Combinatorial approaches for targeting hematopoietic cells

Abstract

Provided herein are polypeptide compositions and chimeric antigen receptors comprising natural ligands of hematopoietic cell receptors and are thereby able to specifically bind to hematopoietic cell receptors. Also provided are methods and compositions for using the polypeptide compositions and chimeric antigen receptors in the treatment of disease, such as cancer, for editing a gene in a cell, and for culturing populations of cells.

Description

Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 COMBINATORIAL APPROACHES FOR TARGETING HEMATOPOIETIC CELLS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 575,589, filed April 5, 2024, the contents of which are incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted electronically in XML format and hereby is incorporated by reference in its entirety. Said XML copy, created on March 28, 2025, is named 079445-1484474-013910WO_SL.xml and 82,836 bytes in size. The Sequence Listing filed herewith does not go beyond the disclosure in the international application as filed. BACKGROUND

[0003] Pediatric Acute Myeloid Leukemia (AML) is a heterogenous disease of the hematopoietic system in which hematopoietic stem and progenitor cells (HSPCs) acquire genetic or epigenetic lesions that bias them toward a self-renewing, immature differentiation state at the cost of healthy, balanced hematopoiesis. While significant progress has been made toward improving outcomes in pediatric AML (in part through advances in hematopoietic stem cell transplantation (HSCT)), still, nearly 25% of children with AML relapse and approximately 50% die due to refractory disease or treatment related morbidities within 5 years of relapse. This highlights the need for therapeutic strategies that can simultaneously improve anti-leukemia efficacy while reducing HSCT related morbidities. Powerful immunotherapeutic modalities, such as chimeric antigen receptor (CAR) T cells, have reached impressive clinical milestones in the treatment of certain hematologic malignancies including acute lymphoblastic leukemia (ALL). However, extension of these approaches to AML has been difficult due to the shared antigen expression between AML cells and healthy hematopoietic tissues. This is a unique and significant challenge in AML, as sparing HSPCs can also potentially spare leukemic or pre-leukemic stem cells (LSCs) that 1 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 can reinitiate disease and cause relapse even after HSCT. The present application proposes a strategy that addresses this issue for the treatment of hematologic malignancies, such as AML, and a range of nonmalignant diseases, including blood disorder (e.g., sickle cell anemia, thalassemia, and hemophilia), infectious disease (human immunodeficiency virus (HIV)), neurologic disease (e.g., metachromatic leukodystrophy (MLD)), cardiovascular disease (e.g., Danon disease) and renal disease (e.g., cystinosis)). BRIEF SUMMARY

[0004] This summary provides a high-level overview of various aspects of the disclosure and introduces some of the concepts that are described and illustrated in the present document and the accompanying figures. The summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. Covered embodiments of the disclosure are defined by the claims, not this summary. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all figures, and each claim. Some of the exemplary embodiments of the present disclosure are discussed below.

[0005] In one aspect, provided is a chimeric antigen receptor (CAR) protein, comprising a first polypeptide, a transmembrane domain; and a first intracellular signaling domain. In some embodiments, the first polypeptide comprises a target-binding domain comprising a first cytokine domain that binds to a first hematopoietic cytokine receptor (HCR) and a second cytokine domain that binds to a second HCR. In some embodiments, the first and the second cytokine domains each bind to a different HCR. In some embodiments, the first and the second cytokine domains each bind to the same HCR. In some embodiments, the target- binding domain further comprises a third cytokine domain that binds to a third HCR. In some embodiments, the third HCR is different from the first HCR and the second HCR. In some embodiments, the third HCR is the same as at least one of the first HCR or the second HCR. In some embodiments, the first, second, and third cytokine domains are in any order in relation to each other.

[0006] In some embodiments, the first, second, and third cytokine domains each bind to one of mast / stem cell growth factor receptor Kit (KIT; CD117), myeloproliferative leukemia virus oncogene (MPL; CD110), or FMS-like tyrosine kinase 3 (FLT3; CD135). In some embodiments, the first, second, and third cytokine domains each comprise one of a HCR- binding portion of stem cell factor (SCF), a HCR-binding portion of thrombopoietin (TPO), 2 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 or a HCR-binding portion of FMS-like tyrosine kinase 3 ligand (FLT3LG), wherein the HCR-binding portion of SCF binds to KIT, the HCR-binding portion of TPO binds to MPL, and the HCR-binding portion of FLT3LG binds to FLT3. In some embodiments, the target- binding domain comprises a first linker between the first and second cytokine domains and a second linker between the second and third cytokine domains.

[0007] In some embodiments of the CAR protein, the first polypeptide comprises a target- binding domain comprising (1) a first cytokine domain that binds to a first HCR or a first antibody or antigen-binding fragment that specifically binds to a first HCR, and (ii) a second cytokine domain that binds to a second HCR or a second a first antibody or antigen-binding fragment that specifically binds to a second HCR. In some embodiments, the first polypeptide comprises a target-binding domain comprising a first antibody or antigen-binding fragment that specifically binds to a first HCR and a second a first antibody or antigen-binding fragment that specifically binds to a second HCR. In some embodiments, the first and the second cytokine domains and / or the first and the second antibodies or antigen-binding fragments each bind to a different HCR. In some embodiments, the first and the second cytokine domains and / or the first and the second antibodies or antigen-binding fragments each bind to the same HCR. In some embodiments, the target-binding domain further comprises a third cytokine domain or a third antibody or antigen-binding fragment that binds to a third HCR. In some embodiments, the third HCR is different from the first HCR and the second HCR. In some embodiments, the third HCR is the same as at least one of the first HCR or the second HCR. In some embodiments, the first, second, and third cytokine domains and / or first, second, and third antibodies or antigen-binding fragments each bind to one of KIT, MPL, or FLT3. In some embodiments, the target-binding domain can comprise any combination of two or more cytokine domains that binds to an HCR or antibodies or antigen- binding fragments that specifically binds to an HCR. In some instances, the target-binding domain comprises at least one of a cytokine domain that binds to an HCR and an antibody or antigen-binding fragment that specifically binds to an HCR. In some embodiments, the first, second, and third cytokine domains and / or antibodies or antigen-binding fragments are in any order in relation to each other.

[0008] In some embodiments, the CAR further comprises a hinge region between the target-binding domain and the transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain or a CD8α transmembrane domain. In some embodiments, the hinge domain comprises a CD28 hinge domain, a CD8α 3 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 hinge domain, an IgG4 hinge domain, a CH2 domain, or a CH3 domain. In some embodiments, the CAR comprises a second intracellular signaling domain. In some embodiments, the first intracellular signaling domain comprises a CD28 signaling domain or a 4-1BB signaling domain. In some embodiments, the second intracellular signaling domain comprises a CD3zeta signaling domain. In some embodiments, the first polypeptide comprises an N-terminal CD8α peptide linked to the target-binding domain.

[0009] In another aspect, provided is a polypeptide composition, comprising a first polypeptide linked to a therapeutic agent and / or an imaging agent. In some embodiments, the first polypeptide comprises a target-binding domain comprising a first cytokine domain that binds to a first hematopoietic cytokine receptor (HCR), and a second cytokine domain that binds to a second HCR. In some embodiments, the first and the second cytokine domains each bind to a different HCR. In some embodiments, the target-binding domain further comprises a third cytokine domain that binds to a third HCR, wherein the third HCR is different from the first HCR and the second HCR. In some embodiments, the first, second, and third cytokine domains are in any order in relation to each other.

[0010] In some embodiments, the first, second, and third cytokine domains each bind to one of KIT, MPL, or FLT3. In some embodiments, the first, second, and third cytokine domains each comprise one of a HCR-binding portion of stem cell factor (SCF), a HCR- binding portion of thrombopoietin (TPO), or a HCR-binding portion of FMS-like tyrosine kinase 3 ligand (FLT3LG), wherein the HCR-binding portion of SCF binds to KIT, the HCR- binding portion of TPO binds to MPL, and the HCR-binding portion of FLT3LG binds to FLT3. In some embodiments, the target-binding domain comprises a first linker between the first and second cytokine domains and a second linker between the second and third cytokine domains.

[0011] In some embodiments of the polypeptide composition, the first polypeptide comprises a target-binding domain comprising (1) a first cytokine domain that binds to a first HCR or a first antibody or antigen-binding fragment that specifically binds to a first HCR, and (ii) a second cytokine domain that binds to a second HCR or a second a first antibody or antigen-binding fragment that specifically binds to a second HCR. In some embodiments, the first polypeptide comprises a target-binding domain comprising a first antibody or antigen- binding fragment that specifically binds to a first HCR and a second a first antibody or antigen-binding fragment that specifically binds to a second HCR. In some embodiments, the 4 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 first and the second cytokine domains and / or the first and the second antibodies or antigen- binding fragments each bind to a different HCR. In some embodiments, the first and the second cytokine domains and / or the first and the second antibodies or antigen-binding fragments each bind to the same HCR. In some embodiments, the target-binding domain further comprises a third cytokine domain or a third antibody or antigen-binding fragment that binds to a third HCR. In some embodiments, the third HCR is different from the first HCR and the second HCR. In some embodiments, the third HCR is the same as at least one of the first HCR or the second HCR. In some embodiments, the first, second, and third cytokine domains and / or first, second, and third antibodies or antigen-binding fragments each bind to one of KIT, MPL, or FLT3. In some embodiments, the target-binding domain can comprise any combination of two or more cytokine domains that binds to an HCR or antibodies or antigen-binding fragments that specifically binds to an HCR. In some instances, the target-binding domain comprises at least one of a cytokine domain that binds to an HCR and an antibody or antigen-binding fragment that specifically binds to an HCR. In some embodiments, the first, second, and third cytokine domains and / or antibodies or antigen- binding fragments are in any order in relation to each other.

[0012] In some embodiments, the therapeutic agent comprises a protein or a small molecule. In some embodiments, the protein is a protein toxin or apoptotic protein. In some embodiments, the protein toxin is saporin or diphtheria toxin. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a T cell engager or a natural killer cell engager. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is an anti-CD3 antibody.

[0013] In some embodiments, the small molecule is a cytotoxic agent or a radioactive moiety. In some embodiments, the cytotoxic agent is a small molecule. In some embodiments, the cytotoxic agent is amanitin, pyrrolobenzodiazepine, monomethyl auristatin E (MMAE), Duocarmycin SA (DUO), PNU-159682 (PNU), or SGD-1882 (PBD).

[0014] In some embodiments, the therapeutic agent comprises a cargo molecule containing the therapeutic agent. In some embodiments, the cargo molecule comprises a nanoparticle, a virus or virus-like particle, or a dense body. In some embodiments, the nanoparticle is a lipid nanoparticle, a polymeric nanoparticle, or inorganic nanoparticle. 5 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0015] In some embodiments, the therapeutic agent in the cargo molecule comprises a small molecule or a gene editing system component. In some embodiments, the imaging agent is a fluorophore, a radioactive label, or a heavy metal particle.

[0016] In another aspect, provided is a nucleic acid encoding the first polypeptide of any CAR or polypeptide composition described above.

[0017] In another aspect, provided is a nucleic acid encoding any CAR described above.

[0018] In another aspect, provided is a construct comprising any nucleic acid described above. In some embodiments, the construct further comprises a nucleic acid encoding a second polypeptide. In some embodiments, in the constructs described above, a nucleic acid sequence encoding a P2A peptide is located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide.

[0019] In some embodiments, the second polypeptide comprises a targeting polypeptide, a chimeric costimulatory receptor (CCR), or a fusion protein comprising a human caspase 9 polypeptide attached to a modified human FK506-binding protein (FKB) polypeptide.

[0020] In some embodiments, the targeting polypeptide is CXCR4.

[0021] In some embodiments, the CCR comprises an extracellular target-binding domain that binds specifically to CXCR4. In some embodiments, the extracellular target-binding domain of the CCR comprises a polypeptide comprising an antigen-binding portion of an anti-CXCR4 antibody or a natural ligand of CXCR4. In some embodiments, the antigen- binding portion of an anti-CXCR antibody comprises a light chain portion of MDX1388, a heavy chain portion of MDX1388, or an scFv portion of MDX1388. In some embodiments, the natural ligand of CXCR4 comprises a polypeptide derived from CXCL12α or CXCL12β.

[0022] In another aspect, provided is a vector comprising any nucleic acid or construct described above. In some embodiments, the vector is delivered to cells via lipid nanoparticles or virus-like particles. In some embodiments, the lipid nanoparticles or the virus-like particles comprise a DNA nuclease, and optionally, a guide RNA (gRNA).

[0023] In another aspect, provided is a cell comprising any CAR, polypeptide composition, nucleic acid, construct, or vector described above. In some embodiments, the cell is a human T cell, natural killer (NK) cell, macrophage, monocyte, B cell, gamma / delta T cell, natural 6 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 killer T (NKT) cells induced pluripotent stem cell, hematopoietic stem cell, myeloid progenitor cell, or lymphoid progenitor cell.

[0024] In another aspect, provided is a pharmaceutical composition comprising any vector or cell described above, and a pharmaceutically acceptable carrier.

[0025] In another aspect, provided is a method of introducing a nucleic acid encoding a recombinant polypeptide into a cell and / or expressing a recombinant polypeptide in a cell, the method comprising: (1) providing the cell; and (2) introducing any nucleic acid, construct, vector, or pharmaceutical composition described above to the cell thereby producing a cell expressing the recombinant polypeptide. In another aspect, provided is a method of expressing a recombinant polypeptide in a cell, the method comprising: (1) providing the cell; and (2) introducing any nucleic acid, construct, vector, or pharmaceutical composition of the aspects and embodiments above to the cell thereby producing a cell expressing the recombinant polypeptide.

[0026] In some embodiments, the cell is a human cell, optionally a human T cell, natural killer (NK) cell, macrophage, monocyte, B cell, gamma / delta T cell, natural killer T (NKT) cells induced pluripotent stem cell, hematopoietic stem cell, myeloid progenitor cell, or lymphoid progenitor cell. In some embodiments, the cell is obtained from a cell donor. In some embodiments, the cell donor is a patient

[0027] In some embodiments, the method further comprises introducing the cell expressing the polypeptide into the patient.

[0028] In another aspect, provided is a method of treating a disease in a patient, the method comprising administering to the patient an effective amount of any pharmaceutical composition described above, wherein the disease in the patient is characterized by cells that express at least one of mast / stem cell growth factor receptor Kit (KIT; CD117), myeloproliferative leukemia virus oncogene (MPL; CD110), or FMS-like tyrosine kinase 3 (FLT3; CD135). In some embodiments, the disease is a cancer or a non-malignant disease.

[0029] In some embodiments, the cancer is leukemia, acute myeloid leukemia (AML), B cell acute lymphoblastic leukemia (B-ALL), B cell chronic lymphocytic leukemia (B-CLL), hairy cell leukemia, B-cell prolymphocytic leukemia, mixed phenotype acute leukemia (MPAL), juvenile myelomonocytic leukemia (JMML), myelodysplastic syndrome (MDS), non-Hodgkin’s B cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular 7 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 lymphoma, follicle center lymphoma, mantle cell lymphoma, Burkitt lymphoma (BL), Waldenstrom macroglobulinemia, Hodgkin lymphoma, multiple myeloma (MM), T cell leukemia, anaplastic large cell lymphoma, peripheral T cell lymphoma, cutaneous T cell lymphoma, extranodal natural killer (NK) / T cell lymphoma, Epstein-Barr virus associated T cell lymphoma, or T cell acute lymphoblastic leukemia.

[0030] In some embodiments, the non-malignant disease is a genetic disease, an autoimmune disease, or graft versus host disease (GvHD).

[0031] In some embodiments, the pharmaceutical composition is administered via intravenous injection, subcutaneous injection, or direct injection into a diseased tissue.

[0032] In some embodiments, the pharmaceutical composition comprises any cell described above, and the cell is an allogeneic cell. In some embodiments, the pharmaceutical composition comprises any cell described above, and the cell is an autologous cell.

[0033] In some embodiments, the method further comprises administering a combination therapy. In some embodiments, the combination therapy comprises at least one of surgery, radiation therapy, or administration of a therapeutically effective amount of a therapeutic agent.

[0034] In some embodiments, the therapeutic agent is a chemotherapeutic agent, a photosensitizer, an immunosuppressive agent, or small molecule that binds to a fusion protein comprising a human caspase 9 polypeptide attached to a modified human FK506-binding protein (FKB) polypeptide.

[0035] In some embodiments, after the administering step, the method further comprises transplanting of an allogeneic hematopoietic stem cell graft or a modified hematopoietic stem cell graft.

[0036] In another aspect, provided is a diagnostic composition comprising any polypeptide composition described above and a pharmaceutically acceptable carrier.

[0037] In another aspect, provided is a method for detecting presence of cells expressing HCRs in a biological sample, comprising: (1) contacting the biological sample with the diagnostic composition described above, and (2) detecting an amount of binding of the polypeptide composition as an indicator of the presence of the cells expressing HCRs. In some embodiments, the cells expressing HCRs express at least one of mast / stem cell growth 8 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 factor receptor Kit (KIT; CD117), myeloproliferative leukemia virus oncogene (MPL; CD110), or FMS-like tyrosine kinase 3 (FLT3; CD135).

[0038] In another aspect, provided is a method for editing a target gene in a cell expressing at least two HCRs. In some embodiments, the method comprises contacting a target cell expressing HCRs with a polypeptide composition of the present disclosure linked to a therapeutic agent. In some embodiments, the therapeutic agent of the polypeptide composition comprises a cargo molecule containing the therapeutic agent, and the therapeutic agent comprising one or more components of a gene editing system is capable of editing the target gene in the target cell. In some embodiments, the contacting of the target cell with the polypeptide composition of the present disclosure linked to a therapeutic agent results in introduction of the one or more components of a gene editing system into the target cell, and thereby results in a genetic modification of the target gene in the target cell.

[0039] In some embodiments, the one of more components of the gene editing system comprise (i) a DNA nuclease component and (ii) a gRNA with a sequence of at least 70% identity to a sequence of the target gene. In some embodiments, the cargo molecule can be, but is not limited to, a virus-like particle (VLP), a lipid nanoparticle (LNP), an adeno- associated virus (AAV), or a lentivirus (LV). Other cargo molecules routinely used for delivery of components to cells can also be used.

[0040] In some embodiments, the genetic modification comprises an insertion, a deletion, or a substitution of one or more nucleotides in the target gene. In some embodiments, the genetic modification disrupts expression of a polypeptide encoded by the target gene. In some embodiments, the genetic modification results in expression of a truncated version of the polypeptide encoded by the target gene. In some embodiments, the genetic modification introduces a transgene into the target gene. In some embodiments, the genetic modification replaces all or a portion of the target gene with a transgene. In some embodiments, the genetic modification produces a polynucleotide encoding a fusion protein at the site of the target gene.

[0041] In another aspect, provided is a method for culturing a population of hematopoietic stem and progenitor cells (HSPCs). In some embodiments, the method comprises contacting a population of HSPCs with a polypeptide composition of the present disclosure, wherein the HSPCs in the population increase in number and / or differentiate into one or more specific cell types. 9 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0042] In some embodiments, the polypeptide composition is added to cell culture media at an amount ranging from 10 pg / mL to 10 μg / mL.

[0043] In some embodiments, the increased number of cells and / or the differentiated cells are administered to a patient in need of HSPC transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1A shows schematics for exemplary therapeutic proteins with a target- binding domain (top portion) according to various aspects of this disclosure. Shown are an exemplary polypeptide composition comprising a therapeutic agent or an imaging agent (bottom left portion), an exemplary polypeptide composition comprising a cargo particle (bottom middle portion), and an exemplary chimeric antigen receptor (CAR) (bottom right portion). While the exemplary target-binding domain shown comprises three cytokine domains, a target-binding domain may comprise two, three, four, or more different cytokine domains. In some embodiments, the cytokine domains can each bind to a different hematopoietic cell receptor (HCR), thereby providing binding specificity for different HCRs. TM = transmembrane.

[0045] Figure 1B shows a three-dimensional model of an exemplary therapeutic protein that has a target-binding domain covalently linked to a T cell engager according to various aspects of this disclosure. The target-binding domain may comprise, as shown, three different cytokine domains, e.g., TPO, SCF, and FLT3LG. While the exemplary target-binding domain shown comprises three cytokine domains, a target-binding domain may comprise two, three, four, or more different cytokine domains. In the depicted exemplary protein, the T cell engager is an anti-CD3 scFv antibody.

[0046] Figures 1C-1G show an overview of natural trivalent ligand (NaTriL) CAR T cells. Figure 1C shows a schematic of NaTriL CARs. Stem Cell Factor (SCF) is on the N-terminus and is followed by a first (G4S)3 linker, thrombopoietin (TPO), a second (G4S)3 linker, Flt-3 ligand (FLT3LG), a CD28 hinge domain, a CD28 hinge transmembrane domain, and a CD28 hinge intracellular (IC) signaling domain, and CD3ζ domain. Figure 1D shows an Alphafold- predicted structure of extracellular domain of NaTriL CAR, with representative binding modes to KIT, MPL, and FLT3. Figure 1E shows a comparison of in vitro T cell expansion after viral transduction of Mock and NaTriL CAR (Mock on left and NaTriL on right for each day) (2-way ANOVA, p < 0.0001). Figure 1F shows transduction efficiency (left) and NaTriL CAR MFI (right) of transduced CAR T cells after viral transduction. Figure 1G, left 10 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 panel, shows a representative flow cytometry plot indicating expression of NaTriL CARS in T cells. Figure 1G, right panel, shows human SCF (hSCF) detection by murine anti-IGG1-PE fluorescent antibody.

[0047] Figures 2A-2E show overexpression of single cytokine CAR and NaTriL CAR constructs. Figure 2A shows a schematic of single cytokine CAR constructs for assessment of maintenance of on-target specificity of NaTriL CAR. In Figures 2B-2E, NALM6 (N6) GFP expression was measured over time via IncyCyte Live Imaging. In Figure 2B, only the NaTriL CAR was able to exhibit tumor control when tested in a heterogenous mixture of GFP-expressing NALM6 (N6) cells expressing all three KIT, MPL, and FLT3 individually. GFP-expressing NALM6 cells overexpressing KIT (Figure 2C), MPL (Figure 2D), or FLT3 (Figure 2E) were individually co-cultured with single cytokine CARs or the NaTriL CAR. Only cognate ligand single cytokine CARs and the NaTriL CAR showed tumor clearance, indicating on-target specificity. OE = overexpression.

[0048] Figures 2F-2H show GFP+ NALM6 (N6) cells overexpressing (OE) KIT (D), MPL (E), or FLT3 (F). In each figure, the x-axis values are -103, 0, 103, 104, and 105, and the y-axis values are 0, 20, 40, 60, 80, and 100.

[0049] Figures 2I-2K show an overview of NaTriL / ELECTRIC CAR. NaTriL / ELECTRIC CAR-T cells were co-cultured across a range of E:T ratios with N6 KIT OE (Figure 2I), N6 MPL OE (Figure 2J), and N6 FLT3 OE (Figure 2K) and monitored via IncuCyte Live Imaging.

[0050] Figure 2L shows monovalent natural ligand CARs expressing SCF, TPO, or FLT3LG and NaTriL / ELECTRIC CARs were co-cultured with N6 KIT OE, N6 MPL OE, and N6 FLT3 OE at 1:1 E:T ratios for 24 hours, and supernatants were harvested for cytokine production measurement by ELISA. Cytokine production was compared between Mock and CAR T cells for each type of target cell (2-way ANOVA, Tukey’s multiple comparison test). For each condition, data is shown from left to right for Mock, SCF CAR, TPO CAR, FLT3LG CAR, and ELECTRIC CAR.

[0051] Figures 3A-3B show that NaTriL CARs target AML cell lines in vitro. Figure 3A shows that NaTriL CARs reduced the relative frequency of live cells in the AML cell lines GDM-1, Kasumi-6, MOLM-13, NOMO1, OCI-AML3, and the B-ALL Line NALM6 (control) (2-way ANOVA, p<0.0001). For each condition, Mock is on the left and NaTriL is 11 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 on the right. In Figure 3B, NaTriL CARs were co-cultured with GFP expressing OCI-AML3 across a range of effector:target (E:T) ratios.

[0052] Figure 3C shows flow cytometric measurement of KIT, MPL, and FLT3 expression among a panel of AML cell lines (GDM-1, Kasumi-6, MOLM13, NOMO1, OCI-AML3) and B-ALL cell line NALM6 (N6). In each figure, the x-axis values are -103, 0, 103, 104, and 105.

[0053] Figure 4A shows that NaTriL CARs reduced the relative frequency of live cells in cord blood (CB) CD34+ hematopoietic stem and progenitor cell (HSPCs) populations (2-way ANOVA, p<0.0001). In Figure 4B, bulk CD34+ CD90+ cells from Figure 4A were enumerated and normalized to Mock controls (2-way ANOVA, p<0.0001). In Figure 4C, NaTriL CARs reduced the relative frequency of live cells in CB cells and two different primary pediatric AML cells (SU4239, SU4034) (2-way ANOVA, p<0.0001). In Figures 4A- 4C, for each condition, Mock is on the left and NaTriL is on the right. Figure 4D shows live cell count of remaining primary pediatric AML cells (pediatric AML cells comprising CBFB- MYH11 rearrangement) after 1:1 co-culture with single RTK-targeting or NaTriL CARs generated from either cord blood (CB) or peripheral blood donors (Donor A and Donor 52).

[0054] Figure 4E shows flow cytometric analysis of Mock (top two panels) or NaTriL / ELECTRIC CAR T (bottom two panels) cells co-cultured with SU4239 after 48 hours. The FACS plots are visual representations of SU4239 sample in Figure 4C. Boxed areas of the FACS plots indicate AML cells. A decrease in frequency of CD34+ cells were observed in the AML cells, which correspond to AML blasts. For each graph, the x-axis values and the y-axis values are -103, 0, 103, 104, and 105.

[0055] Figure 4F shows a summary of flow cytometric measurements of KIT, MPL, and FLT3 expression in CB, SU4239, and SU4034. For each of CB, SU4239, and SU4034, from left to right, data is shown for KIT, MPL, and FLT3.

[0056] In Figure 4G, CBFA2T3-GLIS2 primary AML cells (Tumor) were transduced with red fluorescent protein and co-cultured with Mock or NaTriL / ELECTRIC CAR T cells in the presence of Caspase-3 / 7 Green Dye and monitored via IncuCyte Live imaging.

[0057] In Figure 4H, Mock or NaTriL / ELECTRIC CAR T cells were co-cultured with MOLM13, OCI-AML3, or SJAMLM7007 at 1:1 E:T ratios, and supernatants were harvested for cytokine production via for bead-based multiplex cytokine detection using the Luminex 12 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 platform (2-way ANOVA, p < 0.0001, Šídák’s multiple comparison test). In all the graphs, for each condition, Mock is on the left and ELECTRIC is on the right.

[0058] In Figure 4I, Mock or NaTriL / ELECTRIC CAR T cells were co-cultured with CD34+ Cord Blood at 1:1 E:T ratios, and supernatants were harvested for cytokine production via for bead-based multiplex cytokine detection using the Luminex platform (2- way ANOVA, ***, P < 0.001). In all the graphs, for each condition, Mock is on the left and ELECTRIC is on the right.

[0059] Figure 5A shows an exemplary NaTriL CAR construct that comprises an iCaspase9 (iC9) safety switch (iC9 CAR). Figures 5B-5E show that coexpression of NaTriL CAR iC9 (iC9 CAR) induces death in transduced T cells that were treated with AP1903, a chemical inducer of dimerization. Figure 5B shows % T cells that express NaTriL CAR (CAR+). Figure 5C shows mean fluorescence intensity (MFI) for T cells that express NaTriL CAR (CAR+) (one-way ANOVA, p<0.01). Figure 5D shows % cell viability. Figure 5E shows live number of cells per mL of culture (one-way ANOVA, p<0.0001). Figure 5F shows fluorescence intensity (total green object integrated intensity) of NALM6 cells after co- culture with T cells that express NaTriL CAR (“STF”) or T cells that co-express NaTriL CAR and iC9. AP1903 or DMSO (“vehicle”) was added to the cells where indicated.

[0060] Figures 6A-6D show that CXCR4 overexpression improves in vivo activity of NaTriL CAR without perturbing NaTriL CAR activity. Figure 6A shows the CXCR4 overexpression (“OE”) construct that has full-length CXCR4 located adjacent to the C- terminal end of the NaTriL CAR protein and a P2A ribosomal skip sequence. Figure 6B shows flow cytometry results of T cells transduced with the NaTriL CAR construct (Figure 1C) or the NaTriL CAR and CXCR4 (CXCR4 OE) construct (Figure 6A). In Figure 6C, CXCR4 OE CAR T cells had equivalent cytotoxicity in GFP-expressing NALM6 cells (“N6”) expressing one KIT, MPL, or FLT3, compared to NaTriL CAR T cells. Figure 6D shows that NSG™ mice (The Jackson Laboratory Strain #: 005557) (n=8 per group); transplanted with 1E5 MOLM13 cells had improved survival when treated with CXCR4 OE CAR T cells (“STF-CX”) compared to NaTriL T cells (“STF”) and Mock T cells.

