Membrane-anchored cytokines, engineered immune cells, and their uses

Abstract

Systems comprising a nucleic acid molecule, a polypeptide, a protein, a cell, or a cytokine, optionally a targeting moiety (e.g., a chimeric antigen receptor), and an anchor structure capable of binding to a cell surface (e.g., an engineered immune cell), as well as methods for preparing them and using them to treat a disease or disorder, are provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of International Patent Application No. PCT / CN2023 / 071969, filed January 12, 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Lymphocyte depletion, currently part of CAR (chimeric antigen receptor) T-cell therapy, may involve administering cyclophosphamide or fludarabine to patients before infusion of CAR-T cells. This process prevents autologous lymphocytes from attacking the transferred CAR-T cells, prolonging the effectiveness of immune cell therapy. However, the process of removing many autologous lymphocytes makes the medical procedure more complicated, increases the patient's medical burden, and increases the risk of infection after treatment.

[0003] Some researchers have attempted to co-administer various cytokines to stimulate proliferation and help improve immune cell survival in vivo, but the drawback of this approach is that it may stimulate other unwanted lymphocytes to proliferate, increasing the risk of cytokine release syndrome (CRS).

[0004] There is a need in the art for new agents that can target and improve the proliferation and survival of selected cells. Summary of the Invention

[0005] Disclosed herein, in some embodiments, is a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising a signal peptide, a cytokine peptide, and a non-peptide anchor binding signal, wherein the cytokine peptide comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

[0006] Disclosed herein, in some embodiments, is an immune cell comprising an exogenous nucleic acid sequence encoding a polypeptide comprising a cytokine peptide and a non-peptide anchor binding signal, wherein the cytokine peptide comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

[0007] Disclosed herein, in some embodiments, is an immune cell comprising an exogenous nucleic acid sequence encoding a polypeptide comprising a cytokine peptide and a non-peptide anchor-binding signal, wherein the cytokine peptide is at least a portion of IL-12p40 or at least a portion of IL-12p35, and the exogenous nucleic acid sequence does not encode both IL-12p40 and IL-12p35, and the immune cell does not comprise a PDE5-derived stimulus response element (SRE). Optionally, the immune cell does not comprise a stimulus response element (SRE), and the cytokine peptide is IL-12p40.

[0008] In some embodiments, disclosed herein are immune cells comprising a protein comprising a cytokine peptide and a non-peptide anchor, wherein the cytokine peptide comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. Optionally, the protein is processed from a polypeptide comprising the cytokine peptide and a non-peptide anchor binding signal, and the non-peptide anchor binding is replaced with the non-peptide anchor during protein processing. Optionally, the polypeptide is encoded by an exogenous nucleic acid sequence. Optionally, the polypeptide or protein comprises a signal peptide. Optionally, the signal peptide, cytokine peptide, and non-peptide anchor binding signal are operably linked from the N-terminus of the polypeptide to the C-terminus of the polypeptide. Optionally, the non-peptide anchor binding signal comprises a glycolipid binding signal.

[0009] Disclosed herein, in some embodiments, is a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising a cytokine peptide and a peptide anchor, wherein the cytokine peptide comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

[0010] Disclosed herein, in some embodiments, is a cell comprising a polypeptide comprising a cytokine peptide and a peptide anchor, wherein the cytokine peptide comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

[0011] In any one of the preceding or related embodiments, the polypeptide comprises a signal peptide. Optionally, the signal peptide, cytokine peptide, and peptide anchor are operably linked in a direction from the N-terminus of the polypeptide to the C-terminus of the polypeptide. Optionally, the peptide anchor comprises a transmembrane peptide sequence. Optionally, the signal peptide comprises a CD4 signal peptide, a Cd8α signal peptide, a CD28 signal peptide, a CD33 signal peptide, a CD137 (4-1BB) signal peptide, an IL-2 signal peptide, an IgE signal peptide, an IgG1 signal peptide, a GM-CSF signal peptide, an HLA-A signal peptide, an HLA signal peptide, a TCR signal peptide, or a β2M signal peptide, or a combination thereof. Optionally, the signal peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 2-7. Optionally, the signal peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 52-57. Optionally, the signal peptide is a naturally occurring signal peptide of a wild-type cytokine. Optionally, the signal peptide comprises an IL-2 signal peptide, an IL-4 signal peptide, an IL-7 signal peptide, an IL-9 signal peptide, an IL-10 signal peptide, an IL-12p40 signal peptide, an IL-15 signal peptide, an IL-18 signal peptide, an IL-21 signal peptide, an IL-23 signal peptide, an IL-27 signal peptide, an IL-36γ signal peptide, an IL-23p19 signal peptide, or an IL-1α signal peptide, or a functional variant thereof. Optionally, the signal peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO:113 or 114.Optionally, the signal peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 58 or 59. Optionally, the cytokine peptide comprises at least a portion of IL-2 or a variant thereof. Optionally, the cytokine peptide comprises at least a portion of IL-4 or a variant thereof. Optionally, the cytokine peptide comprises at least a portion of IL-7 or a variant thereof. Optionally, the cytokine peptide comprises at least a portion of IL-9 or a variant thereof. Optionally, the cytokine peptide comprises at least a portion of IL-10 or a variant thereof. Optionally, the cytokine peptide comprises at least a portion of IL-18 or a variant thereof. Optionally, the cytokine peptide comprises at least a portion of IL-23 or a variant thereof. Optionally, the cytokine peptide comprises at least a portion of IL-27 or a variant thereof. Optionally, the cytokine peptide comprises at least a portion of IL-36γ or a variant thereof. Optionally, the cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the cytokine peptide comprises at least a portion of IL-1α or a variant thereof. Optionally, the variant of the cytokine peptide comprises an alteration, substitution, deletion, addition, or chemical modification of one or more amino acids, one or more unnatural amino acids, or any combination thereof. Optionally, the cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-104, 107, or 109-112. Optionally, the signal peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-154, 157, or 159-164.

[0012] In any one of the preceding or related embodiments, the polypeptide or protein further comprises a peptide linker. Optionally, the peptide linker connects the cytokine peptide to the peptide anchor. Optionally, the peptide linker connects the cytokine peptide to a non-peptide anchor binding signal. Optionally, the peptide linker connects the peptide anchor to a cleavable linker. Optionally, the peptide linker connects the non-peptide anchor binding signal to the cleavable linker.

[0013] In any of the above or related embodiments, the cytotoxicity of the immune cells is increased compared to comparable immune cells that do not comprise an exogenous nucleic acid sequence encoding a polypeptide comprising a cytokine peptide and a non-peptide anchor binding signal, that do not comprise a nucleic acid sequence encoding a polypeptide comprising a signal peptide, a cytokine peptide, and a non-peptide anchor binding signal, that do not comprise a nucleic acid sequence encoding a polypeptide comprising a cytokine peptide and a peptide anchor, or that do not comprise a protein comprising a cytokine peptide and (i) a non-peptide anchor or (ii) a peptide anchor. Optionally, the increase in cytotoxicity of the immune cells is measured by an in vitro cytotoxicity assay described in Examples A-D. Optionally, the increase in cytotoxicity of the immune cells is measured in vitro or in vivo. Optionally, the increase in cytotoxicity of the immune cells is at least about 5%, 10%, 20%, 30%, 40%, 50%, or more.

