Examples 38 to 42, wherein the CCR is encoded by a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 16 - SEQ ID NO: 22.

[0431] Example 44. The system of any one of Examples 38 to 43, wherein the CCR is encoded by a sequence of any one of SEQ ID NO: 16 - SEQ ID NO: 22.

[0432] Example 45. The system of any one of Examples 1 to 44, wherein the engineered polypeptide is conjugated to a single small molecule activator.

[0433] Example 46. The system of any one of Examples 1 to 44, wherein the engineered polypeptide is conjugated to a plurality of small molecule activators.

[0434] Example 47. The system of any one of Examples 1 to 46, wherein the first composition further comprises a second engineered polypeptide conjugated to the small molecule activator or to the engineered polypeptide, and wherein the second engineered polypeptide has a molecular weight less than or equal to 10 kDa.

[0435] Example 48. The system of Example 47, wherein the second engineered polypeptide is the same as the engineered polypeptide.

[0436] Example 49. The system of Example 47, wherein the second engineered polypeptide is different from the engineered polypeptide.

[0437] Example 50. The system of any one of Examples 47 to 49, wherein the first composition further comprises a second small molecule activator conjugated to the second engineered polypeptide.

[0438] Example 51. The system of any one of Examples 1 to 50, further comprising a third composition comprising the engineered polypeptide conjugated to a second small molecule activator.

[0439] Example 52. The system of Example 51, wherein the immune cell expresses a second engineered receptor that binds to the second small molecule activator.

[0440] Example 53. The system of Example 52, wherein the engineered receptor is a CAR and the second engineered receptor is a CCR.

[0441] Example 54. The system of any one of Examples 1 to 53, further comprising a third composition comprising a second engineered polypeptide conjugated to the small molecule activator, wherein the second engineered polypeptide has a molecular weight less than or equal to 10 kDa.

[0442] Example 55. The system of Example 54, wherein the second engineered polypeptide binds to a different marker than the engineered polypeptide.10443] Example 56. A system for immunotherapy, the system comprising: a first pharmaceutical composition comprising the first composition of the system of any one of Examples 1 to 55 and a pharmaceutically acceptable carrier; and a second pharmaceutical composition comprising the second composition of the system of any one of Examples 1 to 55 and a pharmaceutically acceptable carrier.

[0444] Example 57. A method for treating a disease, the method comprising administering the system of any one of Examples 1 to 56 to a subject.

[0445] Example 58. A method for treating a disease, the method comprising: administering a first composition to a subject, the first composition comprising an engineered polypeptide conjugated to a small molecule activator, wherein the engineered polypeptide has a molecular weight less than or equal to 10 kDa, and wherein the engineered polypeptide binds to a marker on a target cell; and administering a second composition to the subject, the second composition comprising an immune cell expressing an engineered receptor that binds to the small molecule activator.

[0446] Example 59. The method of Example 58, wherein the molecular weight of the engineered polypeptide is less than or equal to 9 kDa, 8 kDa, 7 kDa, 5 kDa, 4 kDa, 3 kDa, 2 kDa, or 1 kDa.{0447] Example 60. The method of Example 58 or 59, wherein the molecular weight of the engineered polypeptide is within a range from 1 kDa to 10 kDa, 1 kDa to 5 kDa, 1 kDa to 2 kDa, 2 kDa to 8 kDa, 2 kDa to 5 kDa, 3 kDa to 7 kDa, 4 kDa to 6 kDa, 5 kDa to 8 kDa, 5 kDa to 10 kDa, or 8 kDa to 10 kDa.

[0448] Example 61. The method of any one of Examples 58 to 60, wherein the engineered polypeptide comprises no more than 100 amino acid residues, 90 amino acid residues, 80 amino acid residues, 70 amino acid residues, 60 amino acid residues, 50 amino acid residues, 40 amino acid residues, 30 amino acid residues, 20 amino acid residues, or 10 amino acid residues.[0449| Example 62. The method of any one of Examples 58 to 61, wherein the engineered polypeptide comprises from 10 amino acid residues to 100 amino acid residues, 10 amino acid residues to 50 amino acid residues, 20 amino acid residues to 80 amino acid residues, 30 amino acid residues to 70 amino acid residues, or 40 amino acid residues to 60 amino acid residues.

[0450] Example 63. The method of any one of Examples 58 to 62, wherein the engineered polypeptide includes a hydrophobic core.