[0061] Figure 6E shows bone marrow human T cell chimerism in NSG mice transplanted with Mock, NaTriL / ELECTRIC, or NaTriL / ELECTRIC CXCR4 CAR T cells via tail vein injection, as measured on Day 13 via flow cytometry. (*, P < 0.05; **, P < 0.01; Two-way ANOVA, Tukey’s multiple comparison test). 13 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0062] Figures 6F-6G relate to CXCR4 expression reanalysis of previously reported bulk RNA-seq data and surface expression quantification of cell lines. Figure 6F shows bioinformatic analysis of bulk RNA-seq data from Fornerod et al. for pediatric patients with AML (n=435). Violin plot showing CXCR4, ACTB (positive control), and CLDN1 (negative control) expression distribution among patients. In Figure 6F, the y-axis is “Expression” (from bottom to top, markers at -5, 0, 5, 10, 15) and the x-axis is “Gene” (from left to right, CXCR4, ACTB, and CLDN1). Figure 6G shows CXCR4 protein expression quantified on different AML and ALL cell lines using flow cytometry.

[0063] Figure 6H shows the structure of anti-CXCR4 CAR T cells. From N- to C-terminus, CARs were designed with the extracellular domain(s) connected via (G4S)3 linker if applicable, followed by a FLAG-tag for CAR detection (ligand-based CAR T cells), CD28 hinge and transmembrane region, a CD28 costimulatory domain, and CD3ζ activation domain. Extracellular domains were as follows: A - Alpha splice variant of CXCL12, B - Beta splice variant of CXCL12, Vk - light chain of the MDX1338 antibody, Vh - heavy chain of the MDX1338 antibody.

[0064] Figure 6I shows CAR-T cell fold-expansion post transduction on day 0 with the same labeling aforementioned.

[0065] Figure 6J shows CAR expression on the surface of ligand CAR T cells quantified via flow cytometry. The data is shown from top to bottom for Beta_Alpha.fcs, Alpha_Beta.fcs, Beta.fcs, Alpha.fcs, and Mock.fcs.

[0066] Figure 6K shows mock transduced, ELECTRIC (NaTriL), Alpha, Beta, Alpha-Beta, or Beta-Alpha CAR T cells were co-cultured with c-6 (top) or Kasumi-1 (bottom) cells stably expressing GFP at a 1:1 Effector:Target ratio. GFP expression was measured in an IncuCyte machine over time for 63 hours, and normalized to initial expression. * - P < 0.05, ** - P < 0.01, *** - P < 0.001, **** - P < 0.0001, ns - P not significant as compared between a construct and Mock (n=3, two-way ANOVA).

[0067] Figure 6L shows Mock transduced (M), Alpha (A), Beta (B), Alpha-Beta (AB), or Beta-Alpha (BA) CAR T cells were co-cultured with NALM-6 cells stably expressing GFP at different Effector:Target ratios. GFP expression was quantified via flow cytometry after 72 hours, (n=3, two-way ANOVA). 14 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0068] Figure 6M shows CXCR4 expression analysis on different internally developed CAR-T cells as quantified by flow cytometry.

[0069] Figure 6N shows CXCR4 expression comparison among Mock transduced (“M”), Alpha (“A”), Beta (“B”), Alpha-Beta (“AB”), or Beta-Alpha (“BA”) CAR T cells, and Nalm- 6 ALL cell line quantified via flow cytometry. P < 0.0001 for Nalm-6 expression levels versus Mock, Alpha, Beta, Alpha-Beta, and Beta-Alpha (one-way ANOVA) and P < 0.0001 for Mock versus Alpha, Beta, Alpha-Beta, and Beta-Alpha (one-way ANOVA).

[0070] Figure 6O shows Structure of bicistronic or truncated anti-CXCR4 CAR T cells. Bicistronic constructs include a full CXCR4 protein sequence after a P2A ribosomal skip sequence. Truncated constructs had the same extracellular domain but were lacking CD28 signaling and CD3ζ activation domains.

[0071] Figure 6P shows Live cell count post transduction for CAR-T cells overexpressing CXCR4.

[0072] Figure 6Q shows CXCR4 expression on the surface of Alpha, Alpha overexpressing CXCR4, truncated Alpha, and Mock CAR-T cells quantified via flow cytometry.

[0073] Figure 6R shows CXCR4 expression on the surface of Alpha and Electric CAR-T cells stratified by CAR positivity. For each graph, the x-axis values are -103, 0, 103, 104, and 105. For the bottom two graphs, the y-axis values are 0, 20, 40, 60, 80, and 100.

[0074] Figure 6S shows CXCR4 expression on the surface of Alpha and Electric CAR-T cells overexpressing CXCR4 stratified by CAR positivity. For both graphs, the x-axis values are -103, 0, 103, 104, and 105, and the y-axis values are 0, 20, 40, 60, 80, and 100.

[0075] Figure 6T shows Mock (M) transduced, Alpha (A), Alpha overexpressing CXCR4 (A_OE), and Alpha truncated (A_T) were co-cultured with Nalm-6 cells stably expressing GFP at a 1:1 Effector:Target ratio. GFP expression was quantified via flow cytometry after 72 hours. (OE) indicates the overexpression via addition of CXCR4 and (T) indicates the truncation via removal of CD28 costimulatory and CD3ζ domains. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001 (n=3, one-way ANOVA).

[0076] Figure 6U shows (A) Mock (M) transduced, Beta (B), Alpha-Beta (AB), or Beta- Alpha (BA) and their engineered derivative CAR T cells were co-cultured with Nalm-6 cells 15 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 stably expressing GFP at a 1:1 Effector:Target ratio. GFP expression was quantified via flow cytometry after 72 hours. (OE) indicates the overexpression via addition of CXCR4 and (T) indicates the truncation via removal of CD28 costimulatory and CD3ζ domains. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001 (n=3, one- way ANOVA).

[0077] Figure 6V shows a model hypothesis for the attenuation of CAR activity. A flexible linker allows extracellular CXCL12 to bind to surface CXCR4. This may lead either to receptor internalization which prevents fluorescent antibody binding (top) or blockage of the fluorescent antibody (bottom) leading to lower apparent CXCR4 expression.

[0078] Figures 6W-6Y relate to blocking experiments performed with the CXCL12 ligand- based CAR T cells and Nalm-6 cells. Figure 6W: CAR-T blocking experiments using low, medium, and high levels of blocking antibody as determined from the concentration necessary for staining. Figure 6X: CAR-T blocking experiments using low, medium, and high levels of CXCL12 as determined by the Kd of CXCL12 (low). Figure 6Y: CXCL12 effects when adding directly to Nalm-6 in culture.

[0079] Figure 6Z.1 shows CD4:CD8 ratio of CD3 positive CAR T cells (extracellular domain left to right: Mock, Alpha (x3), Beta (x3), Alpha-Beta (x3), Beta-Alpha (x3), HK (x2), KH, STF (x2)) in culture was quantified via flow cytometry. Mock transduced, Alpha, Beta, Alpha-Beta, Beta-Alpha, or HK and their engineered derivative CAR T cells are shown. (OE) indicates the overexpression via addition of CXCR4 and (T) indicates the truncation via removal of CD28 costimulatory and CD3ζ domains. Phenotypic analysis of CD3 (APC-Cy7) positive CAR T cells.

[0080] Figure 6Z.2 shows the analysis strategy for identifying CD3+ cells and various populations that were used to stratify and gate observed populations by CD45RA positivity (BV-605) and CD62-Ligand positivity (APC). Stem Cell Memory T cells (CD45RA+CD62Lig+), Central Memory T cells (CD45RA-CD62Lig+), Effector Memory T cells (CD45RA-CD62Lig-), and Effector T cells (CD45RA+CD62Lig-) determined through cell surface co-expression. These FACS plots show the gating strategy used to identify the cell types in Figure 6Z.3. In the top left graph, the x-axis values (FSC-A (x 1000)) are 0, 65.5, 131., 196.6, and 262.1, and the y-axis values (SSC-A (x 1000)) are -0.1, 65.5, 131, 196.6, and 262.1; the lymphocytes are denoted in the boxed area (53.37%). In the top middle graph, the x-axis values (FSC-A (x 1000)) are 0, 65.5, 131.1, 196.6, and 262.1, and the y-axis values 16 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 (FSC-H (x 1000)) are -0.1, 65.5, 131, 196.6, and 262.1; the single cells are denoted in the boxed area (95.89%). In the top right graph, the x-axis values (APC-Cy7-A) are 10-1, 100, 101, 102, 103, 104, and 105, and the y-axis values (SSC-A (x 1000)) are -0.1, 65.5, 131, 196.6, and 262.1; the CD3+ cells are denoted in the boxed area (99.45%). In the bottom three graphs, the x-axis values (BV605-A) are 100, 101, 102, 103, 104, and 105, and the y-axis values (APC-A) are 10-1,100, 101, 102, 103, 104, and 105. In the bottom left graph, the four quadrants are labeled from top left clockwise: 15.31%, 38,84%, 27.29%, and 18.56%. In the bottom middle graph, the four quadrants are labeled from top left clockwise: 47.73%, 39.02%, 4.42%, and 8.82%. In the bottom right graph, the four quadrants are labeled from top left clockwise: 50.54%, 20.83%, 5.82%, and 22.80%.

[0081] Figure 6Z.3 shows Mock, Alpha (x3), HK (x2), KH in culture quantified via flow cytometry. (OE) indicates the overexpression via addition of CXCR4 and (T) indicates the truncation via removal of CD28 costimulatory and CD3ζ domains.

[0082] Figure 6Z.4 shows exhaustion marker (Lag3 and CD39) analysis of CD3 positive CAR T cells in culture was quantified via flow cytometry. Mock transduced, Alpha, Beta, Alpha_Beta, Beta_Alpha, or HK and their engineered derivative CAR T cells are shown. (OE) indicates the overexpression via addition of CXCR4 and (T) indicates the truncation via removal of CD28 costimulatory and CD3ζ domains.

[0083] Figure 6Z.5 shows Mock, Beta, Alpha-Beta, and Beta-Alpha (x3) in culture quantified via flow cytometry. (OE) indicates the overexpression via addition of CXCR4 and (T) indicates the truncation via removal of CD28 costimulatory and CD3ζ domains. Stem Cell Memory T cells (CD45RA+CD62Lig+), Central Memory T cells (CD45RA-CD62Lig+), Effector Memory T cells (CD45RA-CD62Lig-), and Effector T cells (CD45RA+CD62Lig-) determined through cell surface co-expression.

[0084] Figures 7A-7G show NaTriL / ELECTRIC CAR T cells target cognate HCRs in vivo. Figure 7A shows a schematic of the NaTriL / ELECTRIC CAR construct with an IGG4 short hinge and CD28 transmembrane (TM) and intracellular (IC) signaling domain (NaTriL / ELECTRIC IGG4). Figure 7B shows flow cytometry detection of CAR using an anti-G4S Linker antibody in Mock or NaTriL / ELECTRIC IGG4 CAR T cells. Figure 7C shows the experimental design of heterogeneous HCR-expressing NALM6 model. Mice were injected with a 1:1:1 heterogenous mixture of engineered NALM6 (1E6 total cells per mouse) expressing KIT, MPL, or FLT3. Bioluminescence was monitored via IVIS imaging 1-2x per 17 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 week, and peripheral blood measurements were acquired at Day 13 and Day 20. Mice were subsequently monitored for survival. Figure 7D shows average bioluminescence of engineered NALM6-engrafted mice (n=10) treated with Mock (n=5) or NaTriL / ELECTRIC IGG4 (n=5); P = 0.0204 at 17 days post-treatment for Mock vs. NaTriL / ELECTRIC IGG4 (One-way ANOVA). Figure 7E shows overall survival of mice at 45 days after treatment (P = 0.0032, log-rank Mantel-Cox test). Figure 7F shows measurements of CD4+ and CD8+ T cell expansion at Day 13 and Day 20 after CAR T cell injection. CD4+ and CD8+ T cells / ul were enumerated in peripheral blood by flow cytometry with counting beads (Two-way ANOVA). Figure 7G shows flow cytometry measurement of tumor burden in peripheral blood at Day 13 (left panel) and Day 20 (right panel) (Kruskal-Wallis test).

[0085] Figure 8 – NaTriL / ELECTRIC CAR-T cells were co-cultured across a range of E:T ratios with N6 KIT OE (Top Panel), N6 MPL OE (Middle Panel), and N6 FLT3 OE (Bottom Panel) and monitored via IncuCyte Live Imaging.

[0086] Figure 9 – Fraction of control area-under-curve at each concentration of the NaTriL / ELECTRIC BiTE (Heterogenous Engineered N6 + BiTE + Mock T cells / Heterogeneous Engineered NALM6 + BITE). Non-linear fitting to a three-parameter inhibitor dose vs. response model (Prism, Graphpad) yielded an EC50 of 0.04104 pM (4.1E- 14 Molar).

[0087] Figures 10A-10H show analysis of Transcriptomic Diversity in pAML. Figure 10A – Transcriptomic Diversity (1 – Simpson Diversity Index) of 22 Oncogenic Driver categories analyzed in Fornerod, et al. Integrative Genomic Analysis of Pediatric Myeloid-Related Acute Leukemias Identifies Novel Subtypes and Prognostic Indicators. Blood Cancer Discov 2021;2(6):586–599. Error bars indicate the standard deviation of each calculation of the Simpson Diversity Index. Low Diversity drivers are shown for CBFB-MYH11, RUNX1r, and ZNF384. High diversity drivers are shown for ETS, HOXr, T Txn, PRC2, Ph / Ph Like, FLT3 ITD, WT1 FLT3 ITD, KMT2Ar, NUP-r, Unknown, and Other. Figure 10B shows a circos plot highlighting the transcriptomic identity composition of each oncogenic driver. Figure 10C shows a circos plot highlighting the leukemic subtype composition of each oncogenic driver. Figure 10D shows overall survival of patients with low diversity oncogenic drivers and high diversity oncogenic drivers (P < 0.0001, Log-rank (Mantel-Cox) test). Figure 10E shows circos plot highlighting the oncogenic driver composition of each pLSC6- based risk category. See Fornerod et al. Figure 10F shows a comparison of pLSC6 scores 18 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 between low diversity and high diversity oncogenic drivers (P < 0.0001, unpaired t test). Figure 10G – HOX-family and MEIS1 expression among low diversity and high diversity oncogenic drivers (P < 0.0001, unpaired t test). Figure 10H – RBFOX2 and IRX1 expression among low diversity and high diversity oncogenic drivers (P < 0.0001, unpaired t test). PTD, partial tandem duplication; ITD, internal tandem duplication; Txn, transcription; Ph-like, Philadelphia chromosome–like acute lymphoblastic leukemia; MK, mixed karyotype; AMKL, acute megakaryoblastic leukemia; AML, acute myeloid leukemia; AUL, acute undifferentiated leukemia; ETP, early T-precursor; MPAL, mixed-phenotype acute leukemia; B / M, B-lymphoid and myeloid co-expression; T / B, T-lymphoid and myeloid co-expression; T / B / M, T-lymphoid, B-lymphoid, and myeloid co-expression; T / M T-lymphoid and myeloid co-expression.

[0088] Figures 11A-11G show that diverse oncogenic drivers modify probability of survival despite transcriptomic similarity. Figure 11A shows proportions of transcriptomic identities within low and high diversity oncogenic drivers (pie charts) and distribution of patients across transcriptomic identities (bar plot). Figure 11B shows overall survival of patients with low diversity oncogenic drivers and high diversity oncogenic drivers (proportions shown in pie charts) (P = 0.0028, Log-rank (Mantel-Cox) test). Figure 11C shows overall survival of patients harboring NPM1, KMT2Ar, and NUP-r in the entire pediatric cohort (P = 0.0056, Log-rank (Mantel-Cox) test). Figure 11D shows overall survival of patients harboring NPM1, KMT2Ar, and NUP-r within the MK-V transcriptomic cluster (P = 0.0002, Log-rank (Mantel-Cox) test). Figure 11E shows HOX10 and HOPX expression among patients harboring NPM1, KMT2Ar, and NUP-r within the MK-V transcriptomic cluster (P < 0.0001, One way ANOVA). Figure 11F shows HOPX expression among patients harboring KMT2A rearrangements across transcriptomic identities (**: P = 0.0037, ***: P = 0.0002, One way ANOVA). Figure 11G shows HOPX expression among patients harboring NUP rearrangements across transcriptomic identities (****: P < 0.0001, One way ANOVA).

[0089] Figure 12A shows HOXA10 expression among low diversity and high diversity oncogenic drivers in MPAL (***: P <0.0006, unpaired t test). Figure 12B shows overall survival patients with MPAL who harbor low or high diversity oncogenic drivers (P = 0.0394, Log-rank (Mantel-Cox) test).

[0090] Figure 13A, top portion, shows the proportion of cells within various cell types that express KIT / MPL / FLT3 (OR-gated), CLL1, CD123, and CD33 (data for each is shown from 19 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 left to right, respectively, for each cell type graph). Each dot represents a patient within this cohort. (2-way ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, Tukey’s multiple comparison test).

[0091] Figure 13A, bottom portion, shows the proportion of malignant cells within each cell type annotation that expresses KIT / MPL / FLT3 (OR-gated), CLL1, CD123, and CD33 (data for each is shown from left to right, respectively, for each cell type graph) and in Lambo et. al. Each dot represents a patient within this cohort. (2-way ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, Tukey’s multiple comparison test). HSC, hematopoietic stem cell; MPP, multipotent progenitor; LymP, lymphoid progenitor; GMP, granulocyte-monocyte progenitors; MEP, megakaryocyte-erythroid progenitor; CLP, common lymphoid progenitor.

[0092] Figures 13B-13F relate to the bioinformatic justification for targeting KIT, MPL, and FLT3 in pediatric AML. Figure 13B shows normalized log2(FPKM) of a subset of genes not highly expressed (KRT15) and highly expressed (B2M) in hematopoietic cells in comparison to KIT, MPL, and FLT3 expression. Figure 13C shows normalized log2(FPKM) of KIT, MPL, and FLT3 in each patient colored by HCR Immunophenotype shown in 3-D cartesian space. Figure 13D shows a circos plot indicating the proportions of each HCR- phenotype within each oncogenic driver represented in this cohort. Figure 13E shows proportions of patients who are positive (normalized log2(FPKM) > 0) for the indicated phenotype (horizontal axis) within each oncogenic driver (vertical axis). HCR phenotype Diversity (1 – Simpson Diversity Index) are indicated on the rightmost side of this heatmap. Figure 13F shows a proportion of patients with the indicated HCR phenotype within pLSC6 categories (top), FAB classification (middle), or flow cytometry based leukemic subtype (bottom). HSC, hematopoietic stem cell; LymP, lymphoid progenitor; MEP, megakaryocyte- erythroid progenitor; MPP, multipotent progenitor; GMP, granulocyte-monocyte progenitors; HCR, hematopoietic cytokine receptor; AMKL, acute megakaryoblastic leukemia; AML, acute myeloid leukemia; AUL, acute undifferentiated leukemia; ETP, early T-precursor; MPAL, mixed-phenotype acute leukemia; B / M, B-lymphoid and myeloid co-expression; T / B, T-lymphoid and myeloid co-expression; T / B / M, T-lymphoid, B-lymphoid, and myeloid co- expression; T / M T-lymphoid and myeloid co-expression; Ph-like, Philadelphia chromosome– like acute lymphoblastic leukemia; PTD, partial tandem duplication; Txn, transcription; ITD, internal tandem duplication. 20 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0093] Figure 14 shows HCR diversity does not correlate with the number of patients in each oncogenic driver group (slope not significantly non-zero in linear regression, P = 0.2147).

[0094] Figures 15A-15B show linear regression of the HCR Diversity score vs. Normalized expression of HOXA5 (Figure 15A) and HOXA10 (Figure 15B) in the bulk RNA-seq dataset. The R^2 for Figure 15A and Figure 15B are 0.32 and 0.36, respectively. The R^2 is calculated from a simple linear regression.

[0095] Figure 16 shows a Kaplan-Meier analysis of overall survival of pediatric AML patients separated by HCR phenotype. (P = 0.0067, Log-rank Mantel-Cox test). A comparison for the overall survival among different HCR phenotypes and revealed that KIT+MPL+FLT3+ / - leukemias had poorest prognoses in this pediatric cohort. DETAILED DESCRIPTION I. Terminology

[0096] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present disclosure. For purposes of the present disclosure, the following terms are defined.

[0097] As used herein, the terms “hematopoietic cytokine receptor” and “HCR” refer to a protein that is embedded in the membrane of a hematopoietic stem and progenitor cell (HSPC) and the protein is exposed to extracellular space. HCRs can function as regulators of cellular processes by binding to extracellular molecules, i.e., ligands, thereby allowing communication between the cell and the extracellular space. HCRs are capable of binding cytokines, and, in some cases, other ligands, e.g., without limitations, growth factors and hormones. Upon ligand binding, HCRs can activate signaling cascades which influence protein expression, metabolism, and other activities of the cell. In HSPCs, HCRs can influence cell self-renewal and differentiation. Non-limiting examples of HCRs include mast / stem cell growth factor receptor Kit (KIT; CD117), myeloproliferative leukemia virus oncogene (MPL; CD110), and FMS-like tyrosine kinase 3 (FLT3; CD135). In some cases, the HCR is a “receptor tyrosine kinase” or “RTK.” Non-limiting examples of RTKs include KIT and FLT3. 21 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0098] As used herein, a “target-binding domain” refers to a protein domain that binds to a hematopoietic cytokine receptor (HCR). In general, a target-binding domain is derived from a known protein ligand of an HCR. In some cases, a target-binding domain is derived from a cytokine domain that binds to an HCR. Non-limiting examples of cytokine domains include human stem cell factor (SCF; GenBank Accession No. AAA85450.1), human thrombopoietin (TPO; GenBank Accession No. AAB08424.1), and human FMS-like tyrosine kinase 3 ligand (FLT3LG; GenBank Accession No. AAI44130.1). In certain embodiments, the target-binding domain can comprise one or more antibodies or antigen-binding fragments that bind specifically to an HCR. In some instances, the target-binding domain can comprise any combination of one or more (e.g., one, two, three, four, or more) cytokine domains disclosed herein and at least one antibody or antigen-binding fragment that specifically to an HCR. In some instances, the target-binding domain comprises or consists of any combination of two or more (e.g., two, three, four, or more) antibodies or antigen-binding fragments that specifically to an HCR.

[0099] As used herein, the terms “natural trivalent ligand triplekine,” “NaTriL,” “STF,” “extracellularly-linked concatemeric trivalent cytokine,” and “ELECTRIC” are synonymous and are used interchangeably to refer to a polypeptide that comprises the following three cytokine domains – SCF, TPO, and FLT3LG. The SCF, TPO, and FLT3LG domains are derived from cytokines that bind to HCRs and are not derived from scFv molecules, antibodies, or fragments thereof. In some cases, the NaTriL is a soluble polypeptide and is not bound to a cell membrane. In some cases, the NaTriL is a membrane-bound protein. In some cases, the NaTriL is a chimeric antigen receptor (NaTriL CAR). The NaTriL polypeptides and NaTriL CARs of the present disclosure are capable of binding to HCRs, e.g., KIT, MPL, and FLT3.

[0100] As used herein, “hematopoietic stem and progenitor cells” or “HSPCs” refer to a population of precursor cells that possess the capacity for self-renewal and multilineage differentiation. HSPCs in the peripheral blood and the bone marrow, for example, can develop into blood cells, including white blood cells, red blood cells, and platelets; and immune cells, such as neutrophils, T cells, B cells, and natural killer (NK) cells. HSPCs in the blood and bone marrow are also known as “blood stem cells.”

[0101] The terms “genetic modification,” “genetic edit,” and “genome edit” can be used interchangeably and refer to a change in a nucleic acid sequence of a host cell, such that the 22 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 nucleic acid sequence of the modified DNA is different from the native, endogenous, previously modified, or wild-type sequence of the target DNA. The term encompasses mutations and variants of the target DNA sequence, and includes insertions, replacements, or deletions of the target polynucleotide sequence, including insertion of a CAR polynucleotide coding sequence at a target genomic DNA locus.

[0102] The term “DNA nuclease” refers to an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of DNA, and may be an endonuclease or an exonuclease. According to the present disclosure, the DNA nuclease may be an engineered (e.g., programmable or targetable) DNA nuclease which can be used to induce genome editing of a target DNA sequence. Any suitable DNA nuclease can be used including, but not limited to, CRISPR-associated protein (Cas) nucleases, other endo- or exo- nucleases, variants thereof, fragments thereof, and combinations thereof.

[0103] As used herein, the terms “polynucleotide,” “nucleotide,” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides. The terms include RNA, DNA, and synthetic forms and mixed polymers of the above. In particular embodiments, a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form or analog of either type of nucleotide. Deoxyribonucleic acids and ribonucleic acids thus include both naturally occurring molecules and synthetic analogues. The polynucleotides of the disclosure also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. Reference to a “polynucleotide,” “nucleotide,” or “nucleic acid” that encodes a polypeptide sequence also includes codon-optimized nucleic acids and nucleic acids that comprise alternative codons that encode the same polypeptide sequence.

[0104] The term “operably linked” refers to two or more genetic sequence elements, such as a polynucleotide coding sequence and a promoter sequence, placed in relative positions in a polynucleotide, cassette or vector that permit the proper biological functioning of the elements, such as the promoter binding an RNA polymerase, transcription and / or translation 23 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 of the polynucleotide coding sequence, localization of the polynucleotide sequence, or stability the polynucleotide sequence.

[0105] As used herein, the term “promoter” refers to nucleic acid regions or sequences located upstream and / or downstream from a start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.

[0106] The terms “DNA construct,” “expression cassette,” or “cassette” refers to a combination of genetic sequence elements that may be introduced as a single element and may function together to achieve a desired result. A DNA construct typically comprises polynucleotides in combinations that are not found in nature. Typically, a DNA construct includes a nucleic acid sequence to be transcribed, where the sequence is operably linked to a promoter. A DNA construct can be a part of a vector for the integration of a nucleotide sequence (e.g., a gene or a coding sequence) into a target nucleic acid of a host cell, and optionally, for the expression of the nucleotide sequence (e.g., a gene or a coding sequence) by that host cell. Vector forms include, for example, plasmids or virus vectors.

[0107] The terms “vector,” and “expression vector” refer to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression vector may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression vector includes a polynucleotide to be transcribed, operably linked to a promoter. The term “promoter” is used herein to refer to an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. Other elements that may be present in an expression vector include those that enhance transcription (e.g., enhancers) and terminate transcription (e.g., terminators).

[0108] As used herein, the term “plasmid” refers to a circular, double-stranded DNA containing one or more sequences of interest, for example, sequences encoding one or more particular proteins. In some embodiments, a plasmid can further include regulatory sequences or other genetic elements that are operatively linked to a sequence encoding a particular protein. 24 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0109] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0110] As used herein, the terms “variant” and “fragment,” refer to a polynucleotide or polypeptide related to a wild-type polynucleotide or polypeptide, for example, either by nucleic acid or amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity) and / or function. Variants and fragments of a polynucleotide or polypeptide can include one or more nucleic acid variations or amino acid variations, including, for example, mutations, insertions, deletions, truncations, modifications, or combinations thereof compared to a wild-type polynucleotide or polypeptide. A variant or fragment can include at least 50%, e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence, structure, activity, and / or function of the corresponding wild-type polynucleotide or polypeptide.

[0111] As used herein, the terms “percent identical,” “percent identity,” “sequence identity,” or equivalents used in the context of two polynucleotides or polypeptides refer to the level of identity between the sequences of the two polynucleotides or polypeptides when aligned, e.g., using a sequence alignment program or algorithm. For example, in certain embodiments, a particular polynucleotide or polypeptide sequence may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence as determined by using standard methods, e.g., BLAST. For sequence comparisons, one sequence is typically used as a reference sequence, to which query sequences are compared. Sequence comparison algorithms are readily available to one of ordinary skill in the art for comparison of test and query sequences. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well-known in the art. 25 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math.2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection. Percent sequence identity and sequence similarity can be determined using the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1977) Nucleic Acids Res.25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site.