[0014] In any of the above or related embodiments, the population of immune cells proliferates for a longer period of time compared to a comparable population of immune cells that does not comprise an exogenous nucleic acid sequence encoding a polypeptide comprising a cytokine peptide and a non-peptide anchor binding signal, does not comprise a nucleic acid sequence encoding a polypeptide comprising a signal peptide, a cytokine peptide, and a non-peptide anchor binding signal, does not comprise a nucleic acid sequence encoding a polypeptide comprising a cytokine peptide and a peptide anchor, or does not comprise a protein comprising a cytokine peptide and (i) a non-peptide anchor or (ii) a peptide anchor. Optionally, the proliferation of the population of immune cells is measured by an in vitro immune cell proliferation assay as described in Examples A-E. Optionally, the proliferation of the population of immune cells is measured in vitro or in vivo. Optionally, the proliferation of the population of immune cells persists for at least about 5%, 10%, 20%, 30%, 40%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or more than the comparable population of immune cells.

[0015] Disclosed herein, in some embodiments, is a nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor binding signal or (ii) a first peptide anchor, and the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor binding signal or (ii) a second peptide anchor, wherein the first cytokine peptide is a pro-inflammatory cytokine peptide and the second cytokine peptide is an anti-inflammatory cytokine peptide.

[0016] Disclosed herein, in some embodiments, is a system comprising a first nucleic acid sequence and a second nucleic acid sequence different from the first nucleic acid sequence, wherein the first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and either (i) a first non-peptide anchor binding signal or (ii) a first peptide anchor, and the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and either (i) a second non-peptide anchor binding signal or (ii) a second peptide anchor, wherein the first cytokine peptide is a pro-inflammatory cytokine peptide and the second cytokine peptide is an anti-inflammatory cytokine peptide.

[0017] In some embodiments, the present disclosure provides a cell comprising a first protein and a second protein, wherein the first protein comprises a first cytokine peptide and (i) a first non-peptide anchor or (ii) a first peptide anchor, and the second protein comprises a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor, wherein the first cytokine peptide is a pro-inflammatory cytokine peptide and the second cytokine peptide is an anti-inflammatory cytokine peptide. Optionally, the first protein is processed from a first polypeptide comprising the first cytokine peptide and (i) a first non-peptide anchor binding signal or (ii) a first peptide anchor, wherein the first non-peptide anchor binding signal is replaced by the first non-peptide anchor during protein processing.

[0018] In any one of the aforementioned or related embodiments, the anti-inflammatory cytokine peptide comprises at least a portion of IL-4, IL-10, or IL-27, or a variant thereof, and the pro-inflammatory cytokine peptide comprises at least a portion of IL-2, IL-7, IL-9, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. Optionally, the anti-inflammatory cytokine peptide comprises at least a portion of IL-4 or a variant thereof, and the pro-inflammatory cytokine peptide comprises at least a portion of IL-2, IL-7, IL-9, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. In some cases, the anti-inflammatory cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 101, 104, or 109, and the pro-inflammatory cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 100, 102, 103, 105-108, or 110-112. In some cases, the anti-inflammatory cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 151, 163, 154, 164, or 159, and the pro-inflammatory cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NO: 150, 152, 153, 155-158, or 160-162.In some cases, the anti-inflammatory cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 101, and the pro-inflammatory cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 100, 102, 103, 105-108, or 110-112. In some cases, the anti-inflammatory cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 151 or 163, and the pro-inflammatory cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 150, 152, 153, 155-158, or 160-162.

[0019] Disclosed herein, in some embodiments, is a nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor binding signal or (ii) a first peptide anchor, and the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor binding signal or (ii) a second peptide anchor, and each of the first cytokine peptide and the second cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. Optionally, the first cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-107 or 109-112. Optionally, the first cytokine peptide is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-157 or 159-164. Optionally, the second cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-107 or 109-112. Optionally, the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-157 or 159-164.

[0020] Disclosed herein, in some embodiments, is a cell comprising a first protein and a second protein, wherein the first protein comprises a first cytokine peptide and (i) a first non-peptide anchor or (ii) a first peptide anchor, and the second protein comprises a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor, and each of the first cytokine peptide and the second cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. Optionally, the first cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-107 or 109-112. Optionally, the first cytokine peptide is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-157 or 159-164. Optionally, the second cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-107 or 109-112. Optionally, the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-157 or 159-164.

[0021] Disclosed herein, in some embodiments, is a system comprising a first nucleic acid sequence and a second nucleic acid sequence that is different, wherein the first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and either (i) a first non-peptide anchor binding signal or (ii) a first peptide anchor, and the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and either (i) a second non-peptide anchor binding signal or (ii) a second peptide anchor, wherein each of the first cytokine peptide and the second cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. Optionally, the first cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-107 or 109-112. Optionally, the first cytokine peptide is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-157 or 159-164. Optionally, the second cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-107 or 109-112. Optionally, the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-157 or 159-164.

[0022] In any one of the aforementioned or related embodiments, the first cytokine peptide comprises at least a portion of IL-4 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-2, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. In some cases, the first cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 101, and the second cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 100, 102-107, or 109-112. In some cases, the first cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 151 or 163, and the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 150, 152-157, 159-162, or 164.

[0023] Disclosed herein, in some embodiments, is a nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor binding signal or (ii) a first peptide anchor, and the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor binding signal or (ii) a second peptide anchor, and each of the first cytokine peptide and the second cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. Optionally, the first cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-105 or 107-112. Optionally, the first cytokine peptide is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-155 or 157-164. Optionally, the second cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-105 or 107-112. Optionally, the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-155 or 157-164.

[0024] Disclosed herein, in some embodiments, is a system comprising a first nucleic acid sequence and a second nucleic acid sequence that is different, wherein the first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and either (i) a first non-peptide anchor binding signal or (ii) a first peptide anchor, and the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and either (i) a second non-peptide anchor binding signal or (ii) a second peptide anchor, wherein each of the first cytokine peptide and the second cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. Optionally, the first cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-105 or 107-112. Optionally, the first cytokine peptide is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-155 or 157-164. Optionally, the second cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-105 or 107-112. Optionally, the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-155 or 157-164.

[0025] Disclosed herein, in some embodiments, is a cell comprising a first protein and a second protein, wherein the first protein comprises a first cytokine peptide and (i) a first non-peptide anchor or (ii) a first peptide anchor, and the second protein comprises a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor, and each of the first cytokine peptide and the second cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. Optionally, the first cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-105 or 107-112. Optionally, the first cytokine peptide is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-155 or 157-164. Optionally, the second cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-105 or 107-112. Optionally, the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-155 or 157-164.