[0451] Example 64. The method of any one of Examples 58 to 63, wherein the engineered polypeptide does not include any disulfide bridges.

[0452] Example 65. The method of any one of Examples 58 to 64, wherein the engineered polypeptide includes 2, 3, 4, 5, 6, or more secondary structure domains.

[0453] Example 66. The method of any one of Examples 58 to 65, wherein the engineered polypeptide has a net charge less than or equal to 0, -0.5, -1, -1.5, -2, -2.5, -3, -3.5, -4, -4.5, -5, -5.5, -6, -6.5, -7, -7.5, -8, -8.5, -9, -9.5, or -10.

[0454] Example 67. The method of any one of Examples 58 to 66, wherein the engineered polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 negatively charged amino acid residues.

[0455] Example 68. The method of any one of Examples 58 to 67, wherein the engineered polypeptide comprises a conjugation site for conjugation to the small molecule activator or to a linker.

[0456] Example 69. The method of Example 68, wherein the conjugation site is a lysine residue, a cysteine residue, a histidine residue, a non-natural amino acid residue, an N-terminus of the engineered polypeptide, or a C-terminus of the engineered polypeptide.

[0457] Example 70. The method of Example 68 or 69, wherein the conjugation site is located no more than 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acidresidues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from an N-terminus of the engineered polypeptide.

[0458] Example 71. The method of any one of Examples 68 to 70, wherein the conjugation site is located at least 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from an N-terminus of the engineered polypeptide.

[0459] Example 72. The method of any one of Examples 68 to 71, wherein the conjugation site is located no more than 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from a C-terminus of the engineered polypeptide.

[0460] Example 73. The method of any one of Examples 68 to 72, wherein the conjugation site is located at least 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from a C-terminus of the engineered polypeptide.

[0461] Example 74. The method of any one of Examples 58 to 73, wherein the engineered polypeptide comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 1 - SEQ ID NO: 5.

[0462] Example 75. The method of any one of Examples 58 to 74, wherein the engineered polypeptide comprises a sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 5.

[0463] Example 76. The method of any one of Examples 58 to 75, wherein the marker is a tumor antigen and the target cell is a cancer cell.

[0464] Example 77. The method of Example 76, wherein the tumor antigen comprises CD19, CD20, CD22, CD123, CD33, CD3, CD4, CD8, CD38, SLAMF7, BCMA, GD2, GPRC5D, MUC16, HER2, EGFR, EGFRvIII, CLL-1, CD44v6, folate receptor-a, mesothelin, CD20, CD37, ROR1, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu,surviving, telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, IL13Ra2, B7-H3 (CD276), EPHA2, GRP78, NKG2D, or CD70.

[0465] Example 78. The method of any one of Examples 58 to 77, wherein the small molecule activator has a molecular weight less than or equal to 5 kDa, 4 kDa, 3 kDa, 2 kDa, 1 kDa, 500 Da, 200 Da, or 100 Da.(0466] Example 79. The method of any one of Examples 58 to 78, wherein the small molecule activator is an exogenous small molecule.