[0112] As used herein, the terms “specifically binds” and “specific binding” and the like refer to a molecule (e.g., target-binding domain, chimeric antigen receptor (CAR), polypeptide composition, or antibody as disclosed herein) that binds to a target with greater affinity, avidity, more readily, and / or with greater duration to that target in a sample than it binds to a non-target molecule. For example, a cytokine domain derived from human SCF specifically binds its cognate receptor KIT with greater affinity, avidity, more readily, and / or with greater duration than to a non-target molecule. For example, in some embodiments, a target-binding domain, CAR, or polypeptide composition comprising a cytokine domain derived from human SCF binds to KIT with at least 2-fold greater affinity than non-target compounds, e.g., at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 25-fold, at least 50-fold or greater affinity. For example, in some embodiments, a target-binding domain, CAR, or polypeptide composition comprising a cytokine domain derived from human SCF specifically binds to KIT typically with at least a 2-fold greater affinity than to a non-human SCF target. While specific increased affinity or avidity examples are provided here with respect to SCF and KIT, such examples are also applicable to other ligand / target interactions as described in this disclosure (e.g., TPO and MPL; FLT3LG and FLT3; epitope and antibody).

[0113] As used herein, the term “antibody” encompasses, but is not limited to, whole immunoglobulin (i.e., an intact antibody) of any class. Native antibodies are usually heterotetrametric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent 26 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VHor VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL or VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. As used herein, the term antibody also encompasses an antibody fragment, for example, an antigen-binding fragment. Antigen- binding fragments comprise at least one antigen-binding domain. One example of an antigen- binding domain is an antigen-binding domain formed by a VH-VL dimer. Antibodies and antigen-binding fragments thereof can be described by the antigen to which they specifically bind. For example, as used herein, the terms “CD3 antibody” and “anti-CD3 antibody” both refer to an antibody or fragment thereof that specifically binds to CD3.

[0114] As used herein, the term “variable” describes certain portions of the antibody domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of 27 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 the antigen-binding site of antibodies. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. Each VH and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The CDRs are involved in antigen-binding and confer antigen specificity and binding affinity to the antibody. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD.) CDR sequences on the heavy chain (VH) may be designated as CDRH1, CDRH2, and CDRH3 (alternatively as VHCDR1, VHCDR2, and VHCDR3), while CDR sequences on the light chain (VL) may be designated as CDRL1, CDRL2, and CDRL3 (alternatively as VLCDR1, VLCDR2, and VLCDR3).

[0115] As used herein, the term “epitope,” refers to a component of an antigen capable of specific binding to an antibody or antigen-binding fragment thereof, for example, the target- binding domain of an antibody or antigen-binding fragment thereof disclosed herein. Such components optionally comprise one or more contiguous amino acid residues and / or one or more non-contiguous amino acid residues. Epitopes frequently consist of surface-accessible amino acid residues and / or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non- conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope can comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. The epitope to which a target-binding domain binds can be determined using known techniques for epitope determination such as, for example, testing for binding to antigen variants with different point mutations.

[0116] “Recombinant” refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism. For example, a recombinant polynucleotide (or a copy or complement of a recombinant polynucleotide) is one that has been manipulated using well known methods. A recombinant expression cassette comprising a promoter operably linked to a second polynucleotide (e.g., a coding sequence) can include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). A recombinant expression cassette (or expression 28 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 vector) typically comprises polynucleotides in combinations that are not found in nature. For instance, human manipulated restriction sites or plasmid vector sequences can flank or separate the promoter from other sequences. A recombinant protein is one that is expressed from a recombinant polynucleotide, and recombinant cells, tissues, and organisms are those that comprise recombinant sequences (polynucleotide and / or polypeptide).

[0117] As used herein, the term “heterologous” refers to biological material that is introduced, inserted, or incorporated into a recipient (e.g., host) organism that originates from another organism. Typically, the heterologous material that is introduced into the recipient organism (e.g., a host cell) is not normally found in that organism. Heterologous material can include, but is not limited to, nucleic acids, amino acids, peptides, proteins, and structural elements such as genes, promoters, and cassettes. A host cell can be, but is not limited to, a bacterium, a yeast cell, a mammalian cell, or a plant cell. The introduction of heterologous material into a host cell or organism can result, in some instances, in the expression of additional heterologous material in or by the host cell or organism. As a non-limiting example, the transformation of a yeast host cell with an expression vector that contains DNA sequences encoding a bacterial protein may result in the expression of the bacterial protein by the yeast cell. The incorporation of heterologous material may be permanent or transient. Also, the expression of heterologous material may be permanent or transient.

[0118] The term “introducing,” as used in the context of a polynucleotide described herein, refers to presenting a polynucleotide to a host cell in such a manner that the polynucleotide gains access to the interior of the cell.

[0119] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. In some embodiments, the subject is human. In some embodiments, the subject is a child. In some embodiments, the subject is an adolescent. In some embodiments, the subject is an adult. In some embodiments, the subject is less than 80 years of age, e.g., less than 70 years of age, less than 60 years of age, less than 50 years of age, less than 40 years of age, less than 30 years of age, less than 20 years of age, or less than 10 years of age. In some embodiments, the subject is above 10 years of age, e.g., above 20 years of age, above 30 years of age, above 40 years of age, above 50 years of age, above 60 years of age, above 70 29 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 years of age, or above 80 years of age. In some embodiments, the subject is male. In some embodiments, the subject is female.

[0120] As used herein, the term “treating” refers to an approach for obtaining beneficial or desired results including, but not limited to, a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.

[0121] As used herein, the singular forms “a,” “an,” and “the” include both singular and plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a target-binding domain sequence” optionally includes a combination of two or more target- binding sequences, and the like.

[0122] As used herein, the term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0123] As used herein, the terms “including,” “comprising,” “having,” “containing,” and variations thereof, are inclusive and open-ended and do not exclude additional, unrecited elements or method steps beyond those explicitly recited. As used herein, the phrase “consisting of” is closed and excludes any element, step, or ingredient not explicitly specified. As used herein, the phrase “consisting essentially of” limits the scope of the described feature to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the disclosed feature.

[0124] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0125] The terms “first,” “second,” “third,” and the like when used herein with reference to elements or properties, are simply to more clearly distinguish or identify multiple elements or properties, and are not intended to indicate an order or other serial or numerical limitation. 30 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0126] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0127] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes, such as variations of + / - 10% or less, + / - 1-5% or less, + / - 1% or less, and + / - 0.1% or less from the specified value. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. II. Introduction

[0128] The present disclosure provides various therapeutic proteins that have a target- binding domain that binds specifically to two or more hematopoietic cytokine receptors (HCRs). In particular, provided herein are chimeric antigen receptors (CARs) and isolated therapeutic polypeptides that comprise such a target-binding domain. See Figures 1A and 1B. By virtue of the target binding domain, the CARs and isolated therapeutic polypeptides provided herein are capable of specifically binding to two or more different HCRs. HCRs can be expressed on the surface of disease-related cells (e.g., cancer cells, cells of hematologic malignancies, leukemia cells, pre-leukemic stem cells (LSCs), and leukemic blasts) and healthy hematopoietic stem and progenitor cells (HSPCs). The CARs and isolated therapeutic polypeptides provided herein can be used to simultaneously eliminate disease-related cells (e.g., cells of hematologic malignancies, leukemia cells, pre-leukemic stem cells (LSCs), and leukemic blasts) and HSPCs with heterogeneous expression profiles of receptors.

[0129] In one aspect, the CARs disclosed herein have an extracellular domain that comprises a target-binding domain that binds specifically to two or more HCRs. See Figure 1A. The CARs can be used in T cell immunotherapy, in which T cells are engineered to 31 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 express the CARs, and the CAR T cells are administered to a patient to help the immune system target and kill cancer cells. The T cells can be a patient’s own T cells (i.e., autologous T cells) or from a donor (i.e., allogeneic T cells). The CAR T cells can also be administered to a patient to help the immune system target and kill HSPCs before a patient receive transplantation of donor cells. This is beneficial because patients can relapse after transplant of donor cells because remaining HSPCs can lead to the production leukemia cells again, even if all leukemia cells were wiped out before transplant. This is known as “conditioning immunotherapy.”

[0130] In another aspect, the therapeutic proteins disclosed herein are isolated polypeptides that comprise a target-binding domain that binds specifically to two or more HCRs, and are linked to at least one therapeutic agent, such as a protein (e.g., an antibody) or a small molecule (e.g., a cytotoxin or a radioactive moiety), or an imaging agent. See Figure 1A. The therapeutic agent component or imaging agent portion of the isolated polypeptide can be delivered to disease-related cells (e.g., cells of hematologic malignancies, leukemia cells, pre- LSCs, and leukemic blasts) and HSPCs via its linkage to the target-binding domain.

[0131] As described in more detail below, the target-binding domain of the provided therapeutic proteins comprise sequences derived from cytokine domains that bind to HCRs expressed in cancer cells, e.g., leukemia cells, and healthy HSPCs. By virtue of the ability of the target-binding domain to bind to multiple HCRs, the therapeutic proteins provided here may be considered multivalent. Exemplary HCRs of interest are mast / stem cell growth factor receptor Kit (KIT; CD117), myeloproliferative leukemia virus oncogene (MPL; CD110), and FMS-like tyrosine kinase 3 (FLT3; CD135). The cognate ligands for KIT, MPL, and FLT3 are cytokines stem cell factor (SCF), thrombopoietin (TPO), and / or FMS-like tyrosine kinase 3 ligand (FLT3LG), respectively. KIT, MPL, and FLT3, and their cognate ligands are important to hematopoiesis in both healthy and malignant cells. In some embodiments, the target-binding domain of the provided therapeutic proteins comprises cytokine domains derived from SCF, TPO, and / or FLT3LG. KIT, MPL, and FLT3 are HCRs that are critical to healthy and malignant hematopoiesis and are often mutated in the malignant setting to confer cells with ligand-independent activation of downstream signaling and chemoresistance. The importance of the natural ligands of KIT, MPL, and FLT3 in both human HSC and LSC signaling indicate that this signaling axis is shared in maintaining stem cell identity inside and outside of the bone marrow. 32 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0132] The CARs and therapeutic proteins disclosed herein exploit an overlap in signaling pathways between HSPCs, pre-leukemic stem cells (LSCs), and leukemic blasts, and can be used as a therapeutic strategy in the treatment of acute myeloid leukemia (AML). Interrogation of a large pediatric AML cohort (n=453) revealed that despite significant transcriptional heterogeneity among AML subtypes, expression of KIT, MPL, or FLT3 was a feature shared by nearly all pediatric AMLs. Further, pediatric AML subtypes that are more transcriptionally heterogeneous were found to have higher diversity of KIT, MPL, and FLT3 expression and expression of stemness-related genes.

[0133] The CARs and therapeutic proteins disclosed herein can be used to provide patients with simultaneous anti-leukemia and HSCT conditioning immunotherapy. For example, in a bridge-to-transplant setting, the CARs and therapeutic proteins can be used to deplete HSPCs in a patient’s bone marrow prior to receiving a transplant to facilitate engraftment of donor cells. Thus, also provided herein are methods of using these CARs and therapeutic proteins for therapeutic and / or diagnostic purposes, as well as for detecting cells that express these HCRs. Also provided herein are nucleic acids, constructs, vectors, cells, and associated methods for producing the CARs and therapeutic proteins. III. Target-Binding Domains

[0134] Provided herein are target-binding domain polypeptides capable of specifically binding to two or more hematopoietic cytokine receptors (HCRs). In some embodiments, the HCR is a receptor tyrosine kinase (RTK) and the target-binding domain is capable of specifically binding to one or more RTKs. In some embodiments, the target-binding domain of the present disclosure comprise amino acid sequences encoding a receptor-binding portion of at least two cytokines and each cytokine-derived polypeptide sequence is herein termed a “cytokine domain.” In some cases, the cytokine is a chemokine. Non-limiting examples of cytokines include stem cell factor (SCF; GenBank Accession No. AAA85450.1), thrombopoietin (TPO; GenBank Accession No. AAB08424.1), FMS-like tyrosine kinase 3 ligand (FLT3LG; GenBank Accession No. AAI44130.1), interleukin 3 (IL-3), IL-6, IL-11, granulocyte-colony stimulating factor (GCSF), leukemia inhibitory factor (LIF), lectins (e.g., a lectin that is capable of binding to CD45, which may be a C-type lectin), stromal cell- derived factor 1 alpha (SDF-1α; also referred to as CXCL12α; GenBank Accession No. AAB39332.1), and stromal cell-derived factor 2 beta (SDF-1β; also referred to as CXCL12β; GenBank Accession No. AAA97434.1). In some cases, the cytokine, e.g., CXCL12α and / or 33 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 CXCL12β, is covalently linked to a CD3 zeta (CD3ζ) domain. A target-binding domain can comprise one or more cytokine domains. In some embodiments, the target-binding domain comprises two or more cytokine domains (e.g., two, three, four, or more) that are covalently linked together as a single polypeptide. In some embodiments, some or all of the cytokine domains in a target-binding domain are different from each other. The presence of different cytokine domains in a target-binding domain provides binding specificity for more than one HCR. In some embodiments, some or all of the cytokine domains in a target-binding domain can bind to different HCRs. In some embodiments, two or more cytokine domains can be arranged in any order relative to each other. In some embodiments, some of the cytokine domains are the same. In some embodiments, the target-binding domain comprises two, three, four, or more cytokine domains that each bind to a different HCR, and the target- binding domain has binding specificity for two, three, four, or more different HCRs.

[0135] In some embodiments, the target-binding domain comprises a first cytokine domain that binds to a first HCR and a second cytokine domain that binds to a second HCR. In some embodiments, the first and second cytokine binding domains are different from each other. In some embodiments, the first and second cytokine domains each bind to a different HCR. In some embodiments, the first and second cytokine binding domains are the same cytokine binding domain. In some embodiments, the first and second cytokine domains both bind to the same HCR. In some embodiments, the target-binding domain further comprises a third cytokine domain that binds to a third HCR. In some embodiments, the first, second, and third cytokine domains, are different from each other. In some embodiments, the first, second, and third cytokine domains each bind to a different HCR. In some embodiments, two or more of the first, second, and third cytokine domains, are the same. In some embodiments, two or more of the first, second, and third cytokine domains bind to the same HCR.

[0136] In some embodiments, the cytokine domain is derived from a cytokine stem cell factor (SCF) sequence, e.g., human SCF (GenBank Accession No. AAA85450.1). In some embodiments, the cytokine domain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 3. In some embodiments, the cytokine domain comprises the sequence of SEQ ID NO: 3. In some embodiments, the cytokine domain is capable of specifically binding to mast / stem cell growth factor receptor Kit (KIT; CD117). 34 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0137] In some embodiments, the cytokine domain is derived from a cytokine thrombopoietin (TPO) sequence, e.g., human TPO (GenBank Accession No. AAB08424.1). In some embodiments, the cytokine domain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the cytokine domain comprises the sequence of SEQ ID NO: 4. In some embodiments, the cytokine domain is capable of specifically binding to myeloproliferative leukemia virus oncogene (MPL; CD110).

[0138] In some embodiments, the cytokine domain is derived from a cytokine FMS-like tyrosine kinase 3 ligand (FLT3LG) sequence, e.g., human FLT3LG (GenBank Accession No. AAI44130.1). In some embodiments, the cytokine domain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the cytokine domain comprises the sequence of SEQ ID NO: 5. In some embodiments, the cytokine domain is capable of specifically binding to FMS-like tyrosine kinase 3 (FLT3; CD135).

[0139] In some embodiments, the target-binding domain polypeptide comprises at least two of (1) a cytokine domain derived from an SCF sequence, (2) a cytokine domain derived from a TPO sequence, or (3) a cytokine domain derived from a FLT3LG sequence. In some embodiments, the target-binding domain polypeptide comprises (1) a cytokine domain derived from an SCF sequence, (2) a cytokine domain derived from a TPO sequence, and (3) a cytokine domain derived from a FLT3LG sequence. In some embodiments, the target- binding domain polypeptide comprises at least two of (1) a cytokine domain comprising the SCF sequence of SEQ ID NO: 3, (2) a cytokine domain comprising the TPO sequence of SEQ ID NO: 4, or (3) a cytokine domain comprising the FLT3LG sequence of SEQ ID NO: 5. In some embodiments, the target-binding domain polypeptide comprises (1) a cytokine domain comprising the SCF sequence of SEQ ID NO: 3, (2) a cytokine domain comprising the TPO sequence of SEQ ID NO: 4, and (3) a cytokine domain comprising the FLT3LG sequence of SEQ ID NO: 5.

[0140] In some embodiments, the target-binding domain polypeptide comprises one or more amino acid substitutions in one or more of the cytokine domain sequences as compared to the native sequence in the full length cytokine protein. Such amino acid substitutions can be conservative in nature such that they do not alter the ability of the cytokine domain to bind 35 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 to its cognate receptor. In some instances, the one or more amino acid substitutions may increase binding affinity of the target-binding domain polypeptide for the cognate HRK. In some instances, a cytokine domain in the target-binding domain polypeptide can comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a corresponding portion of the full length cytokine protein sequence (e.g., one or more of SEQ ID NOs: 3, 4, or 5).

[0141] In some embodiments, two or more (e.g., two, three, four, or more) cytokine domains are linked together as a single polypeptide target-binding domain. In some embodiments, the cytokine domains are first produced as separate polypeptides, and the separate cytokine domains are later linked together to form a single polypeptide target- binding domain. The cytokine domains may be joined by covalent bond, e.g., disulfide bond, or by chemical reactions, e.g., click chemistry. Non-limiting examples of methods for joining molecules together are discussed, for example, in Wang, J et al. “Disulfide linkage: a potent strategy in tumor-targeting drug discovery.” Current Medicinal Chemistry vol.19,18 (2012): 2976-83; and Hein, Christopher D et al. “Click Chemistry, a Powerful Tool for Pharmaceutical Sciences.” Pharmaceutical Research vol.25,10 (2008): 2216-30.

[0142] In some embodiments, the cytokine domains are covalently linked together by peptide linkers and are produced as a single polypeptide target-binding domain. Many different types of peptide linkers may be used to covalently connect the cytokine domains together in a single polypeptide target-binding domain. In some embodiments, a target- binding domain comprising two or more cytokine domains comprises at least one peptide linker. In some embodiments, a target-binding domain comprising three or more cytokine domains comprises two or more peptide linker. In these embodiments, some or all of the peptide linker can be different from each other, or they can be the same. In some embodiments, the peptide linker comprises repeats of glycines and serines, e.g., (GxS)y, where x and y can be any integer. In some embodiments, the peptide linker comprises the amino acid sequence of (G4S)3 (SEQ ID NO: 6) or (G4S)2 (SEQ ID NO: 7). In some embodiments, the peptide linker comprises repeats of alanines, e.g., (AAA)x, where x can be any integer. In some embodiments, the peptide linker comprises the amino acid sequence of AAA (SEQ ID NO: 8). In some embodiments, the peptide linker comprises the amino acid sequence of SIG (SEQ ID NO: 9). In some embodiments, the peptide linker comprises the amino acid sequence of GST (SEQ ID NO: 19). 36 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0143] In some embodiments, three cytokine domains are covalently linked together as a single polypeptide target-binding domain. In some embodiments, the target-binding domain comprises a cytokine domain derived from an SCF sequence, a cytokine domain derived from a TPO sequence, and a cytokine domain derived from a FLT3LG sequence. As discussed above, the cytokine domain sequence is derived from a portion of the cytokine that binds specifically to its cognate receptor. In some embodiments, the target-binding domain comprises, from the N-terminus to the C-terminus, an SCF domain (SEQ ID NO: 3), a TPO domain (SEQ ID NO: 4), and a FLT3LG domain (SEQ ID NO: 5). In some embodiments, the target-binding domain comprises, from the N-terminus to the C-terminus, an SCF domain (SEQ ID NO: 3), a FLT3LG domain (SEQ ID NO: 5), and a TPO domain (SEQ ID NO: 4). In some embodiments, the target-binding domain comprises, from the N-terminus to the C- terminus, a TPO domain (SEQ ID NO: 4), an SCF domain (SEQ ID NO: 3), and a FLT3LG domain (SEQ ID NO: 5). In some embodiments, the target-binding domain comprises, from the N-terminus to the C-terminus, a TPO domain (SEQ ID NO: 4), a FLT3LG domain (SEQ ID NO: 5), and an SCF domain (SEQ ID NO: 3). In some embodiments, the target-binding domain comprises, from the N-terminus to the C-terminus, a FLT3LG domain (SEQ ID NO: 5), an SCF domain (SEQ ID NO: 3), and a TPO domain (SEQ ID NO: 4). In some embodiments, the target-binding domain comprises, from the N-terminus to the C-terminus, a FLT3LG domain (SEQ ID NO: 5), a TPO domain (SEQ ID NO: 4), and an SCF domain (SEQ ID NO: 3). In some embodiments, the target-binding domain comprises a (G4S)3linker (SEQ ID NO: 6) that connects each cytokine domain to the next cytokine domain.

[0144] In certain embodiments, the target-binding domain can comprise one or more antibodies or antigen-binding fragments that specifically bind to an HCR. In some instances, the target-binding domain can comprise any combination of one or more (e.g., one, two, three, four, or more) cytokine domains disclosed herein and at least one antibody or antigen- binding fragment that specifically binds to an HCR. In some instances, the target-binding domain comprises or consists of any combination of two or more (e.g., two, three, four, or more) antibodies or antigen-binding fragments that specifically bind to an HCR. In some embodiments, the one or more antibodies or antigen-binding fragments specifically bind to at least one of KIT, MPL, or FLT3. In this disclosure, it will be apparent to the skilled person that, for certain embodiments where the target-binding domain comprises one or more cytokine domain that binds to an HCR, the target-binding domain may alternatively comprise one or more antibodies or antigen-binding fragments that specifically bind to an HCR. 37 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 IV. Chimeric Antigen Receptors

[0145] Also provided herein are chimeric antigen receptors (CARs; also known as chimeric T cell receptors) comprising a target-binding domain as described above in Section III. In some embodiments, the target-binding domain of the CAR comprises any combination of two or more (e.g., two, three, four, or more) cytokine domains disclosed herein, and the cytokine domains can be arranged in any order relative to each other. The CARs are designed to be expressed in target cells. In some embodiments, the target cells are immune cells or human HSPCs. In some embodiments, the target cells are cell that can induce an immune response against a specific target antigen and cells expressing that antigen, for example, without limitations, a T cell. See, e.g., June, Carl H et al. “CAR T cell immunotherapy for human cancer.” Science (New York, N.Y.) vol.359,6382 (2018): 1361-1365; and Sadelain, Michel et al. “The basic principles of chimeric antigen receptor design.” Cancer Discovery vol.3,4 (2013): 388-98. A CAR typically comprises an extracellular target-binding domain, a transmembrane domain, and an intracellular signaling domain (ICD). The CAR domains can be joined via flexible hinge and / or peptide linkers (also referred to as spacer regions).

[0146] Provided herein are CARs comprising (a) an extracellular target-binding domain comprising two or more (e.g., two, three, four, or more) cytokine domains, (b) a transmembrane domain, and (c) at least one intracellular signaling domain. See Figure 1A.

[0147] The CARs of the present disclosure can comprise any of the target-binding domain polypeptides described in Section III of this disclosure as the extracellular target-binding domain. In some embodiments, a CAR provided herein comprises two or more (e.g., two, three, four, or more) cytokine domains in its extracellular target-binding domain. As discussed above, in some embodiments, each of the two or more cytokine domains binds to a different target hematopoietic cytokine receptor (HCR), thereby providing the CAR with binding specificity for more than one HCR. For example, an extracellular target-binding domain with two, three, or four cytokine domains that each bind to a different HCR confers bi-, tri-, and quad-specificity, respectively, to the CAR. In some embodiments, the extracellular target-binding domain comprises two, three, or more cytokine domains. In these embodiments, each of the cytokine domains binds to a different HCR. In some instances, however, at least two of the cytokine domains bind to the same target HCR. Target-binding domains and cytokine domains are discussed in Section III above. 38 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0148] In certain embodiments, the target-binding domain of the CAR can comprise one or more antibodies or antigen-binding fragments that bind specifically to an HCR. In some instances, the target-binding domain can comprise any combination of one or more (e.g., one, two, three, four, or more) cytokine domains disclosed herein and at least one antibody or antigen-binding fragment that specifically to an HCR. In some instances, the target-binding domain comprises or consists of any combination of two or more (e.g., two, three, four, or more) antibodies or antigen-binding fragments that specifically to an HCR.

[0149] In general, the transmembrane domain of the CAR functions as an anchor for the CAR in the cell membrane. While many transmembrane domains are suitable for use in a CAR, transmembrane domains are typically derived from molecules that regulate T cell function, such as CD8α and CD28. The intracellular signaling domain of the CAR contains at least one signaling domain, for example, a signaling domain of the T cell receptor CD3ζ (or CD3zeta) chain. The intracellular signaling domain can also contain one or more costimulatory domains, for example, a CD28 costimulatory domain or a 4-1BB costimulatory domain. CAR domains are discussed in more detail below.

[0150] In some embodiments, the extracellular domain of a CAR disclosed herein comprises a target-binding domain comprising two, three, or more of any of the cytokine domains disclosed herein. In some embodiments, the extracellular target-binding domain comprises (1) a cytokine domain derived from an SCF sequence, (2) a cytokine domain derived from a TPO sequence, and / or (3) a cytokine domain derived from a FLT3LG sequence. In some embodiments, the extracellular target-binding domain comprises (1) an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to the amino acid sequence of SEQ ID NO: 3, (2) an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to the amino acid sequence of SEQ ID NO: 4, and / or (3) an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the extracellular target-binding domain comprises (1) the amino acid sequence of SEQ ID NO: 3, (2) the amino acid sequence of SEQ ID NO: 4, and (3) the amino acid sequence of SEQ ID NO: 5. As discussed above in Section III, the cytokine domains may be connected by linker 39 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 sequences. In some embodiments, the extracellular target-binding domain comprises a (G4S)3 linker (SEQ ID NO: 6) that connects each cytokine domain to the next cytokine domain.

[0151] The transmembrane domain of a CAR disclosed herein can be derived from a known protein or it can be synthetic transmembrane domain. In embodiments where the transmembrane domain is derived from a known protein, the transmembrane domain can be derived from the transmembrane regions of the α, β, δ, γ, or ζ chain of the T cell receptor, CD8α CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the transmembrane domain is a CD8α transmembrane domain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the transmembrane domain is a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14. In embodiments where the transmembrane domain is a synthetic transmembrane domain, the transmembrane domain can comprise predominantly hydrophobic residues such as leucine, valine, phenylalanine, tryptophan, and / or valine, and the transmembrane domain comprises a sufficient number of amino acids so the transmembrane domain can traverse the cell membrane.

[0152] The intracellular signaling domain of a CAR disclosed herein can be derived from cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that function in concert to initiate signal transduction following antigen receptor engagement. Any derivative or variant of these sequences and any synthetic sequence that has the same capability can also be used. The intracellular signaling domain typically comprises a primary signaling domain that couples antigen-binding with intracellular signaling pathways. Non-limiting examples of primary signaling domains, also known as immunoreceptor tyrosine-based activation motifs or ITAMs, include primary signaling domains derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the CAR intracellular signaling domain comprises a primary signaling domain derived from CD3ζ. For example, the intracellular signaling domain can comprise the CD3ζ intracellular signaling domain by itself or combined with any other suitable intracellular signaling sequence useful in the context of the CAR disclosed herein. In some embodiments, the primary intracellular signaling domain comprises the CD3ζ amino acid sequence of SEQ ID NO: 17. 40 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0153] In some cases, signals generated through a primary signaling domain alone is sufficient for full activation of the T cell. In some cases, T cell activation also requires a secondary signal or costimulatory signal. Thus, in some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, and optionally, a costimulatory signaling sequence. In some embodiments, the intracellular signaling domain comprises a CD3ζ primary intracellular signaling domain and a CD28 costimulatory signaling sequence. In some embodiments, the intracellular signaling domain comprises a CD3ζ primary intracellular signaling domain and a 4-1BB costimulatory signaling sequence. In some embodiments, the primary intracellular signaling domain comprises the CD3ζ amino acid sequence of SEQ ID NO: 17, and the costimulatory domain comprises the CD28 amino acid sequence of SEQ ID NO: 15. In some embodiments, the CD3ζ signaling domain is on the C-terminus of the CAR. In some embodiments, the primary intracellular signaling domain comprises the CD3ζ amino acid sequence of SEQ ID NO: 17, and the costimulatory domain comprises the 4-1BB amino acid sequence of SEQ ID NO: 16.