[0026] In any one of the aforementioned or related embodiments, the first cytokine peptide comprises at least a portion of IL-4 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-2, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. In some cases, the first cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 101, and the second cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 100, 102-105, or 107-112. In some cases, the first cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 151 or 163, and the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 150, 152-155, 157-162, or 164.

[0027] In any one of the above or related embodiments, the cytotoxicity of the immune cells is increased compared to a comparable immune cell that does not include both: (1) a first nucleic acid sequence encoding a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor binding signal or (ii) a first peptide anchor; and (2) a second nucleic acid sequence encoding a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor binding signal or (ii) a second peptide anchor; or that does not include both: (1) a first protein comprising the first cytokine peptide and (i) the first non-peptide anchor or (ii) the first peptide anchor; and (2) a second protein comprising the second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor. Optionally, the increase in cytotoxicity of the immune cells is measured by an in vitro cytotoxicity assay described in Examples A-D. Optionally, the increase in cytotoxicity of the immune cells is measured in vitro or in vivo. Optionally, the increase in cytotoxicity of the immune cells is at least about 5%, 10%, 20%, 30%, 40%, 50%, or more.

[0028] In any one of the above or related embodiments, the population of immune cells proliferates for a longer period of time compared to a comparable population of immune cells in which the first nucleic acid sequence does not encode a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor binding signal or (ii) a first peptide anchor, and / or the second nucleic acid sequence does not encode a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor binding signal or (ii) a second peptide anchor, or the population of immune cells does not encode a first protein comprising a first cytokine peptide and (i) a first non-peptide anchor or (ii) a first peptide anchor, and / or the population of immune cells does not encode a second protein comprising a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor. Optionally, proliferation of the population of immune cells is measured by an in vitro immune cell proliferation assay as described in Examples A-E. Optionally, proliferation of the population of immune cells is measured in vitro or in vivo. In some cases, the expansion of the population of immune cells is sustained for at least about 5%, 10%, 20%, 30%, 40%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold or more than the expansion of the comparable population of immune cells.

[0029] In any one of the above or related embodiments, the variants of the first and second cytokine peptides comprise an alteration, substitution, deletion, addition, or chemical modification of one or more amino acids, one or more unnatural amino acids, or any combination thereof.

[0030] In any one of the above or related embodiments, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-7 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-12 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-15 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-21 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-12 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-15 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-21 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof.Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-12 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-15 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-12 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-21 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-12 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-12 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-15 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-15 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-21 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-21 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof.

[0031] Disclosed herein, in some embodiments, is a nucleic acid molecule comprising a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence, wherein the first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) a first peptide anchor, the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor attachment signal or (ii) a second peptide anchor, and the third nucleic acid sequence encodes a third cytokine peptide. and a third polypeptide comprising the first cytokine peptide and either (i) a third non-peptide anchor binding signal or (ii) a third peptide anchor, wherein each of the first cytokine peptide, the second cytokine peptide, and the third cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

[0032] Disclosed herein, in some embodiments, is a system comprising a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence, wherein the first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) a first peptide anchor, the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor attachment signal or (ii) a second peptide anchor, and the third nucleic acid sequence encodes a third cytokine peptide and (i) a third non-peptide anchor attachment signal or (ii) a second peptide anchor. and (ii) a third polypeptide comprising a peptide anchor and a first cytokine peptide, a second cytokine peptide, and a third peptide anchor, wherein each of the first cytokine peptide, the second cytokine peptide, and the third cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof, and the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are different.

[0033] Disclosed herein, in some embodiments, is a cell comprising a first protein, a second protein, and a third protein, wherein the first protein comprises a first cytokine peptide and (i) a first non-peptide anchor or (ii) a first peptide anchor, the second protein comprises a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor, and the third protein comprises a third cytokine peptide and (i) a third non-peptide anchor or (ii) a third peptide anchor, and each of the first cytokine peptide, second cytokine peptide, and third cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

[0034] In any one of the above or related aspects, the immune cell cytotoxicity is achieved by: (1) a first nucleic acid sequence encoding a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor binding signal or (ii) a first peptide anchor; (2) a second nucleic acid sequence encoding a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor binding signal or (ii) a second peptide anchor; and (3) a third nucleic acid sequence encoding a third cytokine peptide and (i) a third non-peptide anchor binding signal or (ii) a third peptide anchor. and encoding a third polypeptide comprising a first polypeptide and a second polypeptide comprising a peptide anchor, or a third polypeptide comprising a peptide anchor and a cytokine signaling pathway, wherein the cytotoxicity of the immune cells is increased compared to a comparable immune cell that does not comprise all of: (1) a first protein comprising a first cytokine peptide and (i) a first non-peptide anchor or (ii) a first peptide anchor; (2) a second protein comprising a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor; and (3) a third protein comprising a third cytokine peptide and (i) a third non-peptide anchor or (ii) a third peptide anchor. Optionally, the increase in cytotoxicity of the immune cells is measured by an in vitro cytotoxicity assay as described in Examples A-D. Optionally, the increase in cytotoxicity of the immune cells is measured in vitro or in vivo. Optionally, the increase in cytotoxicity of the immune cells is at least about 5%, 10%, 20%, 30%, 40%, 50%, or more.

[0035] In any one of the above or related aspects, the population of immune cells comprises: (1) a first nucleic acid sequence encoding a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor binding signal or (ii) a first peptide anchor; (2) a second nucleic acid sequence encoding a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor binding signal or (ii) a second peptide anchor; and (3) a third nucleic acid sequence encoding a third cytokine peptide and (i) a third non-peptide anchor binding signal or (ii) a third peptide anchor. or proliferates for a prolonged period of time compared to a comparable population of immune cells that does not comprise all of: (1) a first protein comprising a first cytokine peptide and (i) a first non-peptide anchor or (ii) a first peptide anchor; (2) a second protein comprising a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor; and (3) a third protein comprising a third cytokine peptide and (i) a third non-peptide anchor or (ii) a third peptide anchor. Optionally, proliferation of the population of immune cells is measured by an in vitro immune cell proliferation assay as described in Examples A-E. Optionally, proliferation of the population of immune cells is measured in vitro or in vivo. In some cases, the expansion of the population of immune cells is sustained for at least about 5%, 10%, 20%, 30%, 40%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold or more than the expansion of the comparable population of immune cells.