[0467] Example 80. The method of Example 79, wherein the exogenous small molecule is selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), fluorescein 5-maleimide, fluorescein-5-carboxamide, fluorescein-6-carboxamide, 6-FAM phosphoramidite, topiramate hemi succinate, creatine, acetaminophen, ketamine, propofol, lidocaine, ractopamine, salicylate, salicylic acid, sulfasalazine, dapsone, albendazole, ivermectin, levamisole, permethrin, pyrantel, thiabendazole, procainamide, sulfamethazine, amikacin, amoxicillin, ampicillin, cefazolin, cefuroxime, cephalexin, chloramphenicol, chloramphenicol, ciprofloxacin, clenbuterol, cioxacillin, colistin A, dicloxacillin, enrofloxacin, furaltadone, gentamicin, gentamicin, kanamycin, kanamycin, kincomycin, lincomycin, metronidazole, nafcillin, nalidixic acid, neomycin, neomycin, nitrofurazone, norfloxacin, ofloxacin, oxacillin, spectinomycin, streptomycin, streptomycin, sulfabenzamide, sulfacetamide, sulfadiazine, sulfadimidine, sulfametoxydiazine, sulfanilamide, trimethoprim, carbamazepine, ethosuximide, lamotrigine, primidone, cetirizine, chlorpheniramine, diphenhydramine, doxylamine, promethazine, sulfadimethoxine, benzothiazinone, butylated hydroxytoluene, tripelennamine, chlorpromazine, clozapine, haloperidol, olanzapine, paliperidone, quetiapine, ribavirin, meprobamate, acebutolol, atenolol, penbutolol, warfarin, salmeterol, aflatoxin Bl, tetraxetan (DOTA), 4-[(6-methylpyrazin- 2-yl) oxy]benzoate (MPOB), biotin, melamine, methotrexate, amphetamine, diethylpropion, dextromethorphan, pseudoephedrine, dihydrochlorothiazide, hydrochlorothiazide, clonazepam, diazepam, nitrazepam, rhodamine B, fluorescent brightener Ksn, zearalenone, Sudan Redl, acetominophen, acrylamide, benzoic acid, benzophenone, benzothiazine, mercaptobenzothiazole, erythrosine, Sudan, tartrazine, erythromycin, sirolimus, atropine, ethyl glucuronide, aflatoxin Ml, methocarbamol, fentanyl, hydromorphone, morphine, remifentanil, tapentadol, tramadol, pregabalin, gabapentin, amitriptyline, desipramine, imipramine, nortriptyline, venlafaxine, dinitrophenyl, His-tag, PEG methoxy group, etodolac, ibuprofen, ketoprofen, meclofenamic acid, phenylbutazone, acetyl salicylic acid, acetamiprid, acetochlor, carbadazim, carbaryl,chlorothalonil, chlorpyrifos, fenpropathrin, imazalil, imidacloprid, parathion, abscisic acid, dibutyl phthalate, clonazepam, lorazepam, oxazepam, phenobarbital, secobarbital, zaleplon, zolpidem, trazodone, fluoxetine, fluvoxamine, cortisone, dexamethasone, dihydrotestosterone, fluocinolone, methylprednisolone, prednisolone, stanozolol, triamcinolone, mazindol, methamphetamine, methylphenidate, modafinil, chrysoidine, deoxynivalenol, fumonisin, microcystin Lr, ochratoxin, sterigmatocystin, T-2 toxin, sildenafil, tadalafil, scopolamine, florfenicol, pirlimycin, and sulfaquinoxaline.

[0468] Example 81. The method of any one of Examples 58 to 80, wherein the small molecule activator is fluorescein, a fluorescein derivative, or tetraxetan (DOTA).

[0469] Example 82. The method of Example 81, wherein the small molecule activator is the fluorescein derivative, and the fluorescein derivative is fluorescein isothiocyanate (FITC), fluorescein 5-maleimide, fluorescein-5-carboxamide, fluorescein-6-carboxamide, or 6-FAM phosphoramidite.

[0470] Example 83. The method of any one of Examples 58 to 82, wherein the engineered polypeptide is conjugated to the small molecule activator via a linker.

[0471] Example 84. The method of Example 83, wherein the linker is a linear linker.

[0472] Example 85. The method of Example 83, wherein the linker is a branched linker.

[0473] Example 86. The method of any one of Examples 83 to 85, wherein the linker comprises at least 2 repeating units, 5 repeating units, 10 repeating units, 12 repeating units, 15 repeating units, 20 repeating units, or 25 repeating units.

[0474] Example 87. The method of any one of Examples 83 to 86, wherein the linker comprises no more than 25 repeating units, 20 repeating units, 15 repeating units, 12 repeating units, 10 repeating units, 5 repeating units, or 2 repeating units.(0475] Example 88. The method of any one of Examples 83 to 87, wherein the linker has a length of at least 2 atoms, 3 atoms, 4 atoms, 5 atoms, 6 atoms, 7 atoms, 8 atoms, 9 atoms, 10 atoms, 15 atoms, 20 atoms, 25 atoms, 30 atoms, 35 atoms, 40 atoms, 45 atoms, 50 atoms, 60 atoms, 70 atoms, 80 atoms, 90 atoms, or 100 atoms.

[0476] Example 89. The method of any one of Examples 83 to 88, wherein the linker has a length no greater than 100 atoms, 90 atoms, 80 atoms, 70 atoms, 60 atoms, 50 atoms, 45 atoms, 40 atoms, 35 atoms, 30 atoms, 25 atoms, 20 atoms, 15 atoms, 10 atoms, 9 atoms, 8 atoms, 7 atoms, 6 atoms, 5 atoms, 4 atoms, 3 atoms, or 2 atoms.