[0154] In some embodiments, the transmembrane domain is connected to the extracellular domain by a hinge domain. A hinge domain can provide flexibility to the extracellular domain and the target-binding domains therein for positioning and specific binding to cognate HCRs. Hinge domains can be derived from an immunoglobulin heavy chain sequence (for example, an immunoglobulin hinge region alone or in combination with a CH2 and / or CH3 domain) or from a transmembrane protein, e.g., a single-pass type I transmembrane protein. In some embodiments, the hinge domain is a CD8α hinge domain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the hinge domain is a CD28 hinge domain comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the hinge domain is an IgG4 hinge domain comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the hinge domain comprises an immunoglobulin heavy chain hinge region and one or more of a CH2 domain and / or a CH3 domain. In some embodiments, the hinge domain is an IgG4 hinge domain comprising the amino acid sequence of SEQ ID NO: 12, a CH2 domain, and a CH3 domain.

[0155] In some embodiments, a peptide linker is present between any of the domains or polypeptide sequences present in a CAR. A linker domain may comprise, for example 3-50 amino acids, 3-25 amino acids, 3-10 amino acids, 5-12 amino acids, 8-15 amino acids, 3 amino acids, 10 amino acids, 15 amino acids, etc. In some embodiments, each peptide linker comprises repeats of glycines and serines, e.g., (GxS)y, where x and y can be any integer. In 41 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 some embodiments, each peptide linker comprises the amino acid sequence of (G4S)3 (SEQ ID NO: 6) or (G4S)2 (SEQ ID NO: 7). In some embodiments, the peptide linker comprises repeats of alanines, e.g., (AAA)x, where x can be any integer. In some embodiments, the peptide linker comprises the amino acid sequence of AAA (SEQ ID NO: 8). In some embodiments, the peptide linker comprises the amino acid sequence of SIG (SEQ ID NO: 9). In some embodiments, the peptide linker comprises the amino acid sequence of GST (SEQ ID NO: 19).

[0156] In some embodiments, the CAR hinge domain is covalently linked to a peptide linker on the hinge domain’s N-terminus or C-terminus. In some embodiments, the peptide linker is between the hinge domain and a cytokine domain of the CAR. In some embodiments, the peptide linker is between the hinge domain and the transmembrane domain of the CAR. In some embodiments, the peptide linker comprises repeats of alanines, e.g., (AAA)x, where x can be any integer. In some embodiments, the peptide linker comprises the amino acid sequence of AAA (SEQ ID NO: 8).

[0157] In some embodiments, the CAR extracellular domain comprises a peptide linker between the two, three, or more cytokine domains. In some embodiments, each peptide linker comprises repeats of glycines and serines, e.g., (GxS)y, where x and y can be any integer. In some embodiments, each peptide linker comprises the amino acid sequence of (G4S)3 (SEQ ID NO: 6) or (G4S)2(SEQ ID NO: 7).

[0158] The CARs provided herein can also include a leader sequence. The leader sequence may direct transport and localization of the CAR to the T cell surface. In some embodiments, the leader sequence comprises a granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) leader sequence of SEQ ID NO: 1. In some embodiments, the leader sequence comprises a CD8α leader sequence of SEQ ID NO: 2.

[0159] The CARs provided herein can also include a signal peptide to properly orient the extracellular domain of the CAR protein to the surface of the cell.

[0160] In some embodiments, the CAR comprises a leader sequence derived from CD8α, a cytokine domain derived from SCF, a cytokine domain derived from a TPO, a cytokine domain derived from FLT3LG, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, and an intracellular signaling domain derived from CD3ζ. In some embodiments, the CAR also comprises one or more peptide linkers, e.g., a (G4S)3 linker, a AAA linker, a SIG linker, a 42 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 GST linker, or any combination thereof. In some embodiments, the CAR comprises, from the N-terminus to the C-terminus, a CD8α leader sequence (e.g., SEQ ID NO: 2), an SCF domain (e.g., SEQ ID NO: 3), a (G4S)3linker (SEQ ID NO: 6), a TPO domain (e.g., SEQ ID NO: 4), a (G4S)3 linker (SEQ ID NO: 6), a FLT3LG domain (e.g., SEQ ID NO: 5), a AAA linker (e.g., SEQ ID NO: 8), a CD28 hinge domain (e.g., SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), and a CD3ζ domain (e.g., SEQ ID NO: 17). In some embodiments, the CAR comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 22. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 22.

[0161] In some embodiments, the CAR comprises a leader sequence derived from CD8α, a cytokine domain derived from SCF, a cytokine domain derived from TPO, a cytokine domain derived from FLT3LG, a hinge domain derived from IgG4, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, and an intracellular signaling domain derived from CD3ζ. In some embodiments, the CAR also comprises one or more peptide linkers, e.g., a (G4S)3linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR comprises, from the N-terminus to the C-terminus, a CD8α leader sequence (e.g., SEQ ID NO: 2), an SCF domain (e.g., SEQ ID NO: 3), a (G4S)3linker (SEQ ID NO: 6), a TPO domain (e.g., SEQ ID NO: 4), a (G4S)3linker (SEQ ID NO: 6), a FLT3LG domain (e.g., SEQ ID NO: 5), a AAA polypeptide linker (e.g., SEQ ID NO: 8), an IgG4 hinge domain (e.g., SEQ ID NO: 12), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), and a CD3ζ domain (e.g., SEQ ID NO: 17). In some embodiments, the CAR comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 24. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 24.

[0162] In some embodiments, the CAR comprises a leader sequence derived from CD8α, a sequence derived from CXCL12α, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ domain. In some embodiments, the CAR also comprises one or more peptide linkers, e.g., a FLAG-tag, a (G4S)3 linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR comprises, from the N-terminus to the C-terminus, a CD8α leader sequence (e.g., SEQ ID 43 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 NO: 2), a CXCL12α domain (e.g., SEQ ID NO: 29), a FLAG-tag (e.g., SEQ ID NO: 45), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), and a CD3ζ domain (e.g., SEQ ID NO: 17). In some embodiments, the CAR comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 48. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 48.

[0163] In some embodiments, the CAR comprises a leader sequence derived from CD8α, a domain derived from CXCL12β, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ domain. In some embodiments, the CAR also comprises one or more peptide linkers, e.g., a FLAG-tag, a (G4S)3 linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR comprises, from the N-terminus to the C-terminus, a CD8α leader sequence (e.g., SEQ ID NO: 2), a CXCL12β domain (e.g., SEQ ID NO: 30), a FLAG-tag (e.g., SEQ ID NO: 45), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), and a CD3ζ domain (e.g., SEQ ID NO: 17). In some embodiments, the CAR comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 49. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 49.

[0164] In some embodiments, the CAR comprises a leader sequence derived from CD8α, a domain derived from CXCL12α, a domain derived from CXCL12β, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, and an intracellular signaling domain derived from CD3ζ domain. In some embodiments, the CAR also comprises one or more peptide linkers, e.g., a FLAG-tag, a (G4S)3 linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR comprises, from the N-terminus to the C-terminus, a CD8α leader sequence (e.g., SEQ ID NO: 2), a CXCL12α domain (e.g., SEQ ID NO: 29), a (G4S)3linker (e.g., SEQ ID NO: 6), a CXCL12β domain (e.g., SEQ ID NO: 30), a FLAG-tag (e.g., SEQ ID NO: 45), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), and a CD3ζ domain (e.g., SEQ ID NO: 17). In some embodiments, the CAR comprises at least 80%, at 44 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 50. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 50.

[0165] In some embodiments, the CAR comprises a leader sequence derived from CD8α, a domain derived from CXCL12β, a domain derived from CXCL12α, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, and an intracellular signaling domain derived from CD3ζ domain. In some embodiments, the CAR also comprises one or more peptide linkers, e.g., a FLAG-tag, a (G4S)3linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR comprises, from the N-terminus to the C-terminus, a CD8α leader sequence (e.g., SEQ ID NO: 2), a CXCL12β domain (e.g., SEQ ID NO: 30), a (G4S)3 linker (e.g., SEQ ID NO: 6), a CXCL12α domain (e.g., SEQ ID NO: 29), a FLAG-tag (e.g., SEQ ID NO: 45), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), and a CD3ζ domain (e.g., SEQ ID NO: 17). In some embodiments, the CAR comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 51. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 51.

[0166] In some embodiments, the CAR comprises a leader sequence derived from CD8α, a domain derived from an MDX1338 light chain sequence, a domain derived from an MDX1338 heavy chain sequence, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, and an intracellular signaling domain derived from CD3ζ domain. In some embodiments, the CAR also comprises one or more peptide linkers, e.g., a FLAG-tag, a (G4S)3 linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR comprises, from the N-terminus to the C-terminus, a CD8α leader sequence (e.g., SEQ ID NO: 2), an MDX1338 light chain sequence (e.g., SEQ ID NO: 32), a (G4S)3 linker (e.g., SEQ ID NO: 6), an MDX1338 heavy chain sequence (e.g., SEQ ID NO: 31), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), and a CD3ζ domain (e.g., SEQ ID NO: 17). In some embodiments, the CAR comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the 45 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 amino acid sequence of SEQ ID NO: 52. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 52.

[0167] In some embodiments, the CAR comprises a leader sequence derived from CD8α, a domain derived from an MDX1338 heavy chain sequence, a domain derived from an MDX1338 light chain sequence, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, and an intracellular signaling domain derived from CD3ζ domain. In some embodiments, the CAR also comprises one or more peptide linkers, e.g., a FLAG-tag, a (G4S)3 linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR comprises, from the N-terminus to the C-terminus, a CD8α leader sequence (e.g., SEQ ID NO: 2), an MDX1338 heavy chain sequence (e.g., SEQ ID NO: 31), (G4S)3 linker (e.g., SEQ ID NO: 6), an MDX1338 light chain sequence (e.g., SEQ ID NO: 32), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), and a CD3ζ domain (e.g., SEQ ID NO: 17). In some embodiments, the CAR comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 53. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 53.

[0168] In some embodiments, the CAR comprises a leader sequence derived from CD8α, a domain derived from CXCL12α, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from a truncated CD28. In some embodiments, the CAR also comprises one or more peptide linkers, e.g., a FLAG-tag, a (G4S)3linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR comprises, from the N-terminus to the C-terminus, a CD8α leader sequence (e.g., SEQ ID NO: 2), a CXCL12α domain (e.g., SEQ ID NO: 29), a FLAG-tag (e.g., SEQ ID NO: 45), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), and a truncated CD28 intracellular signaling domain (e.g., SEQ ID NO: 28). In some embodiments, the CAR comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 54. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 54. 46 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0169] In some embodiments, the CAR comprises a leader sequence derived from CD8α, a domain derived from CXCL12β, a hinge domain derived from CD28, a transmembrane domain derived from CD28, and an intracellular signaling domain derived from a truncated CD28. In some embodiments, the CAR also comprises one or more peptide linkers, e.g., a FLAG-tag, a (G4S)3 linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR comprises, from the N-terminus to the C-terminus, a CD8α leader sequence (e.g., SEQ ID NO: 2), a CXCL12β domain (e.g., SEQ ID NO: 30), a FLAG-tag (e.g., SEQ ID NO: 45), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), and a truncated CD28 intracellular signaling domain (e.g., SEQ ID NO: 28). In some embodiments, the CAR comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 55. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 55.

[0170] In some embodiments, the CAR comprises a leader sequence derived from CD8α, a domain derived from CXCL12α, a domain derived from a CXCL12β, a hinge domain derived from CD28, a transmembrane domain derived from CD28, and an intracellular signaling domain derived from truncated CD28. In some embodiments, the CAR also comprises one or more peptide linkers, e.g., a FLAG-tag, a (G4S)3 linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR comprises, from the N- terminus to the C-terminus, a CD8α leader sequence (e.g., SEQ ID NO: 2), a CXCL12α domain (e.g., SEQ ID NO: 29), a (G4S)3 linker (e.g., SEQ ID NO: 6), a CXCL12β domain (e.g., SEQ ID NO: 30), a FLAG-tag (e.g., SEQ ID NO: 45), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), and a truncated CD28 intracellular signaling domain (e.g., SEQ ID NO: 28). In some embodiments, the CAR comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 56. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 56.

[0171] In some embodiments, the CAR comprises a leader sequence derived from CD8α, a domain derived from CXCL12α, a domain derived from CXCL12β, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from a truncated CD28. In some embodiments, the CAR also comprises one or more peptide linkers, e.g., a FLAG-tag, a (G4S)3 linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR comprises, from the N-terminus 47 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 to the C-terminus, a CD8α leader sequence (e.g., SEQ ID NO: 2), a CXCL12β domain (e.g., SEQ ID NO: 30), a (G4S)3 linker (e.g., SEQ ID NO: 6), a CXCL12α domain (e.g., SEQ ID NO: 29), a FLAG-tag (e.g., SEQ ID NO: 45), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), and a truncated CD28 intracellular signaling domain (e.g., SEQ ID NO: 28). In some embodiments, the CAR comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 57. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 57.

[0172] In some embodiments, the CAR comprises a leader sequence derived from CD8α, a domain derived from an MDX1338 light chain sequence, a domain derived from an MDX1338 heavy chain sequence, a hinge domain derived from CD28, a transmembrane domain derived from CD28, and an intracellular signaling domain derived from a truncated CD28. In some embodiments, the CAR also comprises one or more peptide linkers, e.g., a FLAG-tag, a (G4S)3 linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR comprises, from the N-terminus to the C-terminus, a CD8α leader sequence (e.g., SEQ ID NO: 2), an MDX1338 light chain sequence (e.g., SEQ ID NO: 32), a (G4S)3linker (e.g., SEQ ID NO: 6), an MDX1338 heavy chain sequence (e.g., SEQ ID NO: 31), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), and a truncated CD28 intracellular signaling domain (e.g., SEQ ID NO: 28). In some embodiments, the CAR comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 58. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 58.

[0173] In some embodiments, the CAR comprises a leader sequence derived from CD8α, a domain derived from an MDX1338 light chain sequence, a domain derived from an MDX1338 heavy chain sequence, a hinge domain derived from CD28, a transmembrane domain derived from CD28, and an intracellular signaling domain derived from a truncated CD28. In some embodiments, the CAR also comprises one or more peptide linkers, e.g., a FLAG-tag, a (G4S)3linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR comprises, from the N-terminus to the C-terminus, a CD8α leader sequence (e.g., SEQ ID NO: 2), an MDX1338 light chain sequence (e.g., SEQ ID NO: 32), a (G4S)3 linker (e.g., SEQ ID NO: 6), an MDX1338 heavy chain sequence (e.g., SEQ ID NO: 31), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane 48 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 domain (e.g., SEQ ID NO: 14), and a truncated CD28 intracellular signaling domain (e.g., SEQ ID NO: 28). In some embodiments, the CAR comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 59. In some embodiments, the CAR comprises the sequence of SEQ ID NO: 59. A. Co-Expressed Proteins

[0174] In some cases, it is useful to express a CAR as provided herein (i.e., a first polypeptide) with another polypeptide (i.e., a second polypeptide) in the same cell. Many polypeptides are suitable for use as a second polypeptide, such as a polypeptide that can provide an additional feature to the CAR-expressing cell. In some embodiments, the CAR and the second polypeptide are expressed as a single polypeptide comprising a self-cleaving peptide (also referred to as a ribosomal skip sequence) that is located between the first and second polypeptides. Self-cleaving peptides allow expression of multiple gene products from a single polynucleotide construct (see, e.g., Chng et al. “Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells,” mAbs 7(2): 403-412 (2015)). Thus, in some embodiments, the single polypeptide comprising the first and second polypeptides self-cleaves to provide the first and second polypeptides as two separate polypeptides. Examples of self-cleaving peptides include, but are not limited to, self-cleaving viral 2A peptides, for example, a porcine teschovirus-1 (P2A) peptide, a Thosea asigna virus 2A-like peptide (T2A), an equine rhinitis A virus (E2A) peptide, or a foot-and-mouth disease virus (F2A) peptide. In some embodiments, the self-cleaving peptide is P2A. Non-limiting examples of second polypeptides that can be co-expressed with a CAR as provided in this disclosure include CXCR4, a chimeric costimulatory receptor (CCR) (e.g., a CXCL12 CCR), and a safety switch protein. A discussion of the second polypeptides that can be co-expressed with the CARs of this disclosure is provided below. Polycistronic constructs for expressing a CAR and a second polypeptide are described in more detail in Section VI. In some embodiments, polypeptides of this disclosure include any of the polypeptides encoded by the polynucleotides, and in particular the CAR constructs, as described in Section VI.

[0175] In some embodiments, the second polypeptide is a targeting polypeptide (also referred to as a localization polypeptide) that can direct the CAR-expressing cell, e.g., a CAR T cell, to a target site when that CAR-expressing cell is present in a subject. For example, the chemokine C-X-C motif receptor 4 (CXCR4) is highly expressed in different hematological 49 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 malignancies and has been implicated in localizing cells to bone marrow. See, e.g., Chatterjee, Samit et al. “The intricate role of CXCR4 in cancer.” Advances in Cancer Research vol.124 (2014): 31-82; and Singh, Pratibha et al. “CXCR4 expression in the bone marrow microenvironment is required for hematopoietic stem and progenitor cell maintenance and early hematopoietic regeneration after myeloablation.” Stem Cells (Dayton, Ohio) vol.38,7 (2020): 849-859. In some embodiments, the second polypeptide comprises CXCR4 (SEQ ID NO: 20), and the cell co-expressing the CAR and CXCR4 is able to localize or traffic to the bone marrow of a subject.

[0176] In some embodiments, the second polypeptide is a protein that allows for the removal of CAR-expressing cells, e.g., CAR T cells, from circulation in a subject. Typically, the second polypeptide is a protein that can be stably and efficiently expressed in CAR- expressing cells, e.g., CAR T cells, without impairing phenotype, function, or target-binding specificity. In some cases, the second polypeptide is referred to as a “safety switch.” In some embodiments, the safety switch (i.e. the second polypeptide) comprises a fusion protein comprising a human caspase 9 polypeptide attached to a modified human FK506-binding protein (FKB) polypeptide (referred to herein as “iCaspase 9”, “iC9” or “iC9 fusion protein”). Dimerization of the iC9 fusion protein is inducible by the chemical agent AP1903 (Rimiducid), which binds to and crosslinks the FKB portions of two iC9 fusion proteins. When cells expressing iC9 are exposed to AP1903, the caspase 9 portion of the iC9 fusion protein becomes activated and can cause apoptosis of the cells. The iC9 safety switch system is discussed, e.g., in Straathof, C. et al. Blood vol.105,11 (2005): 4247-54; Gargett, T. and Brown, M.P.. Frontiers in Pharmacology vol.5235.28 Oct.2014; and Zhou, X. et al. “iCaspase 9 Suicide Gene System.” Methods in Molecular Biology (Clifton, N.J.) vol.1317 (2015): 87-105. In some embodiments, the second polypeptide comprises an iC9 fusion protein comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 21.

[0177] In some embodiments, the second polypeptide is a chimeric costimulatory receptor (CCR). CCRs mimic costimulatory signals but, unlike CARs, do not provide a T cell activation signal. Their purpose is to provide costimulation, e.g., a CD28-like signal,(33) in the absence of the natural costimulatory ligand on an antigen-presenting cell. CCRs thus provide a means for the tumor to direct counterfeit costimulation specifically within the tumor microenvironment. In some clinical studies, mechanisms of treatment failure were associated with the down-regulation of target antigen expression and the limited persistence 50 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 of effective CAR T cells. In some cases, the expression of a CCR in a CAR T cell can circumvent this issue by providing the CAR T cell with targeting capability for an additional antigen, which can enhance T cell cytotoxicity and improve T cell durability. See, e.g., Ramakrishna, Sneha et al. “Modulation of Target Antigen Density Improves CAR T-cell Functionality and Persistence.” Clinical Cancer Research : an Official Journal of the American Association for Cancer Research vol.25,17 (2019): 5329-5341; Majzner, Robbie G et al. “Tuning the Antigen Density Requirement for CAR T-cell Activity.” Cancer Discovery vol.10,5 (2020): 702-723; Watanabe, Keisuke et al. “Target antigen density governs the efficacy of anti-CD20-CD28-CD3ζ chimeric antigen receptor-modified effector CD8+ T cells.” Journal of Immunology (Baltimore, Md., 1950) vol.194,3 (2015): 911-20; and Katsarou, Afroditi et al. “Combining a CAR and a chimeric costimulatory receptor enhances T cell sensitivity to low antigen density and promotes persistence.” Science Translational Medicine vol.13,623 (2021).

[0178] In some embodiments, the CCR comprises an extracellular target-binding domain, an optional hinge domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular target-binding domain binds specifically to a protein that binds to a target that is highly expressed in disease-related cells, e.g., a cancer cells, cells of hematologic malignancies, leukemia cells, pre-LSCs, and leukemic blasts; and HSPCs. In some embodiments, the extracellular target-binding domain binds specifically to a target that is highly expressed in bone marrow. In some embodiments, the hinge domain can be a CD28 hinge domain (e.g., SEQ ID NO:11). In some embodiments, the transmembrane domain can be a CD28 transmembrane domain (e.g., SEQ ID NO:14). In some embodiments, the intracellular signaling domain can be a full length CD28 intracellular signaling domain (e.g., SEQ ID NO:15) or a truncated CD28 intracellular signaling domain (e.g., SEQ ID NO:28). In some embodiments, the CCR further comprises an N’ terminal leader sequence to direct transport and localization of the CCR to the T cell surface. In some embodiments, the leader sequence comprises a GM-CSFR leader sequence (e.g., SEQ ID NO: 1). In some embodiments, the leader sequence comprises a CD8α leader sequence (e.g., SEQ ID NO: 2). In some embodiments, the CCR further comprises a heterologous polypeptide tag, such as a FLAG-tag (SEQ ID NO: 45).

[0179] In some embodiments, the extracellular target-binding domain of the CCR binds specifically to CXCR4. In some embodiments, the extracellular target-binding domain of the CCR comprises at least one of a CXCL12α domain (e.g., SEQ ID NO: 29) or a CXCL12β 51 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 domain (e.g., SEQ ID NO: 30). In some embodiments, the extracellular target-binding domain of the CCR comprises an antibody or a fragment thereof that binds to CXCR4, e.g., the antibody Ulocuplumab (BMS-936564 / MDX1338). In some embodiments, the extracellular target-binding domain of the CCR comprises an anti-CXCR4 heavy chain fragment and / or an anti-CXCR4 light chain fragment, e.g., the heavy chain of Ulocuplumab (BMS-936564 / MDX1338; SEQ ID NO: 31) and / or the light chain of Ulocuplumab (BMS- 936564 / MDX1338; SEQ ID NO: 32). In some embodiments, the extracellular target-binding domain of the CCR comprises an anti-CXCR4 scFv fragment wherein the anti-CXCR4 heavy chain and the anti-CXCR4 light chain of the scFv are covalently linked with linker (e.g., a (G4S)3linker) between them. In some embodiments, the extracellular target-binding domain of the CCR comprises an anti-CXCR4 scFv fragment comprising the heavy chain of Ulocuplumab (SEQ ID NO: 31) (or a sequence at least 90% identical thereto), a (G4S)3 linker (SEQ ID NO: 6), and the light chain of Ulocuplumab (SEQ ID NO: 32) (or a sequence at least 90% identical thereto).

[0180] In some embodiments, the second polypeptide is derived from CXCR4 (SEQ ID NO: 20). CXCR4 is a protein that is widely expressed in patients with AML, e.g., pediatric AML (pAML). In some embodiments, a CAR protein is co-expressed with a CXCR4-derived polypeptide (SEQ ID NO: 20) for the purposes of analyzing the impact of the CAR protein on the CXCR4-derived polypeptide. In some embodiments, for example as discussed in Example 6.1 below, co-expression of a CAR protein with CXCR4 in the same cell can influence the expression and / or detection of CXCR4.

[0181] In some embodiments, the second polypeptide is derived from CXCL12 (UniProt ID: P48061), a natural ligand of CXCR4. In some embodiments, a CAR protein is co- expressed with a CXCL12-derived polypeptide for the purposes of targeting CAR T cells to the CXCR4 receptor, for example as discussed in Example 6.3 below. In some embodiments, the second polypeptide is derived from the globular domain of the Alpha (α) splice variant of CXCL12 (CXCL12α; SEQ ID NO: 29). In some embodiments, the second polypeptide is derived from the globular domain of the Beta (β) splice variant of CXCL12 (CXCL12β; SEQ ID NO: 30).

[0182] In some embodiments, the second polypeptide is derived from MDX1338 (Ulocuplumab / BMS-936564), an anti-CXCR4 antibody that induces cell death in chronic lymphocytic leukemia mediated through a reactive oxygen species-dependent pathway. 52 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 MDX1338 (Ulocuplumab / BMS-936564) is discussed in detail in Kashyap et al. “Ulocuplumab (BMS-936564 / MDX1338): a fully human anti-CXCR4 antibody induces cell death in chronic lymphocytic leukemia mediated through a reactive oxygen species- dependent pathway.” Oncotarget vol.7,3 (2016): 2809-22. In some embodiments, a CAR protein is co-expressed with a MDX1338 (Ulocuplumab / BMS-936564)-derived polypeptide for the purposes of targeting CAR T cells to the CXCR4 receptor, for example as discussed in Example 6.3 below. In some embodiments, the second polypeptide is derived from the MDX1338 scFv. In some embodiments, the second polypeptide is derived from the MDX1338 heavy chain (SEQ ID NO: 31). In some embodiments, the second polypeptide is derived from the MDX1338 light chain (SEQ ID NO: 32). In some embodiments, the second polypeptide comprises MDX1338 heavy chain (SEQ ID NO: 31), the MDX1338 light chain (SEQ ID NO: 32), or both. V. Polypeptide Compositions

[0183] Also provided herein are isolated polypeptide compositions comprising a target- binding domain as described above in Section III that can bind specifically to hematopoietic cytokine receptors (HCRs). In some embodiments, the polypeptide composition is useful as a protein therapeutic or as a diagnostic tool. In some embodiments, the polypeptide composition comprises a target-binding domain that is linked to a therapeutic agent, which can be a protein or a small molecule. See Figure 1A. In some embodiments, the polypeptide composition comprises a target-binding domain that is linked to an imaging agent. Therapeutic agents and imaging agents are discussed in detail below.

[0184] In some embodiments, the polypeptide composition can be attached to a cargo molecule containing a therapeutic agent. See Figure 1A. By recognizing the HCRs expressed on the cell surface, the polypeptide composition can bind specifically to target cells, e.g., HSPCs and disease-related cells such as leukemia cells, thereby delivering the therapeutic agent within the cargo molecule to those cells. In some cases, the therapeutic agent is a therapeutic protein or small molecule that is useful for the treatment of cancer or a non- malignant disease. Non-limiting examples of therapeutic agents include toxins, drugs, and gene-editing systems. Cargo molecules and therapeutic agents of cargo molecules are discussed in detail below.

[0185] The isolated polypeptide compositions of the present disclosure can comprise any of the target-binding domain polypeptides described in Section III of this disclosure. A 53 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 polypeptide composition comprising a target-binding domain of this disclosure can have binding specificity for two or more HCRs. In some embodiments, the polypeptide composition comprises two or more cytokine domains and / or antibodies or antigen-binding fragments that bind to two or more HCRs, and the polypeptide composition has binding specificity for said two or more HCRs. Target-binding domains polypeptides are discussed above in detail.

[0186] In some embodiments, the polypeptide composition comprises a polypeptide comprising a target-binding domain that comprises a first cytokine domain that binds to a first HCR, and a second cytokine domain that binds to a second HCR. In some embodiments, the target-binding domain further comprises a third cytokine domain that binds to a third HCR. In some embodiments, the first, second, and / or third cytokine domains are different from each other. In some embodiments, the first, second, and / or third HCRs are different from each other.

[0187] In some embodiments, the polypeptide composition comprises two or more polypeptides that have different target-binding domains, thereby providing the polypeptide composition with increasing multivalent HCR binding. In some embodiments, some or all of the cytokine domains are different from each other. In some embodiments, some or all of the cytokine domains bind to different HCRs.

[0188] In some embodiments, the polypeptide composition comprises a target-binding domain with two or more cytokine domains disclosed herein. In some embodiments, the polypeptide comprises a cytokine domain derived from an SCF sequence, a cytokine domain derived from a TPO sequence, and / or a cytokine domain derived from a FLT3LG sequence. In some embodiments, the polypeptide composition comprises (1) an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to the amino acid sequence of SEQ ID NO: 3, (2) an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to the amino acid sequence of SEQ ID NO: 4, and / or (3) an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the polypeptide composition comprises (1) a cytokine domain comprising the SCF sequence of SEQ ID 54 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 NO: 3, (2) a cytokine domain comprising the TPO sequence of SEQ ID NO: 4, and / or (3) a cytokine domain comprising the FLT3LG sequence of SEQ ID NO: 5. In some embodiments, the cytokine domains are covalently linked together by peptide linkers. Peptide linkers are discussed in detail in Section III above and any of the disclosed peptide linkers may be used to link the cytokine domains. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 6, 7, 8, 9, or 19.