[0036] In any one of the above or related embodiments, the first protein is processed from a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor binding signal or (ii) a first peptide anchor, wherein the first non-peptide anchor binding signal is replaced by the first non-peptide anchor during protein processing. Optionally, the first polypeptide is encoded by a first nucleic acid sequence. In any one of the above or related embodiments, the second protein is processed from a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor binding signal or (ii) a second peptide anchor, wherein the second non-peptide anchor binding signal is replaced by the second non-peptide anchor during protein processing. Optionally, the second polypeptide is encoded by a second nucleic acid sequence. Optionally, the second nucleic acid sequence and the first nucleic acid sequence are under the control of the same promoter. Optionally, the second nucleic acid sequence and the first nucleic acid sequence are under the control of two different promoters. Optionally, the first nucleic acid sequence and the second nucleic acid sequence are operably linked in the 5' to 3' direction. Optionally, the second nucleic acid sequence and the first nucleic acid sequence are operably linked in the 5' to 3' direction. Optionally, the second nucleic acid sequence and the first nucleic acid sequence are linked via a nucleic acid sequence encoding a cleavable linker. Optionally, the first protein or first polypeptide comprises a first signal peptide. Optionally, the first signal peptide, the first cytokine peptide, and (i) the first non-peptide anchor binding signal or (ii) the first peptide anchor are operably linked in the direction from the N-terminus of the polypeptide to the C-terminus of the polypeptide. Optionally, the second protein or second polypeptide comprises a second signal peptide. Optionally, the second signal peptide, the second cytokine peptide, and (i) the second non-peptide anchor binding signal or (ii) the second peptide anchor are operably linked in the direction from the N-terminus of the polypeptide to the C-terminus of the polypeptide. Optionally, the first non-peptide anchor binding signal comprises a glycolipid binding signal.Optionally, the second non-peptide anchor attachment signal comprises a glycolipid attachment signal.

[0037] In any one of the above or related embodiments, the third protein is processed from a third polypeptide comprising a third cytokine peptide and a third non-peptide anchor binding signal or a third peptide anchor, and the third non-peptide anchor binding signal is replaced by the third non-peptide anchor during protein processing. Optionally, the third polypeptide is encoded by a third nucleic acid sequence. Optionally, the second nucleic acid sequence and the third nucleic acid sequence are under the control of the same promoter. Optionally, the second nucleic acid sequence and the third nucleic acid sequence are under the control of two different promoters. Optionally, the first nucleic acid sequence and the third nucleic acid sequence are under the control of the same promoter. Optionally, the first nucleic acid sequence and the third nucleic acid sequence are under the control of two different promoters. Optionally, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are operably linked in a 5' to 3' direction. Optionally, the first nucleic acid sequence, the third nucleic acid sequence, and the second nucleic acid sequence are operably linked in a 5' to 3' direction. Optionally, the second nucleic acid sequence, the first nucleic acid sequence, and the third nucleic acid sequence are operably linked in a 5' to 3' direction. Optionally, the second nucleic acid sequence, the third nucleic acid sequence, and the first nucleic acid sequence are operably linked in a 5' to 3' direction. Optionally, the third nucleic acid sequence, the second nucleic acid sequence, and the first nucleic acid sequence are operably linked in a 5' to 3' direction. Optionally, the third nucleic acid sequence, the first nucleic acid sequence, and the second nucleic acid sequence are operably linked in a 5' to 3' direction. Optionally, the first nucleic acid sequence and the second nucleic acid sequence are linked via a nucleic acid sequence encoding a cleavable linker. Optionally, the second nucleic acid sequence and the third nucleic acid sequence are linked via a nucleic acid sequence encoding a cleavable linker. Optionally, the first nucleic acid sequence and the third nucleic acid sequence are linked via a nucleic acid sequence encoding a cleavable linker.

[0038] In any one of the preceding or related embodiments, the third protein or third polypeptide comprises a third signal peptide. Optionally, the third signal peptide, the third cytokine peptide, and (i) the third non-peptide anchor attachment signal or (ii) the third peptide anchor are operably linked in a direction from the N-terminus of the polypeptide to the C-terminus of the polypeptide. Optionally, the third non-peptide anchor attachment signal comprises a glycolipid attachment signal.

[0039] In any one of the aforementioned or related embodiments, the first signal peptide, the second signal peptide, and the third signal peptide each independently comprise a CD4 signal peptide, a CD8α signal peptide, a CD28 signal peptide, a CD33 signal peptide, a CD137 (4-1BB) signal peptide, an IL-2 signal peptide, an IgE signal peptide, an IgG1 signal peptide, a GM-CSF signal peptide, an HLA-A signal peptide, an HLA signal peptide, a TCR signal peptide, or a β2M signal peptide, or a combination thereof. Optionally, the first signal peptide, the second signal peptide, and the third signal peptide each independently comprise an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 2-7. Optionally, the first signal peptide, the second signal peptide, and the third signal peptide are each independently encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 52-57.

[0040] In any one of the foregoing or related embodiments, the first cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-112. Optionally, the first cytokine peptide is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-164. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-4 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-9 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-10 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-12 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-18 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-23 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-27 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-36γ or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-1α or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-15 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-21 or a variant thereof.

[0041] In any one of the foregoing or related embodiments, the second cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-112. Optionally, the second cytokine peptide is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-164. Optionally, the second cytokine peptide comprises at least a portion of IL-2 or a variant thereof. Optionally, the second cytokine peptide comprises at least a portion of IL-4 or a variant thereof. Optionally, the second cytokine peptide comprises at least a portion of IL-7 or a variant thereof. Optionally, the second cytokine peptide comprises at least a portion of IL-9 or a variant thereof. Optionally, the second cytokine peptide comprises at least a portion of IL-10 or a variant thereof. Optionally, the second cytokine peptide comprises at least a portion of IL-12 or a variant thereof. Optionally, the second cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the second cytokine peptide comprises at least a portion of IL-18 or a variant thereof. Optionally, the second cytokine peptide comprises at least a portion of IL-23 or a variant thereof. Optionally, the second cytokine peptide comprises at least a portion of IL-27 or a variant thereof. Optionally, the second cytokine peptide comprises at least a portion of IL-36γ or a variant thereof. Optionally, the second cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the second cytokine peptide comprises at least a portion of IL-1α or a variant thereof. Optionally, the second cytokine peptide comprises at least a portion of IL-15 or a variant thereof. Optionally, the second cytokine peptide comprises at least a portion of IL-21 or a variant thereof.