[0477] Example 90. The method of any one of Examples 58 to 89, wherein the immune cell is a chimeric antigen receptor (CAR) T cell.[0478 Example 91. The method of Example 90, wherein the engineered receptor comprises a CAR of the CAR T cell.

[0479] Example 92. The method of Example 91, wherein binding of the small molecule activator to the CAR causes the CAR T cell to kill the target cell.

[0480] Example 93. The method of Example 91 or 92, wherein the CAR is encoded by a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 6 - SEQ ID NO: 8.

[0481] Example 94. The method of any one of Examples 91 to 93, wherein the CAR is encoded by a sequence of any one of SEQ ID NO: 6 - SEQ ID NO: 8.

[0482] Example 95. The method of any one of Examples 58 to 94, wherein the engineered receptor comprises a chimeric cytokine receptor (CCR).(0483] Example 96. The method of Example 95, wherein binding of the small molecule activator to the CCR causes a change in phenotype of the immune cell.

[0484] Example 97. The method of Example 96, wherein the change in phenotype comprises a change to a memory phenotype.

[0485] Example 98. The method of any one of Examples 95 to 97, wherein the CCR comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 9 - SEQ ID NO: 15.

[0486] Example 99. The method of any one of Examples 95 to 98, wherein the CCR comprises a sequence of any one of SEQ ID NO: 9 - SEQ ID NO: 15.

[0487] Example 100. The method of any one of Examples 95 to 99, wherein the CCR is encoded by a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 16 - SEQ ID NO: 22.(0488] Example 101. The method of any one of Examples 95 to 100, wherein the CCR is encoded by a sequence of any one of SEQ ID NO: 16 - SEQ ID NO: 22.

[0489] Example 102. The method of any one of Examples 58 to 101, wherein the engineered polypeptide is conjugated to a single small molecule activator.

[0490] Example 103. The method of any one of Examples 58 to 101, wherein the engineered polypeptide is conjugated to a plurality of small molecule activators.

[0491] Example 104. The method of any one of Examples 58 to 103, wherein the first composition further comprises a second engineered polypeptide conjugated to the small molecule activator or to the engineered polypeptide, and wherein the second engineered polypeptide has a molecular weight less than or equal to 10 kDa.

[0492] Example 105. The method of Example 104, wherein the second engineered polypeptide is the same as the engineered polypeptide.

[0493] Example 106. The method of Example 104, wherein the second engineered polypeptide is different from the engineered polypeptide.

[0494] Example 107. The method of any one of Examples 104 to 106, wherein the first composition further comprises a second small molecule activator conjugated to the second engineered polypeptide.

[0495] Example 108. The method of any one of Examples 58 to 107, wherein the first composition is administered to the subject at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 36 hours, or 48 hours after the second composition is administered to the subject.

[0496] Example 109. The method of any one of Examples 58 to 108, wherein the first composition is administered to the subject concurrently with the second composition.

[0497] Example 110. The method of any one of Examples 58 to 109, wherein the first composition is administered to the subject multiple times.

[0498] Example 111. The method of any one of Examples 58 to 110, further comprising administering a third composition to the subject, the third composition comprising the engineered polypeptide conjugated to a second small molecule activator.

[0499] Example 112. The method of Example 111, wherein the immune cell expresses a second engineered receptor that binds to the second small molecule activator.

[0500] Example 113. The method of Example 112, wherein the engineered receptor is a CAR and the second engineered receptor is a CCR.

[0501] Example 114. The method of any one of Examples 58 to 113, further comprising administering a third composition to the subject, the third composition comprising a secondengineered polypeptide conjugated to the small molecule activator, wherein the second engineered polypeptide has a molecular weight less than or equal to 10 kDa.

[0502] Example 115. The method of Example 114, wherein the second engineered polypeptide binds to a different marker than the engineered polypeptide.

[0503] Example 116. The method of any one of Examples 58 to 115, wherein the disease is a cancer.(0504] Example 117. The method of Example 116, wherein the cancer is a hematological cancer.

[0505] Example 118. The method of Example 117, wherein the hematological cancer is acute myeloid leukemia (AML), multiple myeloma (MM), non-Hodgkin lymphoma, acute lymphoblastic leukemia (ALL), mantle cell lymphoma, or follicular lymphoma.