[0189] In some embodiments, the polypeptide composition comprises a target-binding domain with three cytokine domains are covalently linked together. In some embodiments, the polypeptide composition comprises a cytokine domain derived from an SCF sequence, a cytokine domain derived from a TPO sequence, and a cytokine domain derived from a FLT3LG sequence. Such polypeptide compositions are also referred to in this disclosure as a “NaTriL” or “ELECTRIC” polypeptide. The orientation of the cytokine domains may vary with respect to each other. In some embodiments, the polypeptide composition comprises, from the N-terminus to the C-terminus, an SCF domain (SEQ ID NO: 3), a TPO domain (SEQ ID NO: 4), and a FLT3LG domain (SEQ ID NO: 5). In some embodiments, the polypeptide composition comprises, from the N-terminus to the C-terminus, an SCF domain (SEQ ID NO: 3), a FLT3LG domain (SEQ ID NO: 5), and a TPO domain (SEQ ID NO: 4). In some embodiments, the composition polypeptide comprises, from the N-terminus to the C- terminus, a TPO domain (SEQ ID NO: 4), an SCF domain (SEQ ID NO: 3), and a FLT3LG domain (SEQ ID NO: 5). In some embodiments, the polypeptide composition comprises, from the N-terminus to the C-terminus, a TPO domain (SEQ ID NO: 4), a FLT3LG domain (SEQ ID NO: 5), and an SCF domain (SEQ ID NO: 3). In some embodiments, the polypeptide composition comprises, from the N-terminus to the C-terminus, a FLT3LG domain (SEQ ID NO: 5), an SCF domain (SEQ ID NO: 3), and a TPO domain (SEQ ID NO: 4). In some embodiments, the polypeptide composition comprises, from the N-terminus to the C-terminus, a FLT3LG domain (SEQ ID NO: 5), a TPO domain (SEQ ID NO: 4), and an SCF domain (SEQ ID NO: 3). In some embodiments, the polypeptide composition comprises a (G4S)3 linker (SEQ ID NO: 6) that connects each cytokine domain to the next cytokine domain.

[0190] In some embodiments, the polypeptide composition comprises a target-binding domain that is linked to a therapeutic agent, such as a protein or a small molecule. Because the polypeptide compositions comprise a target-binding domain for specific binding to HCRs, the therapeutic agent can be delivered to a target cell expressing the HCRs. In some 55 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 embodiments, the protein is an antibody, an antigen-binding fragment, an antibody-drug conjugate, or a protein toxin. Antibody therapeutics and antibody-drug conjugates are discussed, for example in Lu, Ruei-Min et al. “Development of therapeutic antibodies for the treatment of diseases.” Journal of Biomedical Science vol.27,11.2 Jan.2020; Shim, Hyunbo. “Bispecific Antibodies and Antibody-Drug Conjugates for Cancer Therapy: Technological Considerations.” Biomolecules vol.10,3360.26 Feb.2020; and Dumontet, Charles et al. “Antibody-drug conjugates come of age in oncology.” Nat. Rev. Drug Discov. vol.22,8 (2023): 641-661. doi:10.1038 / s41573-023-00709-2. In some embodiments, the small molecule is a cytotoxic agent or a radioactive moiety. Therapeutic agents, cytotoxic agents, radiation therapy, chemotherapeutic agents, and immunosuppressive agents are discussed below and in Section VIII.

[0191] In some embodiments, the polypeptide composition comprises a target-binding domain that is covalently linked to an antibody or an antigen-binding fragment, providing a multi-specific polypeptide that can bind specifically to HCRs and the target epitope(s) for the antibody or antigen-binding fragment. Such polypeptide compositions have valency provided by the epitope(s) binding portions of the antibody or an antigen-binding fragment in addition to the target-binding domain capable of binding HCRs. As used herein, such polypeptides are referred to as “multivalent proteins” or “multivalent polypeptides.” In some embodiments, each of the target-binding domain and the antibody or antigen-binding fragment is produced as individual polypeptide chains, and the two polypeptide chains are then linked together as a single polypeptide. In some embodiments, the target-binding domain and the antibody or antigen-binding fragment are linked together by peptide linkers and are produced as a single polypeptide chain. Polypeptide chains can be joined by, for example, disulfide bonds, chemical reactions (e.g., click chemistry), and peptide linkers. Disulfide bonds, chemical reactions, and peptide linkers are discussed above in Section III.

[0192] In some embodiments, the antibody attached to the target-binding domain is a monoclonal antibody. As used herein, the term “monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are the same or substantially similar and that bind the same epitope(s), except for variants that can normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts. A monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the 56 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 selection of a unique clone from a plurality of clones, such as a pool of yeast clones, phage clones, bacterial clones, mammalian cell clones, hybridoma clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the target, for example, by affinity maturation, to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject. In some embodiments, the antibody is a chimeric antibody, a bispecific antibody (BsAb), trispecific or other multi-specific antibody.

[0193] In some embodiments, the multivalent polypeptide composition comprises a target- binding domain that is covalently linked to an antibody that binds to an immune cell, providing a multi-specific antibody that can bind specifically to HCRs and the target epitope(s) for the antibody. See Figure 1B. In some embodiments, the antibody comprises a T cell engager or a natural killer (NK) cell engager that binds specifically to T cells or NK cells, respectively. Such embodiments are similar to a bispecific T cell engager (BiTE) but are multi-specific. A BiTE is a bispecific antibody that binds simultaneously to CD3 on T cells and to tumor-specific antigens and induce tumor lysis via T cell cytotoxicity. A multi-specific T cell engager as provided herein, for example as discussed in Example 7 below, may bind to HCRs (i.e., KIT, MPL, and / or FLT3) expressed on disease-related cells (e.g., cells of hematologic malignancies, leukemia cells, pre-leukemic stem cells (LSCs), and leukemic blasts) or HSPCs and to an epitope on T cells simultaneously, and induce T cell-mediated cytotoxicity of HCR expressing cells (see, e.g., Zhou et al., 2021, Biomarker Research 9:38). In some embodiments, the T cell engager is derived from an anti-CD3 scFv. In some embodiments, the anti-CD3 scFv is derived from blinatumomab. A multi-specific NK cell engager as provided herein may bind to HCRs (i.e., KIT, MPL, and / or FLT3) expressed on disease-related cells or HSPCs and to an epitope on NK cells simultaneously, and induce NK cell-mediated cytotoxicity of HCR expressing cells (see, e.g., Demaria et al., 2021, European Journal of Immunology 51(8):1934-1942). In some embodiments, a linker (e.g., a glycine- serine linker (e.g., (G4S)3)) can connect one cytokine domain to the next, and / or a cytokine domain to the T cell engager or NK cell engager portion of the polypeptide composition.

[0194] In some embodiments, the multivalent polypeptide composition comprises a target- binding domain (e.g., a “NaTriL” or “ELECTRIC” polypeptide) that is covalently linked to an anti-CD3 antibody, e.g., an anti-CD3ε antibody. Such a multi-specific antibody can act by simultaneous binding to HCRs (e.g., KIT, MPL, and / or FLT3) expressed on disease-related cells or HSPCs and to CD3 expressed on a T cell. Crosslinking of these two cell types by the 57 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 antibody composition allows the formation of an immunological synapse, similar to that of a natural T cell receptor (TCR) / peptide–major histocompatibility complex (MHC) complex. This synapse results in T cell activation and thereby the secretion of inflammatory cytokines and cytolytic molecules that are able to kill the tumor cells in the process. It is generally thought that any T cell can serve as an effector cell, regardless of TCR specificity, as TCR signaling for bispecific or multi-specific CD3 antibodies does not require engagement of the antigen-binding domain of the TCR, but is initiated via CD3 (see Middelburg J. et al., 2021, Overcoming Challenges for CD3-Bispecific Antibody Therapy in Solid Tumors, Cancers (Basel). Vol.13, Issue 2, Article 287).

[0195] In some embodiments, the polypeptide composition comprises a target-binding domain that is covalently linked to a cytotoxic agent. In some embodiments, the cytotoxic agent is a protein toxin, such as ricin, saporin, Mistletoe lectin-1 (ML1), diphtheria toxin, ribosome-inactivating proteins (RPS), Pseudomonas endotoxin A (PE), or truncated Pseudomonas exotoxin A (PE38KDEL). In some embodiments, the cytotoxic agent is an apoptotic protein such as, for example, p53 upregulated modulator of apoptosis (PUMA). In some embodiments, the cytotoxic agent is a small molecule toxin, such as amanitin, pyrrolobenzodiazepine, monomethyl auristatin E (MMAE), Duocarmycin SA (DUO), PNU- 159682 (PNU), or SGD-1882 (PBD). Cytotoxic agents are discussed in more detail in Section VIII.A below.

[0196] In some embodiments, the polypeptide composition comprises a target-binding domain that is linked to an imaging agent. The imaging agent can be a heterologous polypeptide, a photosensitizer, or a detectable label such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal particle, a luminescent label, or an affinity tag such as biotin or streptavidin. Heterologous polypeptides also include polypeptides (e.g., enzymes) that are useful as diagnostic or detectable markers (also referred to herein as enzymatic labels), for example, luciferase, a fluorescent protein (e.g., green fluorescent protein (GFP)), or chloramphenicol acetyl transferase (CAT). An exemplary photosensitizer (also referred to as a photoabsorber) is, for example, IRDye700DX (IR700). Suitable radioactive labels include, but are not limited to, e.g.,32P,33P,14C,125I,131I,35S, and3H. Suitable fluorescent labels (also referred to as fluorophores) include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7. Luminescent labels include, but are not limited to, e.g., any 58 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the europium chelate of diethylene triamine pentaacetic acid (DTPA) or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzymatic labels include, e.g., alkaline phosphatase, CAT, luciferase, and horseradish peroxidase. Another labeling technique that may result in greater sensitivity involves coupling the target-binding domain of the polypeptide composition to low molecular weight haptens. These haptens can then be specifically altered by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific antihapten antibodies. Imaging labels are discussed in more detail in Section VIII below.

[0197] In some embodiments, the polypeptide composition comprises a target-binding domain that is linked to a cargo molecule containing a therapeutic agent. In some embodiments, the target-binding domain is linked to the cargo molecule via a peptide linker. Peptide linkers are discussed in detail above in Section III.

[0198] A cargo molecule is a material (particle) that is suitable for containing or packaging a therapeutic agent. Non-limiting examples of cargo molecules include viruses (e.g., Adeno- associated viruses (AAVs) or lentiviruses (LVs)), virus-like particles (VLPs), nanoparticles (e.g., lipid nanoparticles (LNPs), polymeric nanoparticles, or inorganic nanoparticles), and dense bodies, which are discussed below in Section VII. In some embodiments, the cargo molecule can be, but is not limited to, a virus-like particle (VLP), a lipid nanoparticle (LNP), an adeno-associated virus (AAV), or a lentivirus (LV). Other cargo molecules routinely used for delivery of components to cells can also be used. In some embodiments, the polypeptide composition is partially embedded in the membrane of the VLP, LNP, AAV, or LV and the target-binding domain exposed to the exterior of the VLP, LNP, AAV, or LV.

[0199] In some embodiments, the cargo molecules can include lipid particles, e.g., without limitation, nonionizable lipids, neutral lipids, and cationic lipids. Non-limiting examples of ionizable lipids that may be used in a cargo molecule include SM-102 (CAS number: 2089251-47-6), Dlin-MC3-DMAMC3 (also referred to as “MC3;” CAS number: 1224606- 06-7), CL4H6 (CAS number: 2256087-35-9), ssPalm-O-Phe (CAS number: 2377474-67-2), ALC-0315 (CAS number: 2036272-55-4), LP01 (CAS number: 1799316-64-5), and any combination thereof. Non-limiting examples of neutral lipids that may be used in a cargo molecules include 18:1 Δ9-cis phosphoethanolamine (DOPE) (CAS number: 4004-05-1), 59 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 cholesterol (CAS number: 57-88-5), 14:0 PEG2000 phosphoethanolamine (C14-PEG2000; CAS number: 474922-82-2), and any combination thereof. Non-limiting examples of cationic lipids that may be used in a cargo molecules 1,2- dilineoyl-3-dimethylammonium-propane (DLinDAP), l,2-dilinoleyloxy-3-N,N- dimethylaminopropane (DLinDMA), l,2- dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), l,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DlinKC2-DMA), (3- o-[2”- (methoxypolyethyleneglycol 2000) succinoyl]-l,2-dimyristoyl-sn-glycol (PEG-S-DMG), R-3- [(ro-methoxy-poly(ethylene glycol)2000) carbamoyl]-l,2-dimyristyloxlpropyl-3-amine (PEGC-DOMG, and any combination thereof. Preparation of LNPs and the like as well as encapsulation of cargo molecules may be adapted from Rosin et al, Molecular Therapy, vol. 19, no.12, pages 1286-2200, Dec.2011).

[0200] In some embodiments, the cargo molecule contains a nucleic acid, wherein the charge ratio of nucleic acid backbone phosphates to cationic lipid nitrogen atoms of the cargo molecule (e.g., lipids of the VLP, LNP, AAV, or LV) is about 1: 1.5 – 7 or about 1:4.

[0201] In some embodiments, the cargo molecule also includes a shielding compound, which is removable from the lipid composition under in vivo conditions. In some embodiments, the shielding compound is a biologically inert compound. In some embodiments, the shielding compound does not carry any charge on its surface or on the molecule as such. In some embodiments, the shielding compounds are polyethylenglycols (PEGs), hydroxyethylglucose (HEG) based polymers, polyhydroxyethyl starch (polyHES) and polypropylene. In some embodiments, the PEG, HEG, polyHES, and a polypropylene weight between about 500 to 10,000 Da or between about 2000 to 5000 Da. In some embodiments, the shielding compound is PEG 2000 or PEG 5000.

[0202] In some embodiments, the cargo molecule can include one or more helper lipids. In some embodiments, the helper lipid can be a phosphor lipid or a steroid. In some embodiments, the helper lipid is between about 20 mol % to 80 mol % of the total lipid content of the composition. In some embodiments, the helper lipid component is between about 35 mol % to 65 mol % of the total lipid content of the cargo molecule. In some embodiments, the cargo molecule includes lipids at 50 mol% and the helper lipid at 50 mol% of the total lipid content of the cargo molecule.

[0203] In some embodiments, the cargo molecule contains viral particles and / or viral components that are useful for increasing transduction efficiency. In some embodiments, the 60 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 cargo molecule does not contain any viral components, which helps minimize immunogenicity concerns. The cargo molecule may be used for in vitro, ex vivo, and in vivo delivery of a therapeutic agent to a cell. In some embodiments, the cargo molecule can be delivered to a subject in vivo. See, for example, U.S. Patent No.9,737,604 and Zhang et al. “Lipid nanoparticle-mediated efficient delivery of CRISPR / Cas9 for tumor therapy,” NPG Asia Materials Volume 9, page e441 (2017).

[0204] In some embodiments, the therapeutic agent is a therapeutic protein or small molecule. Non-limiting examples of therapeutic agents include toxins, drugs, and components of gene editing systems. In some embodiments, the therapeutic agent is a protein cytotoxic agent (i.e., a protein toxin or apoptotic protein) or a small molecule toxin, as discussed above. Cytotoxic agents are discussed in more detail above and in Section VIII below.

[0205] In some embodiments, the therapeutic agent is a radioactive moiety for use as radiotherapy. Radioactive moieties are discussed in detail below and in Section VIII.

[0206] In some embodiments, the therapeutic agent is a drug, such as, for example, a small molecule or a polynucleotide. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the therapeutic agent is an immunosuppressive agent. Chemotherapeutic agents and immunosuppressive agents are discussed below in Section VIII. In some embodiments, the polynucleotide can encode a therapeutic polypeptide, such as for use in gene therapy, or a therapeutic RNA molecule (e.g., antisense oligonucleotide, small interfering RNA (siRNA), or aptamer).

[0207] In some embodiments, the therapeutic agent is one or more components of a gene editing system. In some embodiments, the gene editing system comprises a DNA nuclease component, such as, for example, a CRISPR-Cas nuclease, a TALEN polypeptide, a MegaTal polypeptide, a meganuclease, and zinc finger nuclease, or an ARCUS nuclease. In some embodiments, the gene editing system comprises a CRISPR-Cas nuclease component and a guide RNA (gRNA) component, one or both of which (or the coding sequences thereof) can be packaged in a cargo molecule. DNA nucleases and gRNAs, components for gene editing, and related methods are discussed below in Section VIII.C. In some embodiments, the gene editing system can comprise a base editor or a prime editor. In some embodiments, the gene editing system can comprise a cytosine base editor (CBE) or adenine base editor (ABE). In some embodiments, the gene editing system can comprise a mobile genetic element (MGE) 61 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 (e.g., DNA gene writer or RNA gene writer; Tessera Therapeutics). In some embodiments, the gene editing system comprises a polynucleotide expressing a DNA nuclease component as described herein.

[0208] In some embodiments, the polypeptide comprising a target-binding domain is covalently linked to a heterologous polypeptide. Suitable heterologous polypeptides include, for example, without limitations, an antigenic tag (e.g., FLAG (DYKDDDDK) (SEQ ID NO: 45), polyhistidine (6-His; HHHHHH (SEQ ID NO: 46)), hemagglutinin (HA; YPYDVPDYA (SEQ ID NO: 47)), glutathione-S-transferase (GST), or maltose-binding protein (MBP)) for use in purifying the antibodies or antigen-binding fragments.

[0209] In some embodiments, the target-binding domain and the therapeutic agent, imaging agent, and / or heterologous polypeptide of the polypeptide composition are produced as two separate molecules, and the two molecules are cross-linked using any of a number of known chemical cross linkers. Examples of such cross linkers are those that link two amino acid residues via a linkage that includes a “hindered” disulfide bond. In these linkages, a disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-a-methyl-a(2- pyridyldithio) toluene (SMPT), forms such a linkage between two proteins utilizing a terminal lysine on one of the proteins and a terminal cysteine on the other. Heterobifunctional reagents that cross-link by a different coupling moiety on each protein can also be used. Other useful cross-linkers include, without limitation, reagents which link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis- maleimidobutane), an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N- hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p- azidosalicylamido]butylamine), and an amino group and a guanidinium group that is present in the side chain of arginine (e.g., p-azidophenyl glyoxal monohydrate).

[0210] Techniques for conjugating a therapeutic agent disclosed herein to the target- binding domain of the polypeptide composition as disclosed herein are well known. In some embodiments, a radioactive label or radioactive moiety can be directly conjugated to the amino acid backbone of the polypeptide composition. Alternatively, the radioactive label can be included as part of a larger molecule (e.g., 125I in meta-[125I]iodophenyl-N- hydroxysuccinimide ([125I]mIPNHS), which binds to free amino groups to form meta- 62 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 iodophenyl (mIP) derivatives of relevant proteins (see, e.g., Rogers et al., 1997, J. Nucl. Med. 38:1221-29) or chelate (e.g., to DOTA or DTPA), which is in turn bound to the protein backbone. Methods of conjugating the radioactive labels or larger molecules / chelates containing them to polypeptide compositions as described herein are known in the art. Such methods involve incubating the proteins with the radioactive label under conditions (e.g., pH, salt concentration, and / or temperature) that facilitate binding of the radioactive label or chelate to the protein (see, e.g., U.S. Patent No.6,001,329). Methods of attaching therapeutic agents and imaging agents to polypeptides are described, for example, in Arnon et al., 1985, Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56; Hellstrom et al., 1987, Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.623-53; Thorpe, 1985, Monoclonal Antibodies '84::Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506; “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy” In: Monoclonal Antibodies For Cancer Detection And Therapy, (Baldwin et al. eds.), pp.303-316 (1985), and Thorpe et al., 1982, Immunol. Rev.62:119-158.

[0211] Methods for conjugating a fluorescent label (sometimes referred to as a fluorophore) or other heterologous moiety to a protein (e.g., an antibody) are known in the art of protein chemistry. For example, fluorophores can be conjugated to free amino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophores. In some embodiments, the fluorophores can be conjugated to a heterobifunctional cross-linker moiety such as sulfo- SMCC. Suitable conjugation methods involve incubating an antibody protein or antigen- binding fragment thereof with the fluorophore under conditions that facilitate binding of the fluorophore to the protein. See, e.g., Welch and Redvanly, (2003), Handbook of Radiopharmaceuticals: Radiochemistry and Applications, John Wiley and Sons.

[0212] Many protein chemistry techniques can be used to covalently link separate polypeptides together. For example, polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyl-oxycarbonyl) or Boc (tert-butyloxycarbonoyl) chemistry (Applied Biosystems, Inc.; Foster City, CA). Those of skill in the art readily appreciate that a polypeptide composition provided herein, for example, can be synthesized by standard chemical reactions. By peptide condensation reactions, two separate polypeptides can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form a polypeptide composition. (Grant GA, 63 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 1992, Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y.; Bodansky M and Trost B., Ed., 1993, Principles of Peptide Synthesis. Springer Verlag Inc., NY). Alternatively, the polypeptides can by independently synthesized in vivo. Once isolated, these independent polypeptides may be linked to form a polypeptide composition via similar peptide condensation reactions.

[0213] For example, enzymatic ligation of cloned or synthetic polypeptides can allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides, or whole protein domains (Abrahmsen et al., 1991, Biochemistry, 30:4151). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct a polypeptide composition from shorter peptide fragments. This method consists of a two-step chemical reaction (Dawson et al., 1994, Science, 266:776779). The first step is the chemoselective reaction of an unprotected synthetic peptide a thioester with another unprotected peptide segment containing an amino terminal Cys residue to give a thioester linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site. Application of this native chemical ligation method to the total synthesis of a protein molecule is illustrated by the preparation of human interleukin 8 (IL-8) (Baggiolini et al., 1992, FEBS Lett.307:97-101; Clark et al., 1994, J. Biol. Chem. 269:16075; Clark et al., 1991, Biochemistry 30:3128; Rajarathnam et al., 1994, Biochemistry 33:6623-30).

[0214] Alternatively, unprotected peptide segments can be chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer et al., 1992, Science 256:221). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle et al., 1992, Techniques in Protein Chemistry IV. Academic Press, New York, pp.257-267).

[0215] In some embodiments, the polypeptide compositions disclosed herein can be modified, e.g., with a moiety that improves the stabilization and / or retention of the polypeptide compositions in circulation, e.g., in blood, serum, or other tissues. For example, the polypeptide compositions can be PEGylated as described in, e.g., Lee et al. (1999) Bioconjug Chem 10(6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476, or 64 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 HESylated (Fresenius Kabi, Germany) (see, e.g., Pavisić et al. (2010) Int J Pharm 387(1- 2):110-119). The stabilization moiety can improve the stability, or retention of, the polypeptide compositions by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.

[0216] In some embodiments, the polypeptide compositions described herein can be glycosylated. In some embodiments, a polypeptide composition described herein can be subjected to enzymatic or chemical treatment, or produced from a cell, such that the polypeptide composition has reduced or absent glycosylation. Methods for producing polypeptide compositions with reduced glycosylation are known in the art and described in, e.g., U.S. Patent No.6,933,368; Wright et al. (1991) EMBO J 10(10):2717-2723; and Co et al. (1993) Mol Immunol 30:1361. VI. Expression of Polypeptides A. Polynucleotides

[0217] Also provided herein are polynucleotides encoding the various polypeptides discussed above in Sections III-V (i.e. target-binding domains, isolated polypeptide compositions, CARs, co-expressed polypeptides) disclosed in this disclosure. In some embodiments, the polynucleotides encode a target-binding domain polypeptide as described in Section III. In some embodiments, the polynucleotides encode a CAR as described in Section IV. In some embodiments, the polynucleotides encode an isolated polypeptide composition as described in Section V. In some embodiments, the polynucleotides encode a CAR or an isolated polypeptide composition as described above and a second polypeptide.

[0218] In some embodiments, the polynucleotide encodes a target-binding domain as disclosed in Section III. In some embodiments, the polynucleotide encodes a target-binding domain comprising two amino acid sequences that are at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to at least two of SEQ ID NO: 3, 4, or 5, respectively. In some embodiments, the polynucleotide encodes a target-binding domain comprising three amino acid sequences that are at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3, 4, and 5, respectively.

[0219] In some embodiments, the polynucleotide encodes a CAR as disclosed in Section IV. In some embodiments, the polynucleotide encodes a CAR comprising a target-binding 65 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 domain comprising two amino acid sequences that are at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3, 4, or 5, respectively. In some embodiments, the polynucleotide encodes a CAR comprising a target-binding domain comprising three amino acid sequences that are at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3, 4, and 5, respectively.

[0220] In some embodiments, the polynucleotide comprises a CAR construct encoding a leader sequence derived from CD8α, a cytokine domain derived from SCF, a cytokine domain derived from a TPO, a cytokine domain derived from FLT3LG, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, and an intracellular signaling domain derived from CD3ζ. In some embodiments, the CAR construct also comprises sequences encoding one or more peptide linkers, e.g., a (G4S)3linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct, from 5’ to 3’, a CD8α leader sequence (e.g., SEQ ID NO: 2), an SCF domain (e.g., SEQ ID NO: 3), a (G4S)3 linker (SEQ ID NO: 6), a TPO domain (e.g., SEQ ID NO: 4), a (G4S)3linker (SEQ ID NO: 6), a FLT3LG domain (e.g., SEQ ID NO: 5), a AAA linker (e.g., SEQ ID NO: 8), a CD28 hinge domain (e.g., SEQ ID NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), and a CD3ζ domain (e.g., SEQ ID NO: 17). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 22. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 22.

[0221] In some embodiments, the polynucleotide comprises a CAR construct encoding a leader sequence derived from CD8α, a cytokine domain derived from SCF, a cytokine domain derived from TPO, a cytokine domain derived from FLT3LG, a hinge domain derived from IgG4, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, and an intracellular signaling domain derived from CD3ζ. In some embodiments, the CAR construct also comprises sequences encoding one or more one or more peptide linkers, e.g., a (G4S)3 linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct encodes, from 5’ to 3’, a CD8α leader sequence (e.g., SEQ ID NO: 2), an SCF domain (e.g., SEQ ID NO: 3), a (G4S)366 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 linker (SEQ ID NO: 6), a TPO domain (e.g., SEQ ID NO: 4), a (G4S)3 linker (SEQ ID NO: 6), a FLT3LG domain (e.g., SEQ ID NO: 5), a AAA polypeptide linker (e.g., SEQ ID NO: 8), an IgG4 hinge domain (e.g., SEQ ID NO: 12), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), and a CD3ζ domain (e.g., SEQ ID NO: 17). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 24. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 24.

[0222] In some embodiments, the polynucleotide encodes a CAR comprising an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 22 or 24.

[0223] In some embodiments, the polynucleotide encodes a CAR (i.e., a first polypeptide) and a second polypeptide as a single CAR construct as described in Section IV. Non-limiting examples of second polypeptides that can be co-expressed with a CAR as provided in this disclosure include CXCR4, a chimeric costimulatory receptor (CCR) (e.g., a CXCL12 CCR), and a safety switch protein. In some instances, the CAR and the second polypeptide are encoded by a polycistronic coding sequence. Exemplary polynucleotides comprising polycistronic coding sequences (i.e. polycistronic constructs) are provided below.

[0224] In some embodiments, the polynucleotide comprises a CAR construct encoding a CAR first polypeptide and a CXCR4 second polypeptide. In some embodiments, the CAR construct encodes a leader sequence derived from CD8α, a cytokine domain derived from SCF, a cytokine domain derived from TPO, a cytokine domain derived from FLT3LG, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from, an intracellular signaling domain derived from CD3ζ, a ribosomal skip sequence, and a CXCR4 polypeptide sequence. In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3 linker, a AAA linker, a SIG linker, a GST polypeptide, or any combination thereof. In some embodiments, the CAR construct encodes, from 5’ to 3’, a CD8α leader sequence (e.g., SEQ ID NO: 2), an SCF domain (e.g., SEQ ID NO: 3), a (G4S)3linker (SEQ ID NO: 6), a TPO domain (e.g., SEQ ID NO: 4), a (G4S)3 linker (SEQ ID NO: 6), a FLT3LG domain (e.g., SEQ ID NO: 5), a AAA linker (e.g., SEQ ID NO: 8), a CD28 hinge domain (e.g., SEQ ID 67 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 NO: 10), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a SIG linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (e.g., SEQ ID NO: 18), a GST linker (e.g., SEQ ID NO: 19), and a CXCR4 polypeptide sequence (e.g., SEQ ID NO: 20). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 23. In some embodiments, the polynucleotide encodes a CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 23.