[0042] In any one of the foregoing or related embodiments, the third cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-112. Optionally, the third cytokine peptide is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-164. Optionally, the third cytokine peptide comprises at least a portion of IL-2 or a variant thereof. Optionally, the third cytokine peptide comprises at least a portion of IL-4 or a variant thereof. Optionally, the third cytokine peptide comprises at least a portion of IL-7 or a variant thereof. Optionally, the third cytokine peptide comprises at least a portion of IL-9 or a variant thereof. Optionally, the third cytokine peptide comprises at least a portion of IL-10 or a variant thereof. Optionally, the third cytokine peptide comprises at least a portion of IL-12 or a variant thereof. Optionally, the third cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the third cytokine peptide comprises at least a portion of IL-18 or a variant thereof. Optionally, the third cytokine peptide comprises at least a portion of IL-23 or a variant thereof. Optionally, the third cytokine peptide comprises at least a portion of IL-27 or a variant thereof. Optionally, the third cytokine peptide comprises at least a portion of IL-36γ or a variant thereof. Optionally, the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the third cytokine peptide comprises at least a portion of IL-1α or a variant thereof. Optionally, the third cytokine peptide comprises at least a portion of IL-15 or a variant thereof. Optionally, the third cytokine peptide comprises at least a portion of IL-21 or a variant thereof.Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-7 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-12 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-7 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-15 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-7 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-21 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-7 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-7 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-12 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-15 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-12 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-21 or a variant thereof.Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-12 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-12 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-15 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-21 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-15 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-15 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-21 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-21 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof.Optionally, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-12 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-15 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-12 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-21 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-12 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-12 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-15 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-21 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-15 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof.Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-15 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-21 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-21 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-12 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-15 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-21 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-12 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-15 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-12 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-15 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof.Optionally, the first cytokine peptide comprises at least a portion of IL-12 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-21 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-12 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-21 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-12 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. In some cases, the first cytokine peptide comprises at least a portion of IL-15 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-21 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. Thus, the first cytokine peptide comprises at least a portion of IL-15 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-21 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-15 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. Optionally, the first cytokine peptide comprises at least a portion of IL-21 or a variant thereof, the second cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof, and the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof.

[0043] In any one of the above or related embodiments, the glycolipid attachment signal comprises a glycosylphosphatidylinositol (GPI) attachment signal. Optionally, the GPI attachment signal comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 200. Optionally, the GPI attachment signal is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 250-252.

[0044] In any one of the above or related embodiments, the first peptide anchor, the second peptide anchor, and the third peptide anchor each independently comprise a transmembrane peptide sequence.

[0045] In any one of the above or related embodiments, the variants of the first cytokine peptide, the second cytokine peptide, and the third cytokine peptide comprise an alteration, substitution, deletion, addition, or chemical modification of one or more amino acids, one or more unnatural amino acids, or any combination thereof.

[0046] In any one of the above or related embodiments, the first peptide anchor, the second peptide anchor, and the third peptide anchor each independently comprise a transmembrane peptide sequence. Optionally, the transmembrane peptide sequence comprises a B7-1 transmembrane amino acid sequence, a B7-2 transmembrane amino acid sequence, a B7-H1 transmembrane amino acid sequence, a B7-H3 transmembrane amino acid sequence, a tumor necrosis factor receptor 2 (TNFR2) transmembrane amino acid sequence, a CD8α transmembrane amino acid sequence, a CD28 transmembrane amino acid sequence, a CD3ζ transmembrane amino acid sequence, a CTLA-4 (CD152) transmembrane amino acid sequence, or a PD-L1 transmembrane amino acid sequence, or any fragment or variant thereof. Optionally, the transmembrane peptide sequence comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 203-204. Optionally, the transmembrane peptide sequence is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 253-255.

[0047] In any one of the above or related aspects, the nucleic acid molecule described herein, the immune cell described herein, the system described herein, or the cell described herein further comprises a targeting sequence encoding a targeting moiety.

[0048] In any one of the above or related embodiments, the targeting sequence is linked to the nucleic acid sequence or exogenous nucleic acid sequence via a nucleic acid sequence encoding a cleavable linker.

[0049] In any one of the above or related embodiments, the targeting sequence and the first nucleic acid sequence are under the control of two different promoters. In any one of the above or related embodiments, the targeting sequence and the first nucleic acid sequence are under the control of the same promoter. Optionally, the targeting sequence and the second nucleic acid sequence are under the control of two different promoters. Optionally, the targeting sequence and the second nucleic acid sequence are under the control of the same promoter. Optionally, the targeting sequence and the third nucleic acid sequence are under the control of two different promoters. Optionally, the targeting sequence and the third nucleic acid sequence are under the control of the same promoter. Optionally, a cleavable linker connects the CAR to the first signal peptide, the second signal peptide, and / or the third signal peptide. Optionally, a cleavable linker connects the CAR to the first peptide anchor, the second peptide anchor, and / or the third peptide anchor. Optionally, a cleavable linker connects the CAR and the first non-peptide anchor binding signal, the second non-peptide anchor binding signal, and / or the third non-peptide anchor binding signal.

[0050] In any one of the foregoing or related embodiments, the first polypeptide, the first protein, the second polypeptide, the second protein, the third polypeptide, and the third protein each independently further comprise a peptide linker. Optionally, the peptide linker connects the first cytokine peptide to the first peptide anchor, the second cytokine peptide to the second peptide anchor, and / or the third cytokine peptide to the third peptide anchor. Optionally, the peptide linker connects the first cytokine peptide to the first non-peptide anchor binding signal, the second cytokine peptide to the second non-peptide anchor binding signal, and / or the third cytokine peptide to the third non-peptide anchor binding signal. Optionally, the peptide linker connects the first peptide anchor, the peptide anchor, and / or the third peptide anchor to a cleavable linker. Optionally, the targeting sequence and the third nucleic acid sequence are present on the same plasmid in the system. Optionally, the targeting sequence and the third nucleic acid sequence are present on two different plasmids in the system. Optionally, the first nucleic acid sequence and the third nucleic acid sequence are present on the same plasmid within the system. Optionally, the first nucleic acid sequence and the third nucleic acid sequence are present on two different plasmids within the system. Optionally, the second nucleic acid sequence and the third nucleic acid sequence are present on the same plasmid within the system. Optionally, the second nucleic acid sequence and the third nucleic acid sequence are present on two different plasmids within the system. Optionally, the targeting sequence and the third nucleic acid sequence are present on the same plasmid within the cell. Optionally, the targeting sequence and the third nucleic acid sequence are present on two different plasmids within the cell. Optionally, the first nucleic acid sequence and the third nucleic acid sequence are present on the same plasmid within the cell. Optionally, the first nucleic acid sequence and the third nucleic acid sequence are present on two different plasmids within the cell. Optionally, the second nucleic acid sequence and the third nucleic acid sequence are present on the same plasmid within the cell. Optionally, the second nucleic acid sequence and the third nucleic acid sequence are present on two different plasmids within the cell. Optionally, the targeting sequence, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are present in the genome of the cell.

[0051] In any one of the above-mentioned or related embodiments, the targeting moiety comprises a chimeric antigen receptor, a T cell receptor, a B cell receptor, or any combination thereof.Optionally, the targeting sequence is under the control of a promoter different from that of the nucleic acid sequence or the exogenous nucleic acid sequence.Optionally, the targeting sequence is under the control of the same promoter as that of the nucleic acid sequence or the exogenous nucleic acid sequence.Optionally, the targeting sequence encodes a chimeric antigen receptor (CAR). In some cases, the chimeric antigen receptor (CAR) comprises a ligand binding domain, and the ligand binding domain is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, CD138, CD171, CD5, CD7, MUC1, AFP, CEA, PSCA, PSMA, HER2, EGFR, IL13Ralpha2, GD2, NKG2D, EGFTvIII, CS1, CCL1, BCMA, mesothelin, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL The targeting domain is selected from the group consisting of -13Ralpha2, PRSS21, VEGR2, LewisY, CD24, PDGFR-beta, SSEA-4, AFP, NCAM, Claudin18.2, GPC3, GM3, TGS5, HMWMAA, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TRAP, WT1, NY-ESO-1, LAGE-1a, and MAGE-A1. In some cases, the targeting domain is selected from the group consisting of CD19, CAR19, and CAR19. In some cases, the targeting domain is selected from the group consisting of CD19, CAR19, and CAR19. Optionally, the CAR comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NO: 4 or 400-407. Optionally, the CAR is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NO: 54 or 450-457.