[0506] Example 119. The method of Example 116, wherein the cancer is gastric cancer, glioma, mesothelioma, ovarian cancer, pancreatic cancer, or prostate cancer.Conclusion

[0507] Although many of the embodiments are described above with respect to compositions and methods for cancer immunotherapy, the technology is applicable to other applications and / or other approaches, such as immunotherapy for other types of diseases and conditions. Moreover, other embodiments in addition to those described herein are within the scope of the technology. Additionally, several other embodiments of the technology can have different configurations, components, or procedures than those described herein. A person of ordinary skill in the art, therefore, will accordingly understand that the technology can have other embodiments with additional elements, or the technology can have other embodiments without several of the features shown and described above with reference to FIGS. 1 A-58D.

[0508] The descriptions of embodiments of the technology are not intended to be exhaustive or to limit the technology to the precise form disclosed above. Where the context permits, singular or plural terms may also include the plural or singular term, respectively. Although specific embodiments of, and examples for, the technology are described above for illustrative purposes, various equivalent modifications are possible within the scope of the technology, as those skilled in the relevant art will recognize. For example, while steps are presented in a given order, alternative embodiments may perform steps in a different order. The various embodiments described herein may also be combined to provide further embodiments.

[0509] Moreover, unless the word “or” is expressly limited to mean only a single item exclusive from the other items in reference to a list of two or more items, then the use of “or” in such a list is to be interpreted as including (a) any single item in the list, (b) all of the items in the list, or (c) any combination of the items in the list. As used herein, the phrase “and / or” as in “A and / or B” refers to A alone, B alone, and A and B. Additionally, the term “comprising” is used throughout to mean including at least the recited feature(s) such that any greater number of the same feature and / or additional types of other features are not precluded. Additionally, the term “comprising” is used throughout to mean including at least the recited feature(s) such that any greater number of the same feature and / or additional types of other features are not precluded.

[0510] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0511] As used herein, the terms “about” and “approximately,” in reference to a number, is used herein to include numbers that fall within a range of 10%, 5%, or 1% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0512] As used herein, the term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, may refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.|0513| For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0514] For purposes herein, percent identity and sequence similarity may be determined by conventional methods, such as using the BLAST algorithm, which is described in Altschul etal. (J. Mol. Biol. 215:403-410 (1990)). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0515] A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0516] As used herein, the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, pigs, poultry, fish, crustaceans, etc.).

[0517] As used herein, the term “effective amount” refers to the amount of a composition sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0518] As used herein, the term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a “prophylactically effective amount” as prophylaxis can be considered therapy.10519] As used herein, the terms “administration” and “administering” refer to the act of giving a drug, prodrug, or other agent, or therapeutic treatment to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal or lingual), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

[0528] As used herein, the term “treatment” means an approach to obtaining a beneficial or intended clinical result. The beneficial or intended clinical result can include alleviation of symptoms, a reduction in the severity of the disease, inhibiting an underlying cause of a disease or condition, steadying diseases in a non-advanced state, delaying the progress of a disease, and / or improvement or alleviation of disease conditions.{0521 ] As used herein, the term “pharmaceutical composition” refers to the combination of an active ingredient with a carrier, inert or active, making the composition especially suitable for therapeutic or diagnostic use in vitro, in vivo or ex vivo.

[0522] The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.[05231 As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), glycerol, liquid polyethylene glycols, aprotic solvents such as dimethylsulfoxide, N-methylpyrrolidone and mixtures thereof, and various types of wetting agents, solubilizing agents, anti-oxidants, bulking agents, protein carriers such as albumins, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), and the like. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 21st Ed., Mack Publ. Co., Easton, Pa. (2005), incorporated herein by reference in its entirety.

[0524] As used herein, the term “transfection” refers to any method for introducing a nucleic acid into a cell, including both viral and non-viral methods, and encompasses both transient and stable modifications.

[0525] To the extent any materials incorporated herein by reference conflict with the present disclosure, the present disclosure controls.

[0526] It will also be appreciated that specific embodiments have been described herein for purposes of illustration, but that various modifications may be made without deviating from the technology. Further, while advantages associated with certain embodiments of the technology have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the technology. Accordingly, the disclosure and associated technology can encompass other embodiments not expressly shown or described herein.

Claims

CLAIMSWhat is claimed is:

1. A system for immunotherapy, the system comprising a first composition comprising an engineered polypeptide conjugated to a small molecule activator, wherein the engineered polypeptide has a molecular weight less than or equal to 10 kDa, and wherein the engineered polypeptide binds to a marker on a target cell; and a second composition comprising an immune cell expressing an engineered receptor that binds to the small molecule activator.