[0225] In some embodiments, the polynucleotide comprises a CAR construct encoding a CAR first polypeptide and a CXCR4 second polypeptide. In some embodiments, the CAR construct encodes a leader sequence derived from CD8α, a cytokine domain derived from SCF, a cytokine domain derived from TPO, a cytokine domain derived from FLT3LG, a hinge domain derived from IgG4, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ, a ribosomal skip sequence, and a CXCR4 polypeptide sequence (SEQ ID NO: 20). In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct encodes, from 5’ to 3’, a CD8α leader sequence (e.g., SEQ ID NO: 2), an SCF domain (e.g., SEQ ID NO: 3), a (G4S)3linker (SEQ ID NO: 6), a TPO domain (e.g., SEQ ID NO: 4), a (G4S)3linker (SEQ ID NO: 6), a FLT3LG domain (e.g., SEQ ID NO: 5), a AAA polypeptide linker (e.g., SEQ ID NO: 8), an IgG4 hinge domain (e.g., SEQ ID NO: 12), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a SIG polypeptide linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (e.g., SEQ ID NO: 18), a GST polypeptide linker (e.g., SEQ ID NO: 19), and a CXCR4 polypeptide sequence (e.g., SEQ ID NO: 20). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 25.

[0226] In some embodiments, the polynucleotide comprises a CAR construct encoding a CAR first polypeptide and a CXCR4 second polypeptide. In some embodiments, the CAR construct encodes a leader sequence derived from CD8α, a cytokine domain derived from 68 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 SCF, a cytokine domain derived from TPO, a cytokine domain derived from FLT3LG, a hinge domain derived from IgG4, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ, a ribosomal skip sequence, and a CXCR4 polypeptide sequence (SEQ ID NO: 20). In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct encodes, from 5’ to 3’, a CD8α leader sequence (e.g., SEQ ID NO: 2), an SCF domain (e.g., SEQ ID NO: 3), a (G4S)3 linker (SEQ ID NO: 6), a TPO domain (e.g., SEQ ID NO: 4), a (G4S)3linker (SEQ ID NO: 6), a FLT3LG domain (e.g., SEQ ID NO: 5), a AAA polypeptide linker (e.g., SEQ ID NO: 8), an IgG4 hinge domain (e.g., SEQ ID NO: 12), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a SIG polypeptide linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (e.g., SEQ ID NO: 18), a GST polypeptide linker (e.g., SEQ ID NO: 19), and a CXCR4 polypeptide sequence (e.g., SEQ ID NO: 20). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 25.

[0227] In some embodiments, the polynucleotide encodes (1) a CAR (i.e., a first polypeptide) comprising an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3, 4, and / or 5; (2) a self-cleaving peptide; and (3) a second polypeptide sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 20. In some embodiments, the polynucleotide encodes (1) a CAR (i.e., a first polypeptide) comprising an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 22 or 24; (2) a self-cleaving peptide; and (2) a second polypeptide sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 20. In some embodiments, the polynucleotide encodes a CAR; a self-cleaving peptide; and a second polypeptide comprising an amino acid sequence that is at least 90% identical (for example, at 69 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 23 or 25.

[0228] In some embodiments, the polynucleotide comprises a CAR construct encoding a CAR first polypeptide and a iCaspase9 (iC9) safety switch second polypeptide. In some embodiments, the polynucleotide comprises a CAR construct encoding a leader sequence derived from CD8α, a cytokine domain derived from SCF, a cytokine domain derived from TPO, a cytokine derived from FLT3LG, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, an intracellular signaling domain derived CD3ζ, a ribosomal skip sequence, and an iCaspase9 (iC9) polypeptide sequence. In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3 linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct encodes, from 5’ to 3’, a CD8α leader sequence (e.g., SEQ ID NO: 2), an SCF domain (e.g., SEQ ID NO: 3), a (G4S)3 linker (SEQ ID NO: 6), a TPO domain (e.g., SEQ ID NO: 4), a (G4S)3 linker (SEQ ID NO: 6), a FLT3LG domain (e.g., SEQ ID NO: 5), a AAA polypeptide linker (e.g., SEQ ID NO: 8), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a SIG polypeptide linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (SEQ ID NO: 18), a GST polypeptide linker (e.g., SEQ ID NO: 19), and an iC9 polypeptide (e.g., SEQ ID NO: 21). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 26. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 26.

[0229] In some embodiments, the polynucleotide comprises a CAR construct encoding a CAR first polypeptide having an IgG4H domain and a iCaspase9 (iC9) safety switch second polypeptide. In some embodiments, the CAR construct encodes a leader sequence derived from CD8α, a cytokine domain derived from SCF, a cytokine domain derived from TPO, a cytokine domain derived from FLT3LG, a hinge domain derived from IgG4, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, an intracellular signaling domain CD3ζ, a ribosomal skip sequence (e.g., SEQ ID NO: 18), and an iC9 polypeptide (e.g., SEQ ID NO: 21). In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3linker, a AAA 70 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct encodes, from 5’ to 3’, a CD8α leader sequence (e.g., SEQ ID NO: 2), an SCF domain (e.g., SEQ ID NO: 3), a (G4S)3linker (SEQ ID NO: 6), a TPO domain (e.g., SEQ ID NO: 4), a (G4S)3 linker (SEQ ID NO: 6), a FLT3LG domain (e.g., SEQ ID NO: 5), a AAA polypeptide linker (e.g., SEQ ID NO: 8), an IgG4 hinge domain (e.g., SEQ ID NO: 12), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a SIG polypeptide linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (SEQ ID NO: 18), a GST polypeptide linker (e.g., SEQ ID NO: 19), and an iC9 polypeptide (e.g., SEQ ID NO: 21). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 27. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 27.

[0230] In some embodiments, the polynucleotide encodes (1) a CAR (i.e., a first polypeptide) comprising an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3, 4, and / or 5; (2) a self-cleaving peptide; and (3) a second polypeptide sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 21. In some embodiments, the polynucleotide encodes (1) a CAR (i.e., a first polypeptide) comprising an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 22 or 24; (2) a self-cleaving peptide; and (3) a second polypeptide sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 21. In some embodiments, the polynucleotide encodes a CAR; a self-cleaving peptide; and a second polypeptide comprising an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 26 or 27.

[0231] In some embodiments, the polynucleotide comprises a CAR construct encoding a CAR first polypeptide and a CCR second polypeptide comprising a CXCL12α signaling domain. In some embodiments, the CAR construct encodes a first leader sequence derived from CD8α, a cytokine domain derived from SCF, a cytokine domain derived from TPO, a cytokine domain derived from FLT3LG, a first hinge domain derived from CD28, a first 71 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 transmembrane domain derived from CD28, a first intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ, a ribosomal skip sequence, a second leader sequence derived from CD8α, a CXCL12α domain, a second hinge domain derived from CD28, a second transmembrane domain derived from CD28, and a second intracellular signaling domain derived from CD28. In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct also comprises a sequence encoding an epitope tag such as a FLAG-tag. In some embodiments, the CAR construct encodes, from 5’ to 3’, a first CD8α leader sequence (e.g., SEQ ID NO: 2), an SCF domain (e.g., SEQ ID NO: 3), a (G4S)3linker (SEQ ID NO: 6), a TPO domain (e.g., SEQ ID NO: 4), a (G4S)3 linker (SEQ ID NO: 6), a FLT3LG domain (e.g., SEQ ID NO: 5), a AAA polypeptide linker (e.g., SEQ ID NO: 8), a first CD28 hinge domain (e.g., SEQ ID NO: 11), a first CD28 transmembrane domain (e.g., SEQ ID NO: 14), a first CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a SIG polypeptide linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (e.g., SEQ ID NO: 18), a GST polypeptide linker (e.g., SEQ ID NO: 19), a second CD8α leader sequence (e.g., SEQ ID NO: 2), a CXCL12α polypeptide sequence (e.g., SEQ ID NO: 29), a FLAG-tag (e.g., SEQ ID NO: 45), a second CD28 hinge domain (e.g., SEQ ID NO: 12), a second CD28 transmembrane domain (e.g., SEQ ID NO: 14), and either (i) a second CD28 intracellular signaling domain (e.g., SEQ ID NO: 15) or (ii) a truncated CD28 intracellular signaling domain (SEQ ID NO: 28). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NOs: 33 or 34. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NOs: 33 or 34.

[0232] In some embodiments, the polynucleotide encodes a CAR construct encoding a CAR first polypeptide and a CCR second polypeptide comprising a CXCL12β signaling domain. In some embodiments, the CAR construct encodes a first leader sequence derived from CD8α, a cytokine domain derived from SCF, a cytokine domain derived from TPO, a cytokine domain derived from FLT3LG, a first hinge domain derived from CD28, a first transmembrane domain derived from CD28, a first intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ, a ribosomal skip sequence, a second leader sequence derived from CD8α, a CXCL12β domain, a second hinge domain 72 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 derived from CD28, a second transmembrane domain derived from CD28, and a second intracellular signaling domain derived from CD28. In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct also comprises a sequence encoding an epitope tag such as a FLAG-tag. In some embodiments, the CAR construct encodes, from 5’ to 3’, a first CD8α leader sequence (e.g., SEQ ID NO: 2), an SCF domain (e.g., SEQ ID NO: 3), a (G4S)3linker (SEQ ID NO: 6), a TPO domain (e.g., SEQ ID NO: 4), a (G4S)3 linker (SEQ ID NO: 6), a FLT3LG domain (e.g., SEQ ID NO: 5), a AAA polypeptide linker (e.g., SEQ ID NO: 8), a first CD28 hinge domain (e.g., SEQ ID NO: 11), a first CD28 transmembrane domain (e.g., SEQ ID NO: 14), a first CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a SIG polypeptide linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (e.g., SEQ ID NO: 18), a GST polypeptide linker (e.g., SEQ ID NO: 19), a second CD8α leader sequence (e.g., SEQ ID NO: 2), a CXCL12β polypeptide sequence (e.g., SEQ ID NO: 30), a FLAG-tag (e.g., SEQ ID NO: 45), a second CD28 hinge domain (e.g., SEQ ID NO: 12), a second CD28 transmembrane domain (e.g., SEQ ID NO: 14), and either (i) a second CD28 intracellular signaling domain (e.g., SEQ ID NO: 15) or (ii) a truncated CD28 intracellular signaling domain (e.g., SEQ ID NO: 28). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NOs: 35 or 36. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NOs: 35 or 36.

[0233] In some embodiments, the polynucleotide comprises a CAR construct encoding a CAR first polypeptide and a CCR second polypeptide comprising, in order, a CXCL12α signaling domain and a CXCL12β signaling domain. In some embodiments, the CAR construct encodes a first leader sequence derived from CD8α, a cytokine domain derived from SCF, a cytokine domain derived from TPO, a cytokine domain derived from FLT3LG, a first hinge domain derived from CD28, a first transmembrane domain derived from CD28, a first intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ, a ribosomal skip sequence, a second leader sequence derived from CD8α, a CXCL12α domain, a CXCL12β domain, a second hinge domain derived from CD28, a second transmembrane domain derived from CD28, and a second intracellular signaling domain derived from CD28. In some embodiments, the CAR construct also 73 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 comprises sequences encoding peptide linkers, e.g., a (G4S)3 linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct also comprises a sequence encoding an epitope tag such as a FLAG-tag. In some embodiments, the CAR construct encodes, from 5’ to 3’, a first CD8α leader sequence (e.g., SEQ ID NO: 2), an SCF domain (e.g., SEQ ID NO: 3), a (G4S)3 linker (SEQ ID NO: 6), a TPO domain (e.g., SEQ ID NO: 4), a (G4S)3linker (SEQ ID NO: 6), a FLT3LG domain (e.g., SEQ ID NO: 5), a AAA polypeptide linker (e.g., SEQ ID NO: 8), a first CD28 hinge domain (e.g., SEQ ID NO: 11), a first CD28 transmembrane domain (e.g., SEQ ID NO: 14), a first CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a SIG polypeptide linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (e.g., SEQ ID NO: 18), a GST polypeptide linker (e.g., SEQ ID NO: 19), a second CD8α leader sequence (e.g., SEQ ID NO: 2), a CXCL12α polypeptide sequence (e.g., SEQ ID NO: 29), a (G4S)3linker (SEQ ID NO: 6), CXCL12β polypeptide sequence (e.g., SEQ ID NO: 30), a FLAG-tag (e.g., SEQ ID NO: 45), a second CD28 hinge domain (e.g., SEQ ID NO: 12), a second CD28 transmembrane domain (e.g., SEQ ID NO: 14), and either (i) a second CD28 intracellular signaling domain (e.g., SEQ ID NO: 15) or (ii) a truncated CD28 intracellular signaling domain (e.g., SEQ ID NO: 28). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NOs: 37 or 38. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NOs: 37 or 38.

[0234] In some embodiments, the polynucleotide comprises a CAR construct encoding a CAR first polypeptide and a CCR second polypeptide comprising, in order, a CXCL12β signaling domain and a CXCL12α signaling domain. In some embodiments, the CAR construct encodes a first leader sequence derived from CD8α, a cytokine domain derived from SCF, a cytokine domain derived from TPO, a cytokine domain derived from FLT3LG, a first hinge domain derived from CD28, a first transmembrane domain derived from CD28, a first intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ, a ribosomal skip sequence, a second leader sequence derived from CD8α, a CXCL12β domain, a CXCL12α domain, a second hinge domain derived from CD28, a second transmembrane domain derived from CD28, and a second intracellular signaling domain derived from CD28. In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3 linker, a AAA linker, a SIG 74 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct also comprises a sequence encoding an epitope tag such as a FLAG-tag. In some embodiments, the CAR construct encodes, from 5’ to 3’, a first CD8α leader sequence (e.g., SEQ ID NO: 2), an SCF domain (e.g., SEQ ID NO: 3), a (G4S)3 linker (SEQ ID NO: 6), a TPO domain (e.g., SEQ ID NO: 4), a (G4S)3 linker (SEQ ID NO: 6), a FLT3LG domain (e.g., SEQ ID NO: 5), a AAA polypeptide linker (e.g., SEQ ID NO: 8), a first CD28 hinge domain (e.g., SEQ ID NO: 11), a first CD28 transmembrane domain (e.g., SEQ ID NO: 14), a first CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a SIG polypeptide linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (e.g., SEQ ID NO: 18), a GST polypeptide linker (e.g., SEQ ID NO: 19), a second CD8α leader sequence (e.g., SEQ ID NO: 2), CXCL12β polypeptide sequence (e.g., SEQ ID NO: 30), a (G4S)3 linker (SEQ ID NO: 6), a CXCL12α polypeptide sequence (e.g., SEQ ID NO: 29), a FLAG-tag (e.g., SEQ ID NO: 45), a second CD28 hinge domain (e.g., SEQ ID NO: 12), a second CD28 transmembrane domain (e.g., SEQ ID NO: 14), and either (i) a second CD28 intracellular signaling domain (e.g., SEQ ID NO: 15) or (ii) a truncated CD28 intracellular signaling domain (e.g., SEQ SEQ ID NO: 28). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NOs: 39 or 40. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NOs: 39 or 40.

[0235] In some embodiments, the polynucleotide comprises a CAR construct encoding a CAR first polypeptide and a CCR second polypeptide comprising a target-binding domain comprising, in order, heavy and light chain sequences from MDX1338. In some embodiments, the target-binding domain of the CCR comprises a scFv. In some embodiments, the CAR construct encodes a first leader sequence derived from CD8α, a cytokine domain derived from SCF, a cytokine domain derived from TPO, a cytokine domain derived from FLT3LG, a first hinge domain derived from CD28, a first transmembrane domain derived from CD28, a first intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ, a ribosomal skip sequence, a second leader sequence derived from CD8α, an MDX1338 heavy chain sequence, MDX1338 light chain sequence, a second hinge domain derived from CD28, a second transmembrane domain derived from CD28, and a second intracellular signaling domain derived from CD28. In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a 75 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 (G4S)3 linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct encodes, from 5’ to 3’, a first CD8α leader sequence (e.g., SEQ ID NO: 2), an SCF domain (e.g., SEQ ID NO: 3), a (G4S)3linker (SEQ ID NO: 6), a TPO domain (e.g., SEQ ID NO: 4), a (G4S)3 linker (SEQ ID NO: 6), a FLT3LG domain (e.g., SEQ ID NO: 5), a AAA polypeptide linker (e.g., SEQ ID NO: 8), a first CD28 hinge domain (e.g., SEQ ID NO: 11), a first CD28 transmembrane domain (e.g., SEQ ID NO: 14), a first CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a SIG polypeptide linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (e.g., SEQ ID NO: 18), a GST polypeptide linker (e.g., SEQ ID NO: 19), a second CD8α leader sequence (e.g., SEQ ID NO: 2), an MDX1338 heavy chain sequence (e.g., SEQ ID NO: 31), a (G4S)3 linker (SEQ ID NO: 6), an MDX1338 light chain sequence (e.g., SEQ ID NO: 32), a second CD28 hinge domain (e.g., SEQ ID NO: 12), a second CD28 transmembrane domain (e.g., SEQ ID NO: 14), and either (i) a second CD28 intracellular signaling domain (e.g., SEQ ID NO: 15) or (ii) a truncated CD28 intracellular signaling domain (e.g., SEQ ID NO: 28). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NOs: 41 or 42. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NOs: 41 or 42.

[0236] In some embodiments, the polynucleotide comprises a CAR construct encoding a CAR first polypeptide and a CCR second polypeptide comprising a target-binding domain comprising, in order, light and heavy chain sequences from MDX1338. In some embodiments, the target-binding domain of the CCR comprises a scFv. In some embodiments, the CAR construct encodes a first leader sequence derived from CD8α, a cytokine domain derived from SCF, a cytokine domain derived from TPO, a cytokine domain derived from FLT3LG, a first hinge domain derived from CD28, a first transmembrane domain derived from CD28, a first intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ, a ribosomal skip sequence, a second leader sequence derived from CD8α, MDX1338 light chain sequence, an MDX1338 heavy chain sequence, a second hinge domain derived from CD28, a second transmembrane domain derived from CD28, and a second intracellular signaling domain derived from CD28. In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some 76 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 embodiments, the CAR construct encodes, from 5’ to 3’, a first CD8α leader sequence (SEQ ID NO: 2), an SCF domain (SEQ ID NO: 3), a (G4S)3 linker (SEQ ID NO: 6), a TPO domain (SEQ ID NO: 4), a (G4S)3linker (SEQ ID NO: 6), a FLT3LG domain (SEQ ID NO: 5), a AAA polypeptide linker (SEQ ID NO: 8), a first CD28 hinge domain (SEQ ID NO: 11), a first CD28 transmembrane domain (SEQ ID NO: 14), a first CD28 intracellular signaling domain (SEQ ID NO: 15), a CD3ζ domain (SEQ ID NO: 17), a SIG polypeptide linker (SEQ ID NO: 9), a P2A ribosomal skip sequence (SEQ ID NO: 18), a GST polypeptide linker (SEQ ID NO: 19), a second CD8α leader sequence (SEQ ID NO: 2), an MDX1338 light chain sequence (SEQ ID NO: 32), a (G4S)3linker (SEQ ID NO: 6), an MDX1338 heavy chain sequence (SEQ ID NO: 31), a second CD28 hinge domain (SEQ ID NO: 12), a second CD28 transmembrane domain (SEQ ID NO: 14), and either (i) a second CD28 intracellular signaling domain (SEQ ID NO: 15) or (ii) a truncated CD28 intracellular signaling domain (e.g., SEQ ID NO: 28). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NOs: 43 or 44. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NOs: 43 or 44.

[0237] In some embodiments, the polynucleotide encodes (1) a CAR (i.e., a first polypeptide) comprising an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3, 4, and / or 5; (2) a self-cleaving peptide; and (3) a second polypeptide sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the CCR portion of the amino acid sequence of any one of SEQ ID NO: 33-44. As discussed above in Section IV above, a CCR (i.e., a second polynucleotide) may comprise (i) a portion that binds to CXCR4 (e.g., a CXCL12α domain (SEQ ID NO: 29), a CXCL12β domain (SEQ ID NO: 30), the heavy chain sequence of Ulocuplumab (BMS- 936564 / MDX1338) (SEQ ID NO: 31), and / or the light chain sequence of Ulocuplumab (BMS-936564 / MDX1338) (SEQ ID NO: 32)), a CD8α leader sequence comprising the amino acid sequence of SEQ ID NO: 2, a CD28 hinge domain comprising the amino acid sequence of SEQ ID NO: 11, a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and a CD28 intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 15; or (ii) a portion that binds to CXCR4 (e.g., a CXCL12α domain (SEQ ID NO: 29), a CXCL12β domain (SEQ ID NO: 30), the heavy chain sequence of 77 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 Ulocuplumab (BMS-936564 / MDX1338) (SEQ ID NO: 31), and / or the light chain sequenceof Ulocuplumab (BMS-936564 / MDX1338) (SEQ ID NO: 32)), a CD8 leader sequencecomprising the amino acid sequence of SEQ ID NO: 2, a CD28 hinge domain comprising the amino acid sequence of SEQ ID NO: 11, a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 14, and a truncated CD28 intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the second polypeptide is at least 90% identical to CXCR4 polypeptide (SEQ ID NO: 20) instead of a CCR portion of the amino acid sequence of any one of SEQ ID NO: 33-44.

[0238] In some embodiments, the polynucleotide encodes (1) a CAR (i.e., a first polypeptide) comprising an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 22 or 24; (2) a self-cleaving peptide; and (3) a second polypeptide sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the CCR portion of the amino acid sequence of any one of SEQ ID NO: 33-44. In some embodiments, the polynucleotide encodes a CAR; a self-cleaving peptide; and a second polypeptide comprising an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any one of SEQ ID NO: 33-44.

[0239] In some embodiments, the polynucleotide encodes (1) a CAR (i.e., a first polypeptide) comprising an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to any one of SEQ ID NO: 48-53; (2) a self-cleaving peptide; and (3) a second polypeptide sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the CXCR4 polypeptide (SEQ ID NO: 20). In some embodiments, the polynucleotide encodes for a CAR protein and a CXCR4 polypeptide, as represented by any oen of SEQ ID NOS: 60-65.

[0240] In some embodiments, the CAR first polypeptide comprises a first leader sequencederived from CD8 , a domain derived from CXCL12 , a hinge domain derived from CD28,a transmembrane domain derived from CD28, an intracellular signaling domain derived fromCD28, an intracellular signaling domain derived from CD3 , a ribosomal skip sequence, anda CXCR4 polypeptide. In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3 linker, a AAA linker, a SIG linker, a GST linker, or US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 any combination thereof. e.g., SEQ ID NO: 29), a FLAG-tag (e.g., SEQ ID NO: 45), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a first short linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (e.g., SEQ ID NO: 18), a second short linker (e.g., SEQ ID NO: 19), and a CXCR4 polypeptide (e.g., SEQ ID NO: 20). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 60. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 60.

[0241] In some embodiments, the CAR first polypeptide comprises a first leader sequence derived from CD8α, a domain derived from CXCL12β, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ, a ribosomal skip sequence, and a CXCR4 polypeptide. In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct encodes, from 5’ to 3’, a CD8α leader sequence (e.g., SEQ ID NO: 2), a CXCL12β domain (e.g., SEQ ID NO: 30), a FLAG-tag (e.g., SEQ ID NO: 45), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a first short linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (SEQ ID NO: 18), a second short linker (e.g., SEQ ID NO: 19), and a CXCR4 polypeptide (e.g., SEQ ID NO: 20). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 61. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 61.

[0242] In some embodiments, the CAR first polypeptide comprises a first leader sequence derived from CD8α, a domain derived from CXCL12α, a domain derived from CXCL12β, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ, a ribosomal skip sequence, and a CXCR4 polypeptide. In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3linker, a 79 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct encodes, from 5’ to 3’, a CD8α leader sequence (e.g., SEQ ID NO: 2), a CXCL12α domain (e.g., SEQ ID NO: 29), a (G4S)3linker (e.g., SEQ ID NO: 6), a CXCL12β domain (e.g., SEQ ID NO: 30), a FLAG-tag (e.g., SEQ ID NO: 45), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a first short linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (e.g., SEQ ID NO: 18), a second short linker (e.g., SEQ ID NO: 19), and a CXCR4 polypeptide (e.g., SEQ ID NO: 20). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 62. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 62.

[0243] In some embodiments, the CAR first polypeptide comprises a first leader sequence derived from CD8α, a domain derived from CXCL12β, a domain derived from CXCL12α, a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ, a ribosomal skip sequence, and a CXCR4 polypeptide. In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct encodes, from 5’ to 3’, a CD8α leader sequence (e.g., SEQ ID NO: 2), a CXCL12β domain (e.g., SEQ ID NO: 30), a (G4S)3 linker (e.g., SEQ ID NO: 6), a CXCL12α domain (e.g., SEQ ID NO: 29), a FLAG-tag (e.g., SEQ ID NO: 45), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (e.g., SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a first short linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (e.g., SEQ ID NO: 18), a second short linker (e.g., SEQ ID NO: 19), and a CXCR4 polypeptide (e.g., SEQ ID NO: 20). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 63. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NO: 63. 80 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0244] In some embodiments, the CAR first polypeptide comprises a first leader sequence derived from CD8α, a domain derived from MDX1338 scFv (i.e., light and heavy chain sequences), a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ, a ribosomal skip sequence, and a CXCR4 polypeptide. In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct encodes, from 5’ to 3’, a CD8α leader sequence (SEQ ID NO: 2), an MDX1338 light chain sequence (e.g., SEQ ID NO: 32), a (G4S)3linker (e.g., SEQ ID NO: 6), an MDX1338 heavy chain sequence (e.g., SEQ ID NO: 31), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a first short linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (SEQ ID NO: 18), a second short linker (e.g., SEQ ID NO: 19), and a CXCR4 polypeptide (e.g., SEQ ID NO: 20). In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NOs: 64. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NOs: 64.

[0245] In some embodiments, the CAR first polypeptide comprises a first leader sequence derived from CD8α, a domain derived from MDX1338 scFv (i.e., light and heavy chain sequences), a hinge domain derived from CD28, a transmembrane domain derived from CD28, an intracellular signaling domain derived from CD28, an intracellular signaling domain derived from CD3ζ, a ribosomal skip sequence, and a CXCR4 polypeptide. In some embodiments, the CAR construct also comprises sequences encoding peptide linkers, e.g., a (G4S)3linker, a AAA linker, a SIG linker, a GST linker, or any combination thereof. In some embodiments, the CAR construct encodes, from 5’ to 3’, a CD8α leader sequence (SEQ ID NO: 2), an MDX1338 heavy chain sequence (e.g., SEQ ID NO: 31), a (G4S)3 linker (e.g., SEQ ID NO: 6), an MDX1338 light chain sequence (e.g., SEQ ID NO: 32), a CD28 hinge domain (e.g., SEQ ID NO: 11), a CD28 transmembrane domain (SEQ ID NO: 14), a CD28 intracellular signaling domain (e.g., SEQ ID NO: 15), a CD3ζ domain (e.g., SEQ ID NO: 17), a first short linker (e.g., SEQ ID NO: 9), a P2A ribosomal skip sequence (SEQ ID NO: 18), a second short linker (e.g., SEQ ID NO: 19), and a CXCR4 polypeptide (e.g., SEQ ID NO: 20). 81 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 In some embodiments, the CAR construct encodes an amino acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NOs: 65. In some embodiments, the CAR construct encodes an amino acid sequence comprising the sequence of SEQ ID NOs: 65.

[0246] In some embodiments, the polynucleotides encode a polypeptide composition comprising an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3, 4, and / or 5. In some embodiments, the polynucleotides encode a polypeptide composition comprising (1) an amino acid sequence that is at least 90% identical (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3, 4, and / or 5; and (2) a therapeutic agent, e.g., an antibody or a cytotoxic protein. Antibodies and cytotoxic proteins that can be used in polypeptide compositions are discussed in Section VIII.

[0247] Polynucleotides encoding the polypeptide compositions provided herein can be provided in expression cassettes for expression in a cell or organism of interest. The cassette will include 5’ and 3’ regulatory sequences operably linked to a polynucleotide encoding a polypeptide composition provided herein that allows for expression of the polynucleotide. The cassette may additionally contain at least one additional gene or genetic element to be cotransformed into the cell or organism. Where additional genes or elements are included, the components are operably linked. Alternatively, the additional gene(s) or element(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and / or recombination sites for insertion of the polynucleotides to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain a selectable marker gene.

[0248] The expression cassette will include in the 5’-3’ direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a polypeptide composition of the present disclosure , and a transcriptional and translational termination region (i.e., termination region) functional in the cell or organism of interest. The promoters are capable of directing or driving expression of a coding sequence in a host cell. A more detailed discussion of promoters is provided in Section VI.B below. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) may be endogenous or heterologous to the host cell or to each other. As used herein, “heterologous” 82 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

[0249] Additional regulatory signals include, but are not limited to, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. See, for example, Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), hereinafter “Sambrook 11”; Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N.Y., and the references cited therein.