[0052] In any one of the above or related embodiments, the targeting sequence is linked to the first nucleic acid sequence or the second nucleic acid sequence via a nucleic acid sequence encoding a cleavable linker.Optionally, the cleavable linker connects the CAR to the first signal peptide and / or the second signal peptide.Optionally, the cleavable linker connects the CAR to the first peptide anchor and / or the second peptide anchor.Optionally, the cleavable linker connects the CAR to the first non-peptide anchor binding signal and / or the second non-peptide anchor binding signal.

[0053] In any one of the above or related embodiments, the cleavable linker comprises a P2A peptide, a T2A peptide, an E2A peptide, an F2A peptide, or an IRES peptide. Optionally, the cleavable linker is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 350-355. Optionally, the cleavable linker connects the CAR and a signal peptide. Optionally, the cleavable linker connects the CAR and a peptide anchor. Optionally, the cleavable linker connects the CAR and a non-peptide anchor binding signal. Optionally, the cleavable linker comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 300-303.

[0054] In any one of the above or related embodiments, the first polypeptide, the first protein, the second polypeptide, and the second protein each independently further comprise a peptide linker. Optionally, the peptide linker connects the first cytokine peptide to the first peptide anchor and / or the second cytokine peptide to the second peptide anchor. Optionally, the peptide linker connects the first cytokine peptide to the first non-peptide anchor binding signal and / or the second cytokine peptide to the second non-peptide anchor binding signal. Optionally, the peptide linker connects the first peptide anchor and / or the second peptide anchor to a cleavable linker. Optionally, the peptide linker connects the first non-peptide anchor binding signal and / or the second non-peptide anchor binding signal to the cleavable linker.

[0055] In any one of the above or related embodiments, the peptide linker comprises a GS linker, an Lr1 linker, or an Lr8 linker. Optionally, the peptide linker comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to a sequence set forth in SEQ ID NO:500, 501, 504, 506, or 507, or any of the sequences of LE, AS, GSG, or EF. Optionally, the peptide linker is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to a sequence of ggctccggc, ggaagcgga, gagttc, the sequence of SEQ ID NO:509, or the sequence of SEQ ID NO:520.

[0056] In any one of the above or related embodiments, the nucleic acid molecule is RNA. In any one of the above or related embodiments, the nucleic acid molecule is DNA. Optionally, the nucleic acid molecule is linear RNA. Optionally, the nucleic acid molecule is circular RNA. Optionally, the nucleic acid molecule is a vector. Optionally, the vector is a viral vector. Optionally, the vector is a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

[0057] In any one of the above-mentioned or related embodiments, the targeting sequence and the first nucleic acid sequence are present in the same plasmid in the system.Optionally, the targeting sequence and the first nucleic acid sequence are present in two different plasmids in the system.Optionally, the targeting sequence and the second nucleic acid sequence are present in the same plasmid in the system.Optionally, the targeting sequence and the second nucleic acid sequence are present in two different plasmids in the system.Optionally, the first nucleic acid sequence and the second nucleic acid sequence are present in the same plasmid in the system.Optionally, the first nucleic acid sequence and the second nucleic acid sequence are present in two different plasmids in the system.

[0058] In any one of the above or related embodiments, the cell is a bacterial cell, a yeast cell, or an insect cell. Optionally, the cell is an immune cell or a tumor cell. Optionally, the immune cell is an engineered immune cell. Optionally, the immune cell is a T cell. Optionally, the immune cell is a tumor-infiltrating lymphocyte (TIL). Optionally, the engineered immune cell is a natural killer (NK) cell. Optionally, the targeting sequence and the nucleic acid sequence are present on the same plasmid within the immune cell. Optionally, the targeting sequence and the nucleic acid sequence are present on two different plasmids within the immune cell. Optionally, the targeting sequence and the nucleic acid sequence are present in the genome of the immune cell.

[0059] In any one of the above-mentioned or related embodiments, the targeting sequence and the first nucleic acid sequence are present in the same plasmid inside the cell. Optionally, the targeting sequence and the first nucleic acid sequence are present in two different plasmids inside the cell. Optionally, the targeting sequence and the second nucleic acid sequence are present in the same plasmid inside the cell. Optionally, the targeting sequence and the second nucleic acid sequence are present in two different plasmids inside the cell. Optionally, the first nucleic acid sequence and the second nucleic acid sequence are present in the same plasmid inside the cell. Optionally, the first nucleic acid sequence and the second nucleic acid sequence are present in two different plasmids inside the cell. Optionally, the targeting sequence, the first nucleic acid sequence and the second nucleic acid sequence are present in the genome of the cell.

[0060] Disclosed herein, in some embodiments, are nucleic acid molecules comprising a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 510-514, 518-519, 521-522, 527-529, 533, 600-602, 606, 611-617, 625-638, 652-654, 658-666, 676-686, or 698.

[0061] Disclosed herein, in some embodiments, are polypeptides comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 515-517, 523-526, 530-532, 534, 603-605, 607, 618-624, 639-651, 655-657, 667-675, 687-697, or 699.

[0062] Disclosed herein, in some aspects, are nucleic acid molecules comprising a nucleic acid sequence that encodes a polypeptide described herein.

[0063] Disclosed herein, in some aspects, are cells comprising the nucleic acid molecules described herein.

[0064] Disclosed herein, in some aspects, is a pharmaceutical composition comprising an immune cell described herein or a cell described herein and a pharmaceutically acceptable excipient or carrier.

[0065] Disclosed herein, in some embodiments, are kits, the kits including (a) an immune cell described herein, a cell described herein, or a pharmaceutical composition described herein, and (b) informational material including instructions for administering to a subject a dose of a dosage form of the immune cell, cell, or pharmaceutical composition.

[0066] Disclosed herein in some embodiments is a method of treating a subject in need thereof, the method comprising administering to the subject an immune cell described herein, a cell described herein, or a pharmaceutical composition described herein. Optionally, the immune cell or cells are allogeneic to the subject. Optionally, the immune cell or cells are autologous to the subject. Optionally, the method further comprises obtaining a population of immune cells and manipulating the population of immune cells or their progeny to produce engineered immune cells. Optionally, the immune cell, cell, or pharmaceutical composition treats cancer in the subject. Optionally, the cancer comprises a solid tumor. Optionally, the cancer comprises a leukemia. Optionally, the cancer comprises a melanoma. Optionally, the cancer comprises a lymphoma. Optionally, the cancer comprises adrenal gland cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, fallopian tube cancer, gastrointestinal cancer, glioma, glioblastoma, head and neck cancer, hematopoietic malignancies, leukemia, liver cancer, lung cancer, lymphoma, myeloma, nasal cancer, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, gastric cancer, squamous cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, or any combination thereof. Optionally, the subject has not undergone lymphodepletion prior to administration.