2. The system of claim 1, wherein the molecular weight of the engineered polypeptide is less than or equal to 9 kDa, 8 kDa, 7 kDa, 5 kDa, 4 kDa, 3 kDa, 2 kDa, or 1 kDa.

3. The system of claim 1 or 2, wherein the molecular weight of the engineered polypeptide is within a range from 1 kDa to 10 kDa, 1 kDa to 5 kDa, 1 kDa to 2 kDa, 2 kDa to 8 kDa, 2 kDa to 5 kDa, 3 kDa to 7 kDa, 4 kDa to 6 kDa, 5 kDa to 8 kDa, 5 kDa to 10 kDa, or 8 kDa to 10 kDa.

4. The system of any one of claims 1 to 3, wherein the engineered polypeptide comprises no more than 100 amino acid residues, 90 amino acid residues, 80 amino acid residues, 70 amino acid residues, 60 amino acid residues, 50 amino acid residues, 40 amino acid residues, 30 amino acid residues, 20 amino acid residues, or 10 amino acid residues.

5. The system of any one of claims 1 to 4, wherein the engineered polypeptide comprises from 10 amino acid residues to 100 amino acid residues, 10 amino acid residues to 50 amino acid residues, 20 amino acid residues to 80 amino acid residues, 30 amino acid residues to 70 amino acid residues, or 40 amino acid residues to 60 amino acid residues.

6. The system of any one of claims 1 to 5, wherein the engineered polypeptide includes a hydrophobic core.

7. The system of any one of claims 1 to 6, wherein the engineered polypeptide does not include any disulfide bridges.

8. The system of any one of claims 1 to 7, wherein the engineered polypeptide includes 2, 3, 4, 5, 6, or more secondary structure domains.

9. The system of any one of claims 1 to 8, wherein the engineered polypeptide has a net charge less than or equal to 0, -0.5, -1, -1.5, -2, -2.5, -3, -3.5, -4, -4.5, -5, -5.5, -6, -6.5, -7, - 7.5, -8, -8.5, -9, -9.5, or -10.

10. The system of any one of claims 1 to 9, wherein the engineered polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 negatively charged amino acid residues.

11. The system of any one of claims 1 to 10, wherein the engineered polypeptide comprises a conjugation site for conjugation to the small molecule activator or to a linker.

12. The system of claim 11, wherein the conjugation site is a lysine residue, a cysteine residue, a histidine residue, a non-natural amino acid residue, an N-terminus of the engineered polypeptide, or a C-terminus of the engineered polypeptide.

13. The system of claim 11 or 12, wherein the conjugation site is located no more than 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from an N-terminus of the engineered polypeptide.

14. The system of any one of claims 11 to 13, wherein the conjugation site is located at least 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from an N-terminus of the engineered polypeptide.

15. The system of any one of claims 11 to 14, wherein the conjugation site is located no more than 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from a C-terminus of the engineered polypeptide.

16. The system of any one of claims 11 to 15, wherein the conjugation site is located at least 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, or 20 amino acid residues away from a C-terminus of the engineered polypeptide.

17. The system of any one of claims 1 to 16, wherein the engineered polypeptide comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 1 - SEQ ID NO: 5.

18. The system of any one of claims 1 to 17, wherein the engineered polypeptide comprises a sequence of any one of SEQ ID NO: 1 - SEQ ID NO: 5.

19. The system of any one of claims 1 to 18, wherein the marker is a tumor antigen and the target cell is a cancer cell.

20. The system of claim 19, wherein the tumor antigen comprises CD 19, CD20, CD22, CD 123, CD33, CD3, CD4, CD8, CD38, SLAMF7, BCMA, GD2, GPRC5D, MUC16, HER2, EGFR, EGFRvIII, CLL-1, CD44v6, folate receptor-a, mesothelin, CD20, CD37, ROR1, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, surviving, telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, IL13Ra2, B7-H3 (CD276), EPHA2, GRP78, NKG2D, or CD70.

21. The system of any one of claims 1 to 20, wherein the small molecule activator has a molecular weight less than or equal to 5 kDa, 4 kDa, 3 kDa, 2 kDa, 1 kDa, 500 Da, 200 Da, or100 Da.