[0250] In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved. B. Vectors

[0251] Several possible vector systems are available for the expression of cloned target- binding domains, CARs, and polypeptide compositions from nucleic acids in mammalian cells. One class of vectors relies upon the integration of the desired gene sequences into the host cell genome. Cells that have stably integrated DNA can be selected by simultaneously introducing drug resistance genes such as E. coli gpt (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072) or Tn5 neo (Southern and Berg, 1982, Mol. Appl. Genet.1:327). The selectable marker gene can be either linked to the DNA gene sequences to be expressed or introduced into the same cell by co-transfection (Wigler et al., 1979, Cell 16:77). A second class of vectors utilizes DNA elements that confer autonomously replicating capabilities to an extrachromosomal plasmid. These vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al., 1982, Proc. Natl. Acad. Sci. USA, 79:7147), CMV, polyoma 83 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 virus (Deans et al., 1984, Proc. Natl. Acad. Sci. USA 81:1292), or SV40 virus (Lusky & Botchan, 1981, Nature 293:79).

[0252] A polynucleotide encoding the target-binding domain, CAR, and polypeptide composition can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences. The regulatory sequences include a promoter and transcriptional start and stop sequences. In addition, the expression vector can include more than one replication system, such that it can be maintained in two different organisms, for example, in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. In some cases, the target-binding domain polypeptide composition is purified from the cells.

[0253] In some embodiments, the expression vector is a plasmid or a viral vector that transports the disclosed nucleic acids into the cell without undesired degradation and include a promoter (i.e., a constitutive or an inducible promoter, a ubiquitous promoter such as CMV or EF1α, or other inducible or cell-type-specific promoter) yielding expression of the polynucleotide and / or adapter polypeptide in the cells into which it is delivered. Viral vectors are, for example, Adenovirus, Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general are described by Coffin et al., 1997, Retroviruses, Cold Spring Harbor Laboratory Press, which is incorporated by reference herein for the vectors and methods of making them. The construction of replication-defective adenoviruses has been described (Berkner et al., 1987, J. Virology 61:1213-20; Massie et al., 1986, Mol. Cell. Biol.6:2872-83; Haj-Ahmad et al., 1986, J. Virology 57:267-74; Davidson et al., 1987, J. Virology 61:1226-39; Zhang et al., 1993, BioTechniques 15:868-72). The benefit and the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infections viral particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors. In some instances, the polynucleotides encoding the target-binding 84 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 domains, CARs, and polypeptide compositions can be delivered via nanoparticles (e.g., lipid nanoparticles) or virus-like particles.

[0254] In some embodiments, the expression vector is delivered to a cell via non-viral based delivery methods, for example, transfection, nucleofection, electroporation, and the like. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs – or viral vectors that have not been packaged into functional and transducible viral particles. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clonetech (Pal Alto, CA), Stratagene (La Jolla, CA), and Invitrogen / Life Technologies (Carlsbad, CA). Vectors typically contain one or more regulatory regions. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5’ and 3’ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.

[0255] Also provided herein are vectors, comprising a DNA construct comprising a promoter that drives expression in a host cell operably linked to a recombinant polynucleotide comprising a nucleotide sequence that encodes a target-binding domain, CAR, or polypeptide composition as described in Section III-V above. In some embodiments, the promoter is capable of directing expression in a bacterial cell, a eukaryotic cell, or a mammalian cell. In some embodiments, the promoter is capable of directing expression in a human cell, e.g., a human immune cell.

[0256] Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters (e.g., β-actin promoter or EF1α promoter), or from hybrid or chimeric promoters (e.g., CMV promoter fused to the β-actin promoter). Promoters from the host cell or related species are also useful herein.

[0257] The term “enhancer” generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5’ or 3’ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 bp in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain 85 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0258] The promoter and / or the enhancer can be inducible (e.g., chemically or physically regulated). A chemically regulated promoter and / or enhancer can, for example, be regulated by the presence of alcohol, a steroid, an antibiotic (for example tetracyclin), or a metal. A physically regulated promoter and / or enhancer can, for example, be regulated by environmental factors, such as temperature and light. Optionally, the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize the expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter and / or enhancer region can be active in a cell type specific manner. Optionally, in certain vectors, the promoter and / or enhancer region can be active in all eukaryotic cells, independent of cell type. Preferred promoters of this type are the CMV promoter, the SV40 promoter, the beta-actin promoter, the EF1α promoter, and the retroviral long terminal repeat (LTR).

[0259] The vectors also can include, for example, origins of replication and / or markers. A marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell. The marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S- transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak; New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus. 86 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 C. Cells and Expression

[0260] Also provided herein are cells, including bacterial host cells, eukaryotic host cells, human cells, e.g., human immune cells or human HSPCs, comprising a recombinant polynucleotide encoding the various polypeptides (i.e. a target-binding domain, CAR (with or without a second polypeptide), or polypeptide composition) as described in this disclosure (i.e., any of the polynucleotides described in Section VIII.A). The various polypeptides discussed above in Sections III-V (i.e. target-binding domains, isolated polypeptide compositions, CARs, co-expressed polypeptides) may be produced by recombinant expression in a human or non-human cell using a variety of techniques known in the art of molecular biology and protein chemistry. In some cases, the CAR disclosed herein is expressed (alone or in combination with a co-expressed polypeptide) in a cell to be administered to a subject. In some cases, the polypeptide composition disclosed herein is expressed in a cell and subsequently isolated from the cell.

[0261] In some embodiments, the CARs disclosed herein are expressed on the surface of cells, such as human immune cells or human HSPCs. In some embodiments, a CAR provided herein is co-expressed in a cell with a second polypeptide. CARs and second polypeptides are discussed in Section IV above.

[0262] In some embodiments, the polypeptide compositions disclosed herein are expressed in a cell as a single polypeptide comprising a target-binding domain provided herein and optionally comprising a therapeutic agent, an imaging agent, and / or a heterologous polypeptide. Polypeptide compositions are discussed in Section V above. In some embodiments, each component in the polypeptide composition (i.e., the target-binding domain provided, therapeutic agent, imaging agent, and / or heterologous polypeptide) is produced separately, and the components are later covalently linked to form a single molecule. Methods for linking a target-binding domain with a therapeutic agent, imaging agent, and / or heterologous polypeptide are discussed in detail in Section V above.

[0263] The expression vectors can be introduced into cells in a manner suitable for subsequent expression of the polynucleotide. The method of introduction is largely dictated by the targeted cell type, discussed below. Exemplary methods include CaPO4precipitation, liposome fusion, cationic liposomes, electroporation, nucleoporation, viral infection, dextran- mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct 87 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 microinjection. In some embodiments, the vector is delivered to a cell via nanoparticles (e.g., lipid nanoparticles) or virus-like particles.

[0264] Appropriate host cells for the expression of a target-binding domain, CAR (with or without a second polypeptide), and polypeptide composition disclosed herein include yeast, bacteria, insect, plant, and mammalian cells. Of particular interest are bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), as well as primary cell lines. Also provided herein are populations of any such cells.

[0265] In some embodiments, the target-binding domain, CAR (with or without a second polypeptide), or polypeptide composition can be produced from the cells by culturing a host cell transformed with the expression vector containing polynucleotide encoding the target- binding domain, CAR, or polypeptide, under conditions, and for an amount of time, sufficient to allow expression of the proteins. Such conditions for protein expression vary with the choice of the expression vector and the host cell and are easily ascertained by one skilled in the art through routine experimentation. For example, polypeptides expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al., 1998, Cytokine 10:319-30). Bacterial expression systems and methods for their use are known in the art (see Ausubel et al., 1988, Current Protocols in Molecular Biology, Wiley & Sons; and Green and Sambrook, 2012, Molecular Cloning--A Laboratory Manual, 4th Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The choice of codons, suitable expression vectors, and suitable host cells vary depending on a number of factors and may be easily optimized as needed. A target- binding domain, CAR, or polypeptide composition described herein can be expressed in mammalian cells or in other expression systems including but not limited to yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al., 2000, Protein Expression and Purification 18:213-20). Additional discussion of expression vectors for use in eukaryotic cells (e.g., for treating a subject with cancer), along with suitable delivery systems, is provided in Section VIII, below.

[0266] Also provided herein are human immune cells or human HSPCs for expressing a CAR (with or without a second polypeptide) described herein. In some embodiments, the cell expresses the CAR on the cell surface. In some embodiments, the cell comprises a polynucleotide encoding the CAR, wherein the CAR is expressed from the polynucleotide and localized to the cell surface. In some embodiments, the cell comprises a polynucleotide 88 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 encoding the CAR and a second polypeptide, wherein the CAR the second polypeptide are expressed from the polynucleotide and are localized to the cell surface. In some embodiments, the immune cell is a T cell, natural killer (NK) cell, macrophage, monocyte, B cell, gamma / delta T cell, thymocyte, dendritic cell, monocyte, macrophage, granulocyte, eosinophil, basophil, neutrophil, peripheral blood mononuclear cell, or natural killer T (NKT) cell. In some embodiments, the HSPC is an induced pluripotent stem cell (iPSC), embryonic stem cell (ESC), myelomonocytic cell, megakaryocyte, myeloid progenitor cell, or lymphoid progenitor cell. In some embodiments, the cell is a T cell. In some embodiments, the T cell is a cytotoxic T cell, a helper T cell, a natural killer T cell, a suppressor T cell, a CD8+ T cell, a CD4+ T cell, a CD8+ / CD4+ T cell, γδ T cell, or a T-regulatory (T-reg) cell. In some embodiments, the T cell is derived from primary cells, a T cell line, ESCs, or iPSCs.

[0267] In some embodiments, immune cells or HSPCs expressing a CAR (with or without a second polypeptide) as provided herein are obtained from a subject. Where the immune cells or HSPCs are used to treat (e.g., according to the treatment methods described in Section VIII below) the same subject from which the cells are obtained, they are referred to as autologous cells. Where the cells are obtained from a different subject, they are referred to as heterologous or allogeneic cells. Immune cells and HSPCs can be isolated from peripheral blood using techniques well known in the art, include Ficoll density gradient centrifugation followed by negative selection to remove undesired cells. In some embodiments, immune cells useful for the methods provided herein comprise allogeneic T cells, as described in, e.g., Bedoya et al., 2021, Front. Immunol.12:640082.

[0268] In some embodiments, the immune cells, e.g., T cells, can be activated and expanded using cell culture methods. For example, isolated T cells can be activated and expanded generally using methods as described, for example, in U.S. Patent Noss 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7, 144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No.2006 / 0121005. Generally, the T cells are expanded by culturing the cells in contact with a surface providing an agent that stimulates a CD3 TCR complex associated signal (e.g., an anti-CD3 antibody) and an agent that stimulates a co- stimulatory molecule on the surface of the T cells (e.g., an anti-CD28 antibody). Conditions appropriate for T cell culture are well known in the art Lin, et al. (2009) Cytotherapy 11(7):912-922 (Optimization and validation of a robust human T-cell culture method for monitoring phenotypic and polyfunctional antigen-specific CD4 and CD8 T-cell responses); 89 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 Smith, et al. (2015) Clinical & Translational Immunology 4:e31 published online 16 January 2015 (“Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement”). The orthogonal CAR-T cells are prepared by introducing the nucleic acid sequences encoding the orthogonal receptor and CAR as described below. The T cells and CAR-T cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

[0269] In some embodiments, HSPCs can be expanded (proliferated) and / or differentiated to using cell culture methods. Methods of culturing HSPCs are discussed in Section VIII.D below.

[0270] Following expression, the polypeptide compositions or polypeptide composition components can be isolated. Polypeptide compositions or polypeptide composition components can be isolated or purified in a variety of ways known in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes, 1994, Protein Purification, 3rd edition, Springer-Verlag, New York City, New York. The degree of purification necessary varies depending on the desired use. In some instances, no purification of the target-binding domain or polypeptide composition is necessary.

[0271] Methods for determining the yield or purity of a purified target-binding domain or polypeptide composition are known in the art and include, e.g., Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoretic methods (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain). VII. Pharmaceutical Compositions, Kits, and Packaging

[0272] The target-binding domains, CARs (with or without second polypeptides), polypeptide compositions, and polynucleotides described in the preceding sections, as well as the cells comprising the target-binding domains, CARs (with or without second polypeptides), polypeptide compositions, or polynucleotides, are suitable for administration in vitro or in vivo as part of a composition comprising a carrier or delivery vehicle (such as a 90 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 liquid, for example, such as phosphate-buffered saline prepared in a research lab). In some embodiments, the composition comprising a polynucleotide disclosed herein further comprises a DNA nuclease and a gRNA. DNA nucleases and gRNAs are discussed in detail in Section VI above.

[0273] In some embodiments, the compositions can also be pharmaceutically acceptable compositions. In some embodiments, the compositions comprise a target-binding domain, CAR (with or without a second polypeptide), or polypeptide composition of the present disclosure and a pharmaceutically acceptable carrier (excipient). A pharmaceutically acceptable carrier (excipient) is a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained. The carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject. The compositions may further comprise a diluent, solubilizer, emulsifier, preservative, and / or adjuvant to be used with the methods disclosed herein. Such compositions can be used, for example, in a subject with a disease, such as a cancer, or a non-malignant disease such as a genetic disease, an autoimmune disease, or graft versus host disease (GvHD), that would benefit from any of the target-binding domains, CARs, and polypeptide compositions described herein.

[0274] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21stEdition, Philip P. Gerbino, ed., Lippincott Williams & Wilkins (2006). In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for subcutaneous and / or intravenous administration. In certain embodiments, the formulation comprises an appropriate amount of a pharmaceutically- acceptable salt to render the formulation isotonic. In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as 91 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. In certain embodiments, the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington: The Science and Practice of Pharmacy, 22ndEdition, Lloyd V. Allen, Jr., ed., The Pharmaceutical Press (2014). In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and / or rate of in vivo clearance of the target-binding domain, CAR, or polypeptide composition.

[0275] In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be sterile water for injection, physiological saline solution, buffered solutions like Ringer’s solution, dextrose solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In certain embodiments, the saline comprises isotonic phosphate- buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, pharmaceutical compositions comprise a pH controlling buffer such phosphate-buffered saline or acetate- buffered saline. In certain embodiments, a composition comprising a target-binding domain, CAR, or polypeptide composition of the present disclosure can be prepared for storage by mixing the selected composition having the desired degree of purity with optional 92 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 formulation agents (see Remington: The Science and Practice of Pharmacy, 22ndEdition, Lloyd V. Allen, Jr., ed., The Pharmaceutical Press (2014)) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising a target- binding domain, CAR, or polypeptide composition of the present disclosure can be formulated as a lyophilizate using appropriate excipients. In some instances, appropriate excipients may include a cryo-preservative, a bulking agent, a surfactant, or a combination of any thereof. Exemplary excipients include one or more of a polyol, a disaccharide, or a polysaccharide, such as, for example, mannitol, sorbitol, sucrose, trehalose, and dextran 40. In some embodiments, the cryo-preservative may be sucrose or trehalose. In some embodiments, the bulking agent may be glycine or mannitol. In one example, the surfactant may be a polysorbate such as, for example, polysorbate-20 or polysorbate-80.

[0276] In certain embodiments, the pharmaceutical composition can be selected for parenteral delivery (e.g., through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneous, intra-ocular, intraarterial, intraportal, or intralesional routes). Preparations for parenteral administration can be in the form of a pyrogen-free, parenterally acceptable aqueous solution (i.e., water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media) comprising a target-binding domain, CAR, or polypeptide composition in a pharmaceutically acceptable vehicle. Preparations for parenteral administration can also include non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives are optionally present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. In certain embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In certain embodiments, hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired molecule. 93 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0277] In certain embodiments, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. Compositions for oral administration include powders or granules, suspension or solutions in water or non-aqueous media, capsules, sachets, or tables. In certain embodiments, the compositions can be formulated as a dry powder for inhalation. In certain embodiments, an inhalation solution can be formulated with a propellant for aerosol delivery. In certain embodiments, solutions can be nebulized. Pulmonary administration is further described in International Application Publication No. WO / 1994 / 020069, which describes pulmonary delivery of chemically modified proteins. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders are optionally desirable.

[0278] In certain embodiments, the compositions can be selected for topical delivery. Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners and the like are optionally necessary or desirable.

[0279] In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. For example, the pH may be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8.6.9, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some instances, the pH of the pharmaceutical composition may be in the range of 6.6-8.5 such as, for example, 7.0-8.5, 6.6-7.2, 6.8-7.2, 6.8-7.4, 7.2-7.8, 7.0-7.5, 7.5-8.0, 7.2-8.2, 7.6-8.5, or 7.8-8.3. In some instances, the pH of the pharmaceutical composition may be in the range of 5.5-7.5 such as, for example, 5.5-5.8, 5.5- 6.0, 5.7-6.2, 5.8-6.5, 6.0-6.5, 6.2-6.8, 6.5-7.0, 6.8-7.2, or 6.8-7.5. In some instances, the pH of the pharmaceutical composition may be in the range of 4.0-5.5 such as, for example, 4.0-4.3, 4.0-4.5, 4.2-4.8, 4.5-4.8, 4.5-5.0, 4.8-5.2, or 5.0-5.5.

[0280] In certain embodiments, a pharmaceutical composition can comprise an effective amount of a target-binding domain, CAR (with or without a second polypeptide), or polypeptide composition, polynucleotide encoding any thereof, or a cell comprising the target-binding domain, CAR (with or without a second polypeptide), polypeptide composition, or polynucleotide encoding any thereof, in a mixture with non-toxic excipients suitable for the manufacture of tablets. In certain embodiments, by dissolving the tablets in 94 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 sterile water or other appropriate vehicle, solutions can be prepared in unit-dose form. In certain embodiments, suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

[0281] Additional pharmaceutical compositions can be selected by one skilled in the art, including formulations involving a target-binding domain, CAR (with or without a second polypeptide), polypeptide composition, polynucleotides, or cells in sustained- or controlled- delivery formulations. In certain embodiments, techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See, for example, International Application Publication No. WO 1993 / 015722, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain embodiments, sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (see, e.g., U.S. Patent No.3,773,919; U.S. Patent No.5,594,091; U.S. Patent No.8,383,153; U.S. Patent No.4,767,628; International Application Publication No. WO 1998 / 043615, Calo et al., 2015, Eur. Polymer J.65:252-67 and European Patent No. EP 058,481), including, for example, chemically synthesized polymers, starch based polymers, and polyhydroxyalkanoates (PHAs), copolymers of L-glutamic acid and gamma ethyl-L- glutamate (Sidman et al., 1993, Biopolymers 22:547-56), poly (2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res.15:167-277; and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Hsu & Langer, 1985, J. Biomed. Materials Res. 19(4):445-60) or poly-D(-)-3-hydroxybutyric acid (European Patent No. EP0133988). In certain embodiments, sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. (See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-92; European Patent No. EP 036,676; and U.S. Patent Nos.4,619,794 and 4,615,885).

[0282] The pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, sterilization is accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization 95 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 and reconstitution. In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0283] In certain embodiments, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0284] In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, the kit can contain both a first container having a dried antibody composition and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes are included.

[0285] In certain embodiments, the effective amount of a pharmaceutical composition comprising any of the target-binding domains, CARs (with or without second polypeptides), or polypeptide compositions, or cells described herein, to be employed therapeutically depends, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, vary depending, in part, upon the molecule delivered, the indication for which a target- binding domain, CAR (with or without a second polypeptide), polypeptide composition, or cell is being used, the route of administration, and the size (body weight, body surface or organ size) and / or condition (the age and general health) of the patient. The clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. Dose selection by a clinician is discussed further below.

[0286] The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the effect desired. The actual dosage amount of a composition of the present embodiments administered to a patient or subject can be determined by physical and physiological factors, such as body 96 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 weight, the age, health, and sex of the subject, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, idiopathy of the patient, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. For example, a dose may also comprise from about 1 µg / kg / body weight to about 1000 mg / kg / body weight (this such range includes intervening doses) or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 µg / kg / body weight to about 100 mg / kg / body weight, about 5 µg / kg / body weight to about 500 mg / kg / body weight, etc., can be administered.

[0287] In certain embodiments, the target-binding domain, CAR (with or without a second polypeptide), polypeptide composition, polynucleotide, or cell disclosed herein can be administered at a dose of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, or 5 mg / kg once every other day at least four times. An exemplary treatment regime may include administration once per day, once per week, twice a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every three to 6 months. In some cases, the treatment comprises administering the target-binding domain, CAR (with or without a second polypeptide), polypeptide composition, or cell according to one of the aforementioned dosing regimens for a first period and another of the aforementioned dosing regimens for a second period. In some cases, the treatment discontinues for a period of time before the same or a different dosing regimen is resumed. For example, a patient may be on a target-binding domain, CAR (with or without a second polypeptide), polypeptide composition, or cell dosing regimen for two weeks, off for a week, on for another two weeks, and so on. Dosage regimens for target-binding domains, CARs (with or without second polypeptides), polypeptide compositions, or cells of this disclosure include 0.1 mg / kg body weight, 0.3 mg / kg body weight, 2 mg / kg body weight, 3 mg / kg body weight, or 10 mg / kg via intravenous administration, with the target-binding domain, CAR (with or without a second polypeptide), polypeptide composition, or cell being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg / kg body weight once followed by 1 mg / kg body weight every three weeks.

[0288] In still another aspect, unit dose forms comprising a target-binding domain, CAR (with or without a second polypeptide), polypeptide composition, or cell as described in this disclosure are provided. A unit dose form can be formulated for administration according to any of the routes described in this disclosure. In one example, the unit dose form is 97 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 formulated for intravenous or intraperitoneal administration. In still another aspect, pharmaceutical packages comprising unit dose forms of a target-binding domain, CAR, or polypeptide composition are provided.

[0289] In some instances, the target-binding domain, polypeptide composition, polynucleotide, or cell as described in this disclosure may be an isolated target-binding domain, polypeptide composition, polynucleotide, or cell. The term “isolated,” as used with reference to a protein (or nucleic acid), denotes that the protein (or nucleic acid) is essentially free of other cellular components with which it is associated in the natural state. The term “isolated,” as used with reference to a cell denotes that the cell is essentially free of other types of cells. In certain aspects, isolated polypeptides according to the present disclosure can consist essentially of the isolated polypeptide, and isolated cells can consist essentially of the isolated cell. The isolated polypeptide or isolated cell is preferably in a homogeneous state. Purity and homogeneity are typically determined using analytical chemistry techniques such as electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high performance liquid chromatography). In some embodiments, an isolated protein (or nucleic acid) is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.

[0290] In some instances, a polynucleotide encoding a target-binding domain, CAR (with or without a second polypeptide), or polypeptide composition may be a formulated into a virus or virus-like particle, such as described above in Section VI. In some embodiments, the polynucleotide is formulated into an Adeno-associated virus (AAV). The AAV is a small, non-enveloped virus of the Parvovirus family. AAVs can be used to target a wide variety of cells, e.g., immune cells, HSPCs, cancer cells, and other disease-related cells. Methods for making and using AAVs are discussed in, e.g., Eichhoff, Anna Marei et al. Molecular Therapy. Methods & Clinical Development vol.15211-220.16 Sep.2019; and Michels, Alexander et al. Molecular Therapy. Methods & Clinical Development vol.23334-347.1 Oct.2021.

[0291] In some instances, a polynucleotide encoding a target-binding domain, CAR (with or without a second polypeptide), or polypeptide composition may be a formulated into a lentivirus (LV) or lentivirus-like particle. In many cases, the LV or LV vector is derived from the human immunodeficiency virus (HIV) and is used in clinical applications for gene therapy. Methods for making and using LVs are discussed in, e.g., Milone, Michael C, and Una O'Doherty. Leukemia vol.32,7 (2018): 1529-1541; Poorebrahim, Mansour et al. Critical 98 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 Reviews in Clinical Laboratory Sciences vol.56,6 (2019): 393-419; and Poletti, Valentina, and Fulvio Mavilio. “Designing Lentiviral Vectors for Gene Therapy of Genetic Diseases.” Viruses vol.13,81526.2 Aug.2021.

[0292] In some instances, a polynucleotide encoding a target-binding domain, CAR (with or without a second polypeptide), or polypeptide composition may be a formulated into virus- like particles (VLPs). As used herein, the terms “virus-like particle” and “VLP” refer to a particle that resembles a virus in its structure but is not infectious because it does not contain viral genetic material. VLPs comprise viral protein(s) derived from the structural proteins of a virus. VLPs can be used to package and deliver a nucleic acid and / or a protein (e.g., a DNA nuclease), wherein the nucleic acid and / or protein may be encapsulated within the VLP. Methods for making and using VLPs are described in, for example, Garcea and Gissmann, 2004, Current Opinion in Biotechnology 15:513-7; and Hamilton, Jennifer R et al. “In vivo human T cell engineering with enveloped delivery vehicles.” Nature Biotechnology, 10.1038 / s41587-023-02085-z.11 Jan.2024; and U.S. Patent Application Publication No. 2022 / 0403379.

[0293] In some instances, a polynucleotide encoding a target-binding domain, CAR (with or without a second polypeptide), or polypeptide composition may be a formulated into nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles (LNPs). As used herein, the terms “lipid particle,” “lipid nanoparticle,” and “LNP” refer to a particle comprising a phospholipid and an ionizable lipid. An LNP may comprise additional lipid components, such as a sterol and / or a conjugated lipid, and may further comprise a nucleic acid and / or a protein (e.g., a DNA nuclease), wherein the nucleic acid and / or protein may be encapsulated within the LNP. LNPs comprise lipids and may comprise phospholipids and / or ionizable lipids. In some cases, LNPs do not contain any viral components, which helps minimize safety and immunogenicity concerns. Methods for making and using LNPs are described, for example, in Pfeiffer, A. et al. In vivo generation of human CD19-CAR T cells results in B-cell depletion and signs of cytokine release syndrome. EMBO Mol. Med.10, e9158 (2018); Smith, T. T. et al. In situ programming of leukemia-specific T cells using synthetic DNA nanocarriers. Nat. Nanotechnol.12, 813–820 (2017); U.S. Patent No. 9,737,604; and Zhang et al. “Lipid nanoparticle-mediated efficient delivery of CRISPR / Cas9 for tumor therapy,” NPG Asia Materials Volume 9, page e441 (2017). 99 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0294] In some embodiments, a polynucleotide encoding a target-binding domain, CAR (with or without a second polypeptide), or polypeptide composition may be a formulated into polymeric nanoparticles. A polymeric nanoparticle can comprise, for example, a polymersome, a dendrimer, a polymer micelle, or a nanosphere. Methods for making and using polymeric nanoparticles are discussed in, e.g., Rai, Raj et al. Polymers vol.11,4745. 25 Apr.2019, doi:10.3390 / polym11040745; and Mangraviti, Antonella et al. ACS Nano vol. 9,2 (2015): 1236-49.

[0295] In some embodiments, a polynucleotide encoding a target-binding domain, CAR (with or without a second polypeptide), or polypeptide composition may be a formulated into inorganic nanoparticles. Methods for making and using inorganic nanoparticles are discussed in, e.g., Lin, Guanyou et al. Advanced Functional Materials vol.31,5 (2021): 2007096.

[0296] In some instances, a polynucleotide encoding a target-binding domain, CAR (with or without a second polypeptide), or polypeptide composition may be a formulated into subviral dense bodies (DBs). DBs transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., 2003, Gene Therapy 10:278-84.

[0297] The target-binding domains, CARs (with or without second polypeptides), polypeptide compositions, cells, or polynucleotides disclosed herein may be used for the preparation of a kit (e.g., a diagnostic test kit or kit for the treatment of a patient). In some embodiments, kits are provided for carrying out any of the methods described herein. The kits of this disclosure may comprise a carrier container being compartmentalized to receive in close confinement one or more containers such as vials, tubes, and the like, each of the containers comprising one of the separate elements to be used in the method.

[0298] In some embodiments, one of the containers may comprise a target-binding domain or polypeptide composition as described in this disclosure that is, or can be, detectably labeled. The kit may also have containers containing buffer(s) and / or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic or fluorescent label. For example, a kit for imaging disease-related cells (e.g., cells of hematologic malignancies, leukemia cells, pre-leukemic stem cells (LSCs), and leukemic blasts) or HSPCs, in vitro or in a subject, with a target- binding domain or polypeptide composition is provided herein. In some embodiments, the kit comprises a container containing a labeled a target-binding domain or polypeptide 100 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 composition. In some embodiments, the kit comprises separate containers containing a target- binding domain or polypeptide composition and a detectable label.