[0067] In some embodiments, disclosed herein is the use of an immune cell described herein, a cell described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for treating a subject. Optionally, the immune cell or cells are allogeneic to the subject. Optionally, the immune cell or cells are autologous to the subject. Optionally, the immune cell or cells treat cancer in the subject. Optionally, the cancer comprises a solid tumor. Optionally, the cancer comprises a leukemia. Optionally, the cancer comprises a melanoma. Optionally, the cancer comprises a lymphoma. In some cases, the cancer comprises adrenal gland cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, fallopian tube cancer, gastrointestinal cancer, glioma, glioblastoma, head and neck cancer, hematopoietic malignancies, leukemia, liver cancer, lung cancer, lymphoma, myeloma, nasal cancer, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, gastric cancer, squamous cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, or any combination thereof.

[0068] Disclosed herein, in some embodiments, are methods for producing engineered immune cells, the methods comprising introducing a nucleic acid molecule described herein or a system described herein into an immune cell.

[0069] Disclosed herein, in some aspects, are methods for producing the immune cells described herein or the cells described herein.

[0070] Disclosed herein, in some aspects, is a method for making a pharmaceutical composition, the method comprising combining an immune cell described herein or a cell described herein with a pharmaceutically acceptable excipient or carrier.

[0071] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0072] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Figure 1A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19+TeIL-2 and CAR19 without TeIL-2 is shown when exposed to tumor cells at an effector-to-target (E:T) ratio of 1:10 for 24 hours. [Figure 1B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+TeIL-2 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-2 when exposed to tumor cells for 12 days. [Figure 2A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19+TeIL-7 and CAR19 without TeIL-7 is shown when exposed to tumor cells at an E:T ratio of 1:10 for 24 hours. [Figure 2B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+TeIL-7 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-7 when exposed to tumor cells for 12 days. [Figure 3A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19+TeIL-9 and CAR19 without TeIL-9 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 3B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+TeIL-9 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-9 upon exposure to tumor cells for 6 days. [Figure 4A]Cytotoxicity of engineered T cells expressing membrane-anchored CAR19+TeIL-12p40 and CAR19 without TeIL-12p40 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 4B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-12p40 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-12p40 when exposed to tumor cells for 9 days. [Figure 5A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-15 and CAR19 without TeIL-15 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 5B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-15 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-15 upon exposure to tumor cells for 9 days. [Figure 6A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19+TeIL-18 and CAR19 without TeIL-18 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 6B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+TeIL-18 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-18 upon exposure to tumor cells for 6 days. [Figure 7A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19+CAR19-TeIL-21 and CAR19 without TeIL-21 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 7B]Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+CAR19-TeIL-21 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-21 when exposed to tumor cells for 6 days. [Figure 8A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19+TeIL-36γ and CAR19 without TeIL-36γ is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 8B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+TeIL-36γ immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-36γ upon exposure to tumor cells for 6 days. [Figure 9A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-12p40+TeIL-7 and CAR19 without TeIL-12p40 or TeIL-7 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 9B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+TeIL-12p40+TeIL-7 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-12p40 or TeIL-7 upon exposure to tumor cells for 9 days. [Figure 10A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-12p40+CAR19-TeIL-15 and CAR19 without TeIL-12p40 or TeIL-15 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 10B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-12p40+CAR19-TeIL-15 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-12p40 or TeIL-15 upon 9 days of exposure to tumor cells. [Figure 11A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-12p40+TeIL-12p40-TeIL-21 and CAR19 without TeIL-12p40 or TeIL-21 is shown when exposed to tumor cells at an E:T ratio of 1:18 for 72 hours. [Figure 11B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-12p40+TeIL-12p40-TeIL-21 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-12p40 or TeIL-21 when exposed to tumor cells for 9 days. [Figure 12A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-15+TeIL-2 and CAR19-expressing engineered T cells without TeIL-15 or TeIL-2 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 12B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-15+TeIL-2 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-15 or TeIL-2 upon 9 days of exposure to tumor cells. [Figure 13A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-15+TeIL-7 and CAR19-expressing engineered T cells without TeIL-15 or TeIL-7 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 13B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-15+TeIL-7 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-15 or TeIL-7 upon exposure to tumor cells for 6 days. [Figure 14A]Cytotoxicity of engineered T cells expressing membrane-anchored CAR19+TeIL-15-TeIL-12p40 and CAR19 without TeIL-12p40 or TeIL-15 is shown when exposed to tumor cells at an E:T ratio of 1:18 for 72 hours. [Figure 14B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+TeIL-15-TeIL-12p40 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-12p40 or TeIL-15 upon 9 days of exposure to tumor cells. [Figure 15A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-15+TeIL-15-TeIL-21 and CAR19 without TeIL-15 or TeIL-21 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 15B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-15+TeIL-15-TeIL-21 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-15 or TeIL-21 when exposed to tumor cells for 9 days. [Figure 16A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-21+TeIL-7 and CAR19 without TeIL-21 or TeIL-7 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 16B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-21+TeIL-7 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-21 or TeIL-7 upon exposure to tumor cells for 13 days. [Figure 17A]Cytotoxicity of engineered T cells expressing membrane-anchored CAR19+TeIL-21-TeIL-12p40 and CAR19 without TeIL-21 or TeIL-12p40 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 17B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+TeIL-21-TeIL-12p40 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-21 or TeIL-12p40 upon exposure to tumor cells for 9 days. [Figure 18A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-21+TeIL-15 and CAR19 without TeIL-21 or TeIL-15 