22. The system of any one of claims 1 to 21, wherein the small molecule activator is an exogenous small molecule.

23. The system of claim 22, wherein the exogenous small molecule is selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), fluorescein 5-maleimide, fluorescein-5-carboxamide, fluorescein-6-carboxamide, 6-FAM phosphoramidite, topiramate hemi succinate, creatine, acetaminophen, ketamine, propofol, lidocaine, ractopamine, salicylate, salicylic acid, sulfasalazine, dapsone, albendazole, ivermectin, levamisole, permethrin, pyrantel, thiabendazole, procainamide, sulfamethazine, amikacin, amoxicillin, ampicillin, cefazolin, cefuroxime, cephalexin, chloramphenicol, chloramphenicol, ciprofloxacin, clenbuterol, cioxacillin, colistin A, dicloxacillin, enrofloxacin, furaltadone, gentamicin, gentamicin, kanamycin, kanamycin, kincomycin, lincomycin, metronidazole, nafcillin, nalidixic acid, neomycin, neomycin, nitrofurazone, norfloxacin, ofloxacin, oxacillin, spectinomycin, streptomycin, streptomycin, sulfabenzamide, sulfacetamide, sulfadiazine, sulfadimidine, sulfametoxydiazine, sulfanilamide, trimethoprim, carbamazepine, ethosuximide, lamotrigine, primidone, cetirizine, chlorpheniramine, diphenhydramine, doxylamine, promethazine, sulfadimethoxine, benzothiazinone, butylated hydroxytoluene, tripelennamine, chlorpromazine, clozapine, haloperidol, olanzapine, paliperidone, quetiapine, ribavirin, meprobamate, acebutolol, atenolol, penbutolol, warfarin, salmeterol, aflatoxin Bl, tetraxetan (DOTA), 4-[(6- methylpyrazin-2-yl) oxy]benzoate (MPOB), biotin, melamine, methotrexate, amphetamine, diethylpropion, dextromethorphan, pseudoephedrine, dihydrochlorothiazide, hydrochlorothiazide, clonazepam, diazepam, nitrazepam, rhodamine B, fluorescent brightener Ksn, zearalenone, Sudan Redl, acetominophen, acrylamide, benzoic acid, benzophenone, benzothiazine, mercaptobenzothiazole, erythrosine, Sudan, tartrazine, erythromycin, sirolimus, atropine, ethyl glucuronide, aflatoxin Ml, methocarbamol, fentanyl, hydromorphone, morphine, remifentanil, tapentadol, tramadol, pregabalin, gabapentin, amitriptyline, desipramine, imipramine, nortriptyline, venlafaxine, dinitrophenyl, His-tag, PEG methoxy group, etodolac, ibuprofen, ketoprofen, meclofenamic acid, phenylbutazone, acetyl salicylic acid, acetamiprid, acetochlor, carbadazim, carbaryl, chlorothalonil, chlorpyrifos, fenpropathrin, imazalil,imidacloprid, parathion, abscisic acid, dibutyl phthalate, clonazepam, lorazepam, oxazepam, phenobarbital, secobarbital, zaleplon, zolpidem, trazodone, fluoxetine, fluvoxamine, cortisone, dexamethasone, dihydrotestosterone, fluocinolone, methylprednisolone, prednisolone, stanozolol, triamcinolone, mazindol, methamphetamine, methylphenidate, modafinil, chrysoidine, deoxynivalenol, fumonisin, microcystin Lr, ochratoxin, sterigmatocystin, T-2 toxin, sildenafil, tadalafil, scopolamine, florfenicol, pirlimycin, and sulfaquinoxaline.

24. The system of any one of claims 1 to 23, wherein the small molecule activator is fluorescein, a fluorescein derivative, or tetraxetan (DOTA).

25. The system of claim 24, wherein the small molecule activator is the fluorescein derivative, and the fluorescein derivative is fluorescein isothiocyanate (FITC), fluorescein 5- maleimide, fluorescein-5-carboxamide, fluorescein-6-carboxamide, or 6-FAM phosphoramidite.

26. The system of any one of claims 1 to 25, wherein the engineered polypeptide is conjugated to the small molecule activator via a linker.

27. The system of claim 26, wherein the linker is a linear linker.

28. The system of claim 26, wherein the linker is a branched linker.

29. The system of any one of claims 26 to 28, wherein the linker comprises at least 2 repeating units, 5 repeating units, 10 repeating units, 12 repeating units, 15 repeating units, 20 repeating units, or 25 repeating units.