[0299] A target-binding domain, CAR (with or without a second polypeptide), or polypeptide composition, or cell of the present disclosure for use in treating a cancer or a non-malignant disease associated with HCR expression (e.g., KIT, MPL, and / or FLT3 expression) may be delivered in a pharmaceutical package or kit to doctors, healthcare providers, treatment facilities, or patients having the cancer or a non-malignant disease. Such packaging is intended to improve patient convenience and compliance with the treatment plan. Typically, the packaging comprises paper (cardboard) or plastic. In some embodiments, the kit or pharmaceutical package further comprises instructions for use (e.g., for administering according to a method as described herein).

[0300] In some embodiments, a pharmaceutical package or kit comprises unit dose forms of a target-binding domain, CAR (with or without a second polypeptide), polypeptide composition, polynucleotide, or cell described herein. In some embodiments, the pharmaceutical package or kit further comprises unit dose forms of one or more of a chemotherapeutic agent, a cytotoxic agent, a radiotherapeutic agent, or an immunotherapeutic agent.

[0301] In one embodiment, the kit or pharmaceutical package comprises a target-binding domain, CAR (with or without a second polypeptide), polypeptide composition, or cell in a defined, therapeutically effective dose in a single unit dosage form or as separate unit doses. The dose and form of the unit dose (e.g., pre-filled syringe, tablet, capsule, immediate release, delayed release, etc.) can be any doses or forms as described herein.

[0302] In one embodiment, the kit or pharmaceutical package includes doses suitable for multiple days of administration, such as one week, one month, or three months.

[0303] In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, kits containing single or multi-chambered pre- filled syringes are included. In certain embodiments, kits containing one or more containers of a formulation described in this disclosure are included. 101 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 VIII. Methods of Use A. Methods of Treatment

[0304] Also provided herein are methods to treat, inhibit, or delay progression of a disease or disorder associated with cells that express at least two hematopoietic cell receptors (HCRs), such as, for example, KIT, MPL, and / or FLT3. In some embodiments, the cells are disease-related cells, e.g., cancer cells, cells of hematologic malignancies, leukemia cells, pre-LSCs, and leukemic blasts. In some embodiments, the cells are HSPCs. In some embodiments, the disease or disorder is cancer¸ e.g., a hematologic cancer. In some embodiments, the disease or disorder is a non-malignant disease.

[0305] In some embodiments, the methods herein comprise treating a subject by administering to the subject a CAR (e.g., a NaTriL / ELECTRIC CAR), a CAR and a co- expressed protein, a polypeptide composition, and / or a multivalent protein (e.g., a NaTriL / ELECTRIC-modified BiTE) as described in Sections III-V of the present disclosure to inhibit, reduce, attenuate, and / or eliminate cells that express at least two HCRs, such as, for example, KIT, MPL, and / or FLT3. In some embodiments, the method comprises eliminating healthy hematopoietic stem and progenitor cells (HSPCs), malignant HSPCs, leukemia cells, pre-leukemic stem cells (LSCs), leukemic blasts, or any combination thereof. In some embodiments, where the CAR is administered to a subject, the CAR is administered as a cell (e.g., a T cell) expressing the CAR. In some embodiments, where the CAR is co-administered to a subject or co-expressed within a subject with another protein, the CAR and the co- expressed protein are administered as a cell (e.g., a T cell) expressing both the CAR and the co-expressed protein.

[0306] In some embodiments, the method comprises administering to a subject a polypeptide composition that is linked to a cargo molecule containing a therapeutic agent. After the administration step, the polypeptide composition contacts a target cell within the subject, thereby trafficking the cargo molecule containing therapeutic agent to the target cell. Examples of therapeutic agents are discussed in detail in Section V above.

[0307] In some embodiments, the method comprises administering a multivalent protein such as, for example, a NaTriL-modified BiTe polypeptide composition. In some embodiments, the multivalent protein specifically binds to four receptors (such as, e.g., KIT, MPL, FLT3, and CD3). In some cases, the multivalent protein (e.g., NaTriL-modified BiTe) is administered to a subject where apheresis is not possible for treatment, such as, for 102 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 example, due to leukemic burden or because the apheresis may enable non-genotoxic conditioning in non-malignant indications for HSCT.

[0308] In some embodiments, the method comprises treating a subject by administering a NaTriL CAR, for example, a second-generation NaTriL CAR, to target cognate KIT, MPL, and FLT3 simultaneously. In some embodiments, the NaTriL CAR is cytotoxic against heterogenous HCR-positive leukemia cells in vitro and / or in vivo, for example in a human subject or an animal xenograft model. In some embodiments, the NaTriL CAR is cytotoxic against healthy HSPCs, LSCs, and / or leukemic blasts. In some embodiments, the NaTriL CAR is administered to a subject in a bridge-to-transplant setting, and the NaTriL CAR can target and / or deplete chemoresistant leukemic stem cells and endogenous HSPCs to prepare the subject for allogeneic HSC transplantation (HSCT). In some cases, administration of the NaTriL CAR is administered to the subject prior to receiving transplant. In some cases, administration of a NaTriL CAR can mitigate risks for long term on-target toxicities to healthy HSPCs in the transplant.

[0309] In some embodiments, the NaTriL CAR is co-administered to a subject or co- expressed within a subject with a safety switch protein. The safety switch protein can be induced (for example, by binding to a chemical agent) to cause apoptosis of the cells expressing the NaTriL CAR such that the NaTriL CAR is eliminated from the subject. Safety switch polypeptides and their co-expression with NaTriL CARs are discussed in Section IV.A above.

[0310] In some embodiments, the NaTriL CAR is co-administered to a subject or co- expressed within a subject with a targeting polypeptide that can localize the NaTriL CAR- expressing cells to the bone marrow. In some embodiments, the NaTriL CAR is co- administered or co-expressed with a polypeptide derived from CXCR4 (e.g., SEQ ID NO: 20), CXCL12α domain (e.g., SEQ ID NO: 29) and / or a CXCL12β domain (e.g., SEQ ID NO: 30).

[0311] As used throughout this disclosure, subject can be a vertebrate, more specifically a mammal (e.g., a human, monkey, horse, cat, dog, cow, pig, sheep, camel, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and any other animal. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, “patient” or “subject” may be used interchangeably and includes human and veterinary subjects. The target-binding domain, CAR (with or 103 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 without a second polypeptide), polypeptide composition, or cell described herein are useful for treating cancer in humans, including, without limitation, pediatric and geriatric populations, and in animals, e.g., veterinary applications. In some embodiments, the subject is a human. In some embodiments, the subject has or is suspected to have cancer or a non- malignant disease associated with the expression of HCRs, such as, KIT, MPL, and / or FLT3. In some embodiments, the subject is diagnosed with a cancer. In some embodiments, a target- binding domain, CAR (with or without a second polypeptide), polypeptide composition, or is useful for treating cancer in humans, including, without limitation, hematologic malignancies such as pediatric acute myeloid leukemia (AML) and including acute lymphoblastic leukemia (ALL). In some embodiments, the subject is a human.

[0312] In some embodiments, the subject has cancer. As used herein the terms “cancer” and “tumor” are used to indicate malignant tissue. The term “cancer” is also used to refer to the disease associated with the presence of malignant tumor cells in an individual, and the term “tumor” is used herein to refer to a plurality of cancer cells that are physically associated with each other. Cancer cells are malignant cells that give rise to cancer, and tumor cells are malignant cells that can form a tumor and thereby give rise to cancer. The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. The term also encompasses a circulating tumor cell.

[0313] In some embodiments, the subject has a hematologic malignancy. In some embodiments, the cancer is leukemia, acute myeloid leukemia (AML), pediatric AML (pAML), acute lymphoblastic leukemia (ALL), B cell acute lymphoblastic leukemia (B- ALL), B cell chronic lymphocytic leukemia (B-CLL), hairy cell leukemia, B-cell prolymphocytic leukemia, mixed phenotype acute leukemia (MPAL), juvenile myelomonocytic leukemia (JMML), myelodysplastic syndrome (MDS), non-Hodgkin’s B cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, follicle center lymphoma, mantle cell lymphoma, Burkitt lymphoma (BL), Waldenstrom macroglobulinemia, Hodgkin lymphoma, multiple myeloma (MM), T cell leukemia, anaplastic large cell lymphoma, peripheral T cell lymphoma, cutaneous T cell lymphoma, extranodal natural killer (NK) / T cell lymphoma, Epstein-Barr virus associated T cell lymphoma, or T cell acute lymphoblastic leukemia.

[0314] In some embodiments, the subject has a non-malignant disease, such as a genetic disease, an autoimmune disease, an infectious disease, a blood disorder, a disease of another 104 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 organ system, or graft versus host disease (GvHD). In some embodiments, the non-malignant disease is a blood disease, such as sickle cell anemia, thalassemia, or hemophilia. In some embodiments, the non-malignant disease is an infectious disease, such as immunodeficiency virus (HIV). In some embodiments, the non-malignant disease is a neurologic disease, such as metachromatic leukodystrophy (MLD). In some embodiments, the non-malignant disease is a cardiovascular disease, such as Danon disease. In some embodiments, the non-malignant disease is a renal disease, such as cystinosis.

[0315] In some embodiments, methods of treatment can include administering to a subject compositions comprising, consisting of, or consisting essentially of one or more target- binding domains, CARs (with or without second polypeptides), polypeptide compositions, polynucleotides, or cells (such as those described in Sections III-VI above) as a monotherapy or part of a combinatorial therapy regime (a treatment schedule comprising more than one treatment modality). In some embodiments, methods of treatment comprise conditioning therapy, wherein a target-binding domain, CAR (with or without a second polypeptide), polypeptide composition, or cell disclosed herein is administered to eliminate a subject’s endogenous cells, e.g., HSPCs, in preparation for (i.e., before) transplant of donor cells, e.g., HSPCs, in the subject.

[0316] Disease-related cells (e.g., cancer cells, cells of hematologic malignancies, leukemia cells, pre-LSCs, and leukemic blasts) or HSPCs expressing two or more HCRs may be reduced, inhibited, or killed by the binding of a target-binding domain, CAR (with or without a second polypeptide), polypeptide composition, or cell disclosed herein. In one aspect, provided is a method of treating a subject with a disease or a disorder, e.g., cancer or non- malignant disease, the method comprising administering to the patient a therapeutically effective amount of a composition comprising (or consisting of or consisting essentially of) a target-binding domain, CAR (with or without a second polypeptide), polypeptide composition, polynucleotides, or cell disclosed herein. In some embodiments, the methods comprise administering to a subject a therapeutically effective amount of a composition comprising an isolated target-binding domain or polypeptide composition described herein. In some embodiments, the methods comprise administering to a subject a therapeutically effective amount of a composition comprising cells comprising a CAR (with or without a second polypeptide) described herein. The composition may further comprise a pharmaceutically acceptable carrier as described above in Section VII above. 105 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403

[0317] Methods of treatment as described herein can reduce one or more symptoms of a disease or disorder (e.g., cancer or a non-malignant disease) by reducing, inhibiting, or killing disease-related cells (e.g., cancer cells, cells of hematologic malignancies, leukemia cells, pre-LSCs, and leukemic blasts) or HSPCs that express two or more HCRs as a result of administration of compositions comprising a target-binding domain, CAR, or polypeptide composition disclosed herein, or cells comprising the target-binding domain, CAR, or polypeptide composition.

[0318] “Treat,” “treatment,” and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. “Treating” or “treatment” may refer to any indicia of success in the treatment or amelioration of a disease or disorder. Treating or treatment of any disease or disorder refers to ameliorating a disease or disorder that exists in a subject or any one or more symptoms thereof. The term ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a cancer or a non- malignant disease, lessening in the severity or progression, promoting remission or durations of remission, or curing thereof. Thus, treating or treatment includes ameliorating at least one physical parameter or symptom. Treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. “Treating” or “treatment” includes the administration of an agent to impede growth of a cancer, to do one or more of the following: cause a cancer to shrink by weight or volume (i.e., shrink from a first weight or volume to a second weight or volume, wherein the second weight or volume is less than the first), delay or prevent metastasis, extend the expected survival time of the subject, or extend the expected time to progression of the tumor, or the like. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for treating a cancer in a subject by administering a pharmaceutical composition as described in this disclosure is considered to be a treatment if there is a 10% reduction in one or more symptoms of the cancer in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. It is 106 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

[0319] “Treating” or “treatment” also includes the administration of an agent to impede progression of a disease or disorder, to do one or more of the following: cause a disease or disorder to decrease progression, delay or prevent a disease or disorder, extend the expected survival time of the subject, or extend the expected time to progression of the disease or disorder, or the like. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or disorder or symptom of the disease or disorder. For example, a method for treating a disease or disorder in a subject by administering a pharmaceutical composition as described in this disclosure is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease or disorder in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, disorder, or symptoms of the disease or disorder.

[0320] The term “administer,” as used herein, refers to a method of delivering agents, compounds, or compositions to the desired site of biological action. The pharmaceutical compositions (e.g., as described above) are prepared for administration in a number of ways, including but not limited to injection, ingestion, transfusion, implantation, or transplantation, depending on whether local or systemic treatment is desired, and on the area to be treated. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art. The compositions are administered via any of several routes of administration, including topical, oral, parenteral, intravenous, intra-articular, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneous, intraarterial, intraportal, intracavity, intralesional, transdermal, intradermal, intrahepatical, intrathecal, intracranial, rectal, transmucosal, intestinal, intra-ocular or ocular, otic, nasal, inhalation, or intrabronchial delivery, or any other method known in the art. In some embodiments, the target-binding domain, CAR (with or without a second polypeptide), polypeptide composition, or cell disclosed herein is administered intravenously, or through local injection. In certain embodiments, administration is by bolus injection, continuously by 107 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 infusion, by sustained release system, or by implantation device. In certain embodiments, individual elements of a combination therapy (as discussed below) may be administered by different routes. In certain embodiments, the composition can be administered locally, e.g., during surgery or topically. Optionally local administration is via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration. Further discussion on routes of administration and formulations is provided in Section VII above.

[0321] In some instances, a target-binding domain or polypeptide composition disclosed herein can be administered as an isolated protein. In some instances, a polynucleotide disclosed herein can be administered via virus-like particles (VLPs), nanoparticles (e.g., lipid nanoparticles), or by subviral dense bodies (DBs), which are discussed above in Section VII.

[0322] As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount of a therapeutic composition that, when administered to a subject, is effective to treat a disease or disorder such that the symptoms of the disease or disorder are ameliorated, or the likelihood of the disease or disorder developing or progressing is decreased. A therapeutically effective amount is not, however, a dosage so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. A suitable dose of a therapeutic composition as described herein, which dose is capable of treating a disease or disorder in a subject, can depend on a variety of factors including the particular therapeutic composition used and whether it is used concomitantly with other therapeutic agents. For example, a different dose of a polypeptide composition disclosed herein may be required to treat a subject with cancer or non-malignant disease as compared to the dose of a cell comprising a CAR disclosed herein required t...

Claims

Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 WHAT IS CLAIMED IS:

1. A chimeric antigen receptor (CAR) protein, comprising: a first polypeptide comprising a target-binding domain comprising a first cytokine domain that binds to a first hematopoietic cytokine receptor (HCR) and a second cytokine domain that binds to a second HCR, wherein the first and the second cytokine domains each bind to a different HCR; a transmembrane domain; and a first intracellular signaling domain.

2. The CAR of claim 1, wherein the target-binding domain further comprises a third cytokine domain that binds to a third HCR, wherein the third HCR is different from the first HCR and the second HCR.

3. The CAR of any one of claims 1 or 2, wherein the first, second, and third cytokine domains are in any order in relation to each other.

4. The CAR of any one of claims 1-3, wherein the first, second, and third cytokine domains each bind to one of mast / stem cell growth factor receptor Kit (KIT; CD117), myeloproliferative leukemia virus oncogene (MPL; CD110), or FMS-like tyrosine kinase 3 (FLT3; CD135).

5. The CAR of any one of claims 1-4, wherein the first, second, and third cytokine domains each comprise one of a HCR-binding portion of stem cell factor (SCF), a HCR-binding portion of thrombopoietin (TPO), or a HCR-binding portion of FMS-like tyrosine kinase 3 ligand (FLT3LG), wherein the HCR-binding portion of SCF binds to KIT, the HCR-binding portion of TPO binds to MPL, and the HCR-binding portion of FLT3LG binds to FLT3.

6. The CAR of any one of claims 1-5, wherein the target-binding domain comprises a first linker between the first and second cytokine domains and a second linker between the second and third cytokine domains.

7. The CAR of any one of claims 1-6, wherein the CAR further comprises a hinge region between the target-binding domain and the transmembrane domain. 209 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 8. The CAR of any one of claims 1-6, wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8α transmembrane domain.

9. The CAR of any one of claims 1-8, wherein the hinge domain comprises a CD28 hinge domain, a CD8α hinge domain, an IgG4 hinge domain, a CH2 domain, or a CH3 domain.

10. The CAR of any one of claims 1-9, wherein the CAR comprises a second intracellular signaling domain.

11. The CAR of any one of claims 1-10, wherein the first intracellular signaling domain comprises a CD28 signaling domain or a 4-1BB signaling domain.

12. The CAR of any one of claims 1-11, wherein the second intracellular signaling domain comprises a CD3zeta signaling domain.

13. The CAR of any one of claims 1-12, wherein the first polypeptide comprises an N-terminal CD8α peptide linked to the target-binding domain.

14. A polypeptide composition, comprising a first polypeptide comprising a target-binding domain comprising a first cytokine domain that binds to a first hematopoietic cytokine receptor (HCR), and a second cytokine domain that binds to a second HCR; wherein the first and the second cytokine domains each bind to a different HCR, and the first polypeptide is linked to a therapeutic agent and / or an imaging agent.

15. The polypeptide composition of claim 14, wherein the target-binding domain further comprises a third cytokine domain that binds to a third HCR, wherein the third HCR is different from the first HCR and the second HCR.

16. The polypeptide composition of claim 14 or 15, wherein the first, second, and third cytokine domains are in any order in relation to each other.

17. The polypeptide composition of any one of claims 14-16, wherein the first, second, and third cytokine domains each bind to one of KIT, MPL, or FLT3.

18. The polypeptide composition of any one of claims 14-17, wherein the first, second, and third cytokine domains each comprise one of a HCR-binding portion of stem cell factor (SCF), a HCR-binding portion of thrombopoietin (TPO), or a HCR-binding portion of 210 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 FMS-like tyrosine kinase 3 ligand (FLT3LG), wherein the HCR-binding portion of SCF binds to KIT, the HCR-binding portion of TPO binds to MPL, and the HCR-binding portion of FLT3LG binds to FLT3.

19. The polypeptide composition of any one of claims 14-18, wherein the target- binding domain comprises a first linker between the first and second cytokine domains and a second linker between the second and third cytokine domains.

20. The polypeptide composition of any one of claims 14-19, wherein the therapeutic agent comprises a protein or a small molecule.

21. The polypeptide composition of claim 20, wherein the protein is a protein toxin or apoptotic protein.

22. The polypeptide composition of claim 21, wherein the protein toxin is saporin or diphtheria toxin.

23. The polypeptide composition of claim 20, wherein the protein is an antibody.

24. The polypeptide composition of claim 23, wherein the antibody is a T cell engager or a natural killer cell engager.

25. The polypeptide composition of claim 23, wherein the antibody is a monoclonal antibody.

26. The polypeptide composition of claim 25, wherein the monoclonal antibody is an anti-CD3 antibody.

27. The polypeptide composition of claim 20, wherein the small molecule is a cytotoxic agent or a radioactive moiety.

28. The polypeptide composition of claim 27, wherein the cytotoxic agent is a small molecule.

29. The polypeptide composition of claim 28, wherein the cytotoxic agent is amanitin, pyrrolobenzodiazepine, monomethyl auristatin E (MMAE), Duocarmycin SA (DUO), PNU-159682 (PNU), or SGD-1882 (PBD). 211 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 30. The polypeptide composition of any one of claims 14-29, wherein the therapeutic agent comprises a cargo molecule containing the therapeutic agent.

31. The polypeptide composition of claim 30, wherein the cargo molecule comprises a nanoparticle, a virus or virus-like particle, or a dense body.

32. The polypeptide composition of claim 31, wherein the nanoparticle is a lipid nanoparticle, a polymeric nanoparticle, or inorganic nanoparticle.

33. The polypeptide composition of claim 30, wherein the therapeutic agent in the cargo molecule comprises a small molecule or a gene editing system component.

34. The polypeptide composition of claim 27, wherein the imaging agent is a fluorophore, a radioactive label, or a heavy metal particle.

35. A nucleic acid encoding the first polypeptide of any one of claims 1-29.

36. A nucleic acid encoding the CAR of any one of claims 1-13.

37. A construct comprising the nucleic acid of claim 35 or 36.

38. The construct of claim 37, further comprising a nucleic acid encoding a second polypeptide.

39. The construct of claim 38, wherein a nucleic acid sequence encoding a P2A peptide is located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide.

40. The construct of claim 38 or 39, wherein the second polypeptide comprises a targeting polypeptide, a chimeric costimulatory receptor (CCR), or a fusion protein comprising a human caspase 9 polypeptide attached to a modified human FK506-binding protein (FKB) polypeptide.

41. The construct of claim 40, wherein the targeting polypeptide is CXCR4.

42. The construct of claim 40, wherein the CCR comprises an extracellular target- binding domain that binds specifically to CXCR4. 212 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 43. The construct of claim 42, wherein the extracellular target-binding domain of the CCR comprises a polypeptide comprising an antigen-binding portion of an anti-CXCR4 antibody or a natural ligand of CXCR4.

44. The construct of claim 43, wherein the antigen-binding portion of an anti- CXCR antibody comprises a light chain portion of MDX1388, a heavy chain portion of MDX1388, or an scFv portion of MDX1388.

45. The construct of claim 43, wherein the natural ligand of CXCR4 comprises a polypeptide derived from CXCL12α or CXCL12β.

46. A vector comprising the nucleic acid of claim 35 or 36 or the construct of any one of claims 37-41.

47. The vector of claim 46, wherein the vector is delivered to cells via lipid nanoparticles or virus-like particles.

48. The vector of claim 46, wherein the lipid nanoparticles or the virus-like particles comprise a DNA nuclease, and optionally, a guide RNA (gRNA).

49. A cell comprising the CAR of any one of claims 1-13, the polypeptide composition of any one of claims 14-34, the nucleic acid of claim 35 or 36, the construct of any one of claims 37-41, or the vector of any one of claims 46-48.

50. The cell of claim 49, wherein the cell is a human T cell, natural killer (NK) cell, macrophage, monocyte, B cell, gamma / delta T cell, natural killer T (NKT) cells induced pluripotent stem cell, hematopoietic stem cell, myeloid progenitor cell, or lymphoid progenitor cell.

51. A pharmaceutical composition comprising the vector of any one of claims 46- 48 or the cell of claim 49 or 50, and a pharmaceutically acceptable carrier.

52. A method of expressing a recombinant polypeptide in a cell, the method comprising: providing the cell; and 213 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 introducing the nucleic acid of claim 35 or 36, the construct of any one of claims 37- 40, the vector of any one of claims 46-48, or the pharmaceutical composition of claim 51 to the cell, thereby producing a cell expressing the recombinant polypeptide.

53. The method of claim 52, wherein the cell is a human cell, optionally a human T cell, natural killer (NK) cell, macrophage, monocyte, B cell, gamma / delta T cell, natural killer T (NKT) cells induced pluripotent stem cell, hematopoietic stem cell, myeloid progenitor cell, or lymphoid progenitor cell.

54. The method of claim 53, wherein the cell is obtained from a cell donor.

55. The method of claim 54, wherein the cell donor is a patient 56. The method of any one of claims 52-55, further comprising introducing the cell expressing the polypeptide into the patient.

57. A method of treating a disease in a patient, the method comprising administering to the patient an effective amount of the pharmaceutical composition of claim 51, wherein the disease in the patient is characterized by cells that express at least one of mast / stem cell growth factor receptor Kit (KIT; CD117), myeloproliferative leukemia virus oncogene (MPL; CD110), or FMS-like tyrosine kinase 3 (FLT3; CD135).

58. The method of claim 57, wherein the disease is a cancer or a non-malignant disease.

59. The method of claim 58, wherein the cancer is leukemia, acute myeloid leukemia (AML), B cell acute lymphoblastic leukemia (B-ALL), B cell chronic lymphocytic leukemia (B-CLL), hairy cell leukemia, B-cell prolymphocytic leukemia, mixed phenotype acute leukemia (MPAL), juvenile myelomonocytic leukemia (JMML), myelodysplastic syndrome (MDS), non-Hodgkin’s B cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, follicle center lymphoma, mantle cell lymphoma, Burkitt lymphoma (BL), Waldenstrom macroglobulinemia, Hodgkin lymphoma, multiple myeloma (MM), T cell leukemia, anaplastic large cell lymphoma, peripheral T cell lymphoma, cutaneous T cell lymphoma, extranodal natural killer (NK) / T cell lymphoma, Epstein-Barr virus associated T cell lymphoma, or T cell acute lymphoblastic leukemia. 214 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 60. The method of claim 58, wherein the non-malignant disease is a genetic disease, an autoimmune disease, or graft versus host disease (GvHD).

61. The method of any one of claims 57-60, wherein the pharmaceutical composition is administered via intravenous injection, subcutaneous injection, or direct injection into a diseased tissue.

62. The method of any one of claims 57-61, wherein the pharmaceutical composition comprises the cell of claim 49 or 50, and the cell is an allogeneic cell.

63. The method of any one of claims 57-61, wherein the pharmaceutical composition comprises the cell of claim 49 or 50, and the cell is an autologous cell.

64. The method of any one of claims 57-63, wherein the method further comprises administering a combination therapy.

65. The method of claim 64, wherein the combination therapy comprises at least one of surgery, radiation therapy, or administration of a therapeutically effective amount of a therapeutic agent.

66. The method of claim 65, wherein the therapeutic agent is a chemotherapeutic agent, a photosensitizer, an immunosuppressive agent, or small molecule that binds to a fusion protein comprising a human caspase 9 polypeptide attached to a modified human FK506-binding protein (FKB) polypeptide.

67. The method of any one of claims 57-66, further comprising, after the administering step, transplanting of an allogeneic hematopoietic stem cell graft or a modified hematopoietic stem cell graft.

68. A diagnostic composition comprising a polypeptide composition of any one of claims 14-34 and a pharmaceutically acceptable carrier.

69. A method for detecting presence of cells expressing HCRs in a biological sample, comprising: contacting the biological sample with the diagnostic composition of claim 68, and detecting an amount of binding of the polypeptide composition as an indicator of the presence of the cells expressing HCRs; 215 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 wherein the cells expressing HCRs express at least one of mast / stem cell growth factor receptor Kit (KIT; CD117), myeloproliferative leukemia virus oncogene (MPL; CD110), or FMS-like tyrosine kinase 3 (FLT3; CD135).

70. A method for editing a target gene in a cell, the method comprising contacting the target cell with the polypeptide composition of any one of claims 14-19, wherein (a) the target cell expresses at least two HCRs; and (b) the polypeptide composition is linked to a therapeutic agent, wherein the therapeutic agent comprises a cargo molecule containing the therapeutic agent and one or more components of a gene editing system capable of editing the target gene in the target cell.

71. The method of claim 70, wherein the contacting of the target cell with the polypeptide composition results in introduction of one or more components of a gene editing system into the target, and thereby results in a genetic modification of the target gene in the target cell.

72. The method of any one of claims 70-71, wherein the one of more components of the gene editing system comprise (i) a DNA nuclease component and (ii) a gRNA with a sequence of at least 70% identity of the target gene.

73. The method of any one of claims 70-72, wherein the cargo molecule comprises a virus-like particle (VLP), a lipid nanoparticle (LNP), an adeno-associated virus (AAV), or a lentivirus (LV).

74. The method of any one of claims 70-73, wherein the genetic modification comprises an insertion, a deletion, or a substitution of one or more nucleotides in the target gene.

75. The method of any one of claims 70-74, wherein the genetic modification disrupts expression of a polypeptide encoded by the target gene, results in expression of a truncated version of the polypeptide encoded by the target gene, replaces all or a portion of the target gene with a transgene, or produces a polynucleotide encoding a fusion protein at the site of the target gene. 216 US2008310953339Attorney Docket No.: 079445-1484474-013910WO Client Reference No.: S23-403 76. A method for culturing a population of hematopoietic stem and progenitor cells (HSPCs), the method comprising contacting a population of HSPCs with the polypeptide composition of any one of claims 14-19, wherein the HSPCs in the population increase in number and / or differentiate into one or more specific cell types.

77. The method of claim 76, wherein the polypeptide composition is added to cell culture media at an amount ranging from 10 pg / mL to 10 μg / mL.

78. The method of any one of claims 74-76, wherein the increased number of cells and / or the differentiated cells are administered to a patient in need of HSPC transplantation. 217 US2008310953339

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