30. The system of any one of claims 26 to 29, wherein the linker comprises no more than 25 repeating units, 20 repeating units, 15 repeating units, 12 repeating units, 10 repeating units, 5 repeating units, or 2 repeating units.

31. The system of any one of claims 26 to 30, wherein the linker has a length of at least 2 atoms, 3 atoms, 4 atoms, 5 atoms, 6 atoms, 7 atoms, 8 atoms, 9 atoms, 10 atoms, 15 atoms, 20 atoms, 25 atoms, 30 atoms, 35 atoms, 40 atoms, 45 atoms, 50 atoms, 60 atoms, 70 atoms, 80 atoms, 90 atoms, or 100 atoms.

32. The system of any one of claims 26 to 31, wherein the linker has a length no greater than 100 atoms, 90 atoms, 80 atoms, 70 atoms, 60 atoms, 50 atoms, 45 atoms, 40 atoms, 35 atoms, 30 atoms, 25 atoms, 20 atoms, 15 atoms, 10 atoms, 9 atoms, 8 atoms, 7 atoms, 6 atoms, 5 atoms, 4 atoms, 3 atoms, or 2 atoms.

33. The system of any one of claims 1 to 32, wherein the immune cell is a chimeric antigen receptor (CAR) T cell.

34. The system of claim 33, wherein the engineered receptor comprises a CAR of the CAR T cell.

35. The system of claim 34, wherein binding of the small molecule activator to the CAR causes the CAR T cell to kill the target cell.

36. The system of claim 34 or 35, wherein the CAR is encoded by a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 6 - SEQ ID NO: 8.

37. The system of any one of claims 34 to 36, wherein the CAR is encoded by a sequence of any one of SEQ ID NO: 6 - SEQ ID NO: 8.

38. The system of any one of claims 1 to 37, wherein the engineered receptor comprises a chimeric cytokine receptor (CCR).

39. The system of claim 38, wherein binding of the small molecule activator to the CCR causes a change in phenotype of the immune cell.

40. The system of claim 39, wherein the change in phenotype comprises a change to a memory phenotype.

41. The system of any one of claims 38 to 40, wherein the CCR comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 9 - SEQ ID NO: 15.

42. The system of any one of claims 38 to 41, wherein the CCR comprises a sequence of any one of SEQ ID NO: 9 - SEQ ID NO: 15.

43. The system of any one of claims 38 to 42, wherein the CCR is encoded by a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 16 - SEQ ID NO: 22.

44. The system of any one of claims 38 to 43, wherein the CCR is encoded by a sequence of any one of SEQ ID NO: 16 - SEQ ID NO: 22.

45. The system of any one of claims 1 to 44, wherein the engineered polypeptide is conjugated to a single small molecule activator.

46. The system of any one of claims 1 to 44, wherein the engineered polypeptide is conjugated to a plurality of small molecule activators.

47. The system of any one of claims 1 to 46, wherein the first composition further comprises a second engineered polypeptide conjugated to the small molecule activator or to the engineered polypeptide, and wherein the second engineered polypeptide has a molecular weight less than or equal to 10 kDa.

48. The system of claim 47, wherein the second engineered polypeptide is the same as the engineered polypeptide.

49. The system of claim 47, wherein the second engineered polypeptide is different from the engineered polypeptide.

50. The system of any one of claims 47 to 49, wherein the first composition further comprises a second small molecule activator conjugated to the second engineered polypeptide.

51. The system of any one of claims 1 to 50, further comprising a third composition comprising the engineered polypeptide conjugated to a second small molecule activator.

52. The system of claim 51, wherein the immune cell expresses a second engineered receptor that binds to the second small molecule activator.

53. The system of claim 52, wherein the engineered receptor is a CAR and the second engineered receptor is a CCR.

54. The system of any one of claims 1 to 53, further comprising a third composition comprising a second engineered polypeptide conjugated to the small molecule activator, wherein the second engineered polypeptide has a molecular weight less than or equal to 10 kDa.

55. The system of claim 54, wherein the second engineered polypeptide binds to a different marker than the engineered polypeptide.

56. A system for immunotherapy, the system comprising: a first pharmaceutical composition comprising the first composition of the system of any one of claims 1 to 55 and a pharmaceutically acceptable carrier; and a second pharmaceutical composition comprising the second composition of the system of any one of claims 1 to 55 and a pharmaceutically acceptable carrier.

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