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 18B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-21+TeIL-7 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without TeIL-21 or TeIL-15 upon exposure to tumor cells for 13 days. [Figure 19A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19+TeIL-2+TeIL-7+TeIL-15 and CAR19 without TeIL-2, TeIL-7, or TeIL-15 is shown when exposed to tumor cells at an E:T ratio of 1:10 for 24 hours. [Figure 19B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+TeIL-2+TeIL-7+TeIL-15 immobilized on the cell membrane and of CD8+ engineered T cells expressing CAR19 without TeIL-2, TeIL-7, or TeIL-15 upon exposure to tumor cells for 12 days. [Figure 20A]Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-12p40+TeIL-7+TeIL-21 and CAR19 without TeIL-12p40, TeIL-7, or TeIL-21 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 20B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+TeIL-12p40+TeIL-7+TeIL-21 immobilized on the cell membrane and of CD8+ engineered T cells expressing CAR19 without TeIL-12p40, TeIL-7, or TeIL-21 upon exposure to tumor cells for 6 days. [Figure 21A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-12p40+TeIL-15+TeIL-21 and CAR19 without TeIL-12p40, TeIL-15, or TeIL-21 is shown when exposed to tumor cells at an E:T ratio of 1:18 for 72 hours. [Figure 21B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-12p40+TeIL-15+TeIL-21 immobilized on the cell membrane and the proliferation of CD8+ engineered T cells expressing CAR19 without TeIL-12p40, TeIL-15, or TeIL-21 when exposed to tumor cells for 9 days. [Figure 22A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-15+TeIL-7+TeIL-21 and CAR19-expressing engineered T cells without TeIL-15, TeIL-7, or TeIL-21 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 22B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-15+TeIL-7+TeIL-21 immobilized on the cell membrane and of CD8+ engineered T cells expressing CAR19 without TeIL-15, TeIL-7, or TeIL-21 upon exposure to tumor cells for 6 days. [Figure 23A]Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-15+TeIL-12p40-TeIL-21 and CAR19 without TeIL-15, TeIL-12p40, or TeIL-21 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 23B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-15+TeIL-12p40-TeIL-21 immobilized on the cell membrane and of CD8+ engineered T cells expressing CAR19 without TeIL-15, TeIL-12p40, or TeIL-21 upon exposure to tumor cells for 9 days. [Figure 24A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-15+TeIL-21-TeIL-12p40 and CAR19 without TeIL-15, TeIL-21, or TeIL-12p40 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 24B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-15+TeIL-21-TeIL-12p40 immobilized on the cell membrane and of CD8+ engineered T cells expressing CAR19 without TeIL-15, TeIL-21, or TeIL-12p40 upon 9 days of exposure to tumor cells. [Figure 25A] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-21+TeIL-7+TeIL-15 and CAR19 without TeIL-21, TeIL-7, or TeIL-15 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 25B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-21+TeIL-7+TeIL-15 immobilized on the cell membrane and of CD8+ engineered T cells expressing CAR19 without TeIL-21, TeIL-7, or TeIL-15 upon exposure to tumor cells for 13 days. [Figure 26A]Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-21+TeIL-12p40+TeIL-15 and CAR19 without TeIL-21, TeIL-12p40, or TeIL-15 is shown when exposed to tumor cells at an E:T ratio of 1:9 for 24 hours. [Figure 26B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19-TeIL-21+TeIL-12p40+TeIL-15 immobilized on the cell membrane and the proliferation of CD8+ engineered T cells expressing CAR19 without TeIL-21, TeIL-12p40, or TeIL-15 upon exposure to tumor cells for 13 days. [Figure 27A] Figure 1 shows the cytotoxicity of engineered T cells expressing CAR19+IL-12p40-Lr1-Ar1-E2A-IL-15-Lr1-Ar2 immobilized on the cell membrane and the cytotoxicity of engineered T cells expressing CAR19 without IL-12p40-Lr1-Ar1-E2A-IL-15-Lr1-Ar2 when exposed to tumor cells at an E:T ratio of 1:3 for 72 hours. [Figure 27B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+IL-12p40-Lr1-Ar1-E2A-IL-15-Lr1-Ar2 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without IL-12p40-Lr1-Ar1-E2A-IL-15-Lr1-Ar2 upon exposure to tumor cells for 12 days. [Figure 28A] Cytotoxicity of engineered T cells expressing CAR19+IL-12p40-Lr1-Ar1-F2A-IL-15-Lr1-Ar2 immobilized on the cell membrane and cytotoxicity of engineered T cells expressing CAR19 without IL-12p40-Lr1-Ar1-F2A-IL-15-Lr1-Ar2 are shown when exposed to tumor cells at an E:T ratio of 1:3 for 72 hours. [Figure 28B]Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+IL-12p40-Lr1-Ar1-F2A-IL-15-Lr1-Ar2 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without IL-12p40-Lr1-Ar1-F2A-IL-15-Lr1-Ar2 upon exposure to tumor cells for 12 days. [Figure 29A] Figure 1 shows the cytotoxicity of engineered T cells expressing CAR19+IL-12p40-Lr1-Ar1-P2A-IL-15-Lr1-Ar2 immobilized on the cell membrane and the cytotoxicity of engineered T cells expressing CAR19 without IL-12p40-Lr1-Ar1-P2A-IL-15-Lr1-Ar2 when exposed to tumor cells at an E:T ratio of 1:3 for 72 hours. [Figure 29B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+IL-12p40-Lr1-Ar1-P2A-IL-15-Lr1-Ar2 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without IL-12p40-Lr1-Ar1-P2A-IL-15-Lr1-Ar2 upon exposure to tumor cells for 12 days. [Figure 30A] Cytotoxicity of engineered T cells expressing CAR19+IL-12p40-Lr1-Ar1-T2A-IL-15-Lr1-Ar2 immobilized on the cell membrane and cytotoxicity of engineered T cells expressing CAR19 without IL-12p40-Lr1-Ar1-T2A-IL-15-Lr1-Ar2 are shown when exposed to tumor cells at an E:T ratio of 1:3 for 72 hours. [Figure 30B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+IL-12p40-Lr1-Ar1-T2A-IL-15-Lr1-Ar2 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without IL-12p40-Lr1-Ar1-T2A-IL-15-Lr1-Ar2 upon exposure to tumor cells for 12 days. [Figure 31A]Cytotoxicity of engineered T cells expressing CAR19+IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar1 immobilized on the cell membrane and cytotoxicity of engineered T cells expressing CAR19 without IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar1 are shown when exposed to tumor cells at an E:T ratio of 1:3 for 72 hours. [Figure 31B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar1 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar1 upon exposure to tumor cells for 12 days. [Figure 32A] Cytotoxicity of engineered T cells expressing CAR19+IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar2 immobilized on the cell membrane and cytotoxicity of engineered T cells expressing CAR19 without IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar2 are shown when exposed to tumor cells at an E:T ratio of 1:3 for 72 hours. [Figure 32B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar2 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar2 upon exposure to tumor cells for 12 days. [Figure 33A] Figure 1 shows the cytotoxicity of engineered T cells expressing CAR19+IL-12p40-Lr8-Ar1-E2A-IL-15-Lr1-Ar2 immobilized on the cell membrane and of engineered T cells expressing CAR19 without IL-12p40-Lr8-Ar1-E2A-IL-15-Lr1-Ar2 when exposed to tumor cells for 72 hours at an E:T ratio of 1:3, and when exposed to tumor cells for 12 and 9 days, respectively. [Figure 33B]Figure 1 shows the proliferation of CD8+ engineered T cells expressing CAR19+IL-12p40-Lr8-Ar1-E2A-IL-15-Lr1-Ar2 immobilized on the cell membrane and CD8+ engineered T cells expressing CAR19 without IL-12p40-Lr8-Ar1-E2A-IL-15-Lr1-Ar2 upon exposure to tumor cells for 12 days. [Figure 34] Cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-4 and CAR19 without TeIL-4 is shown when exposed to tumor cells at E:T ratios of 1:8 and 1:16 for 72 hours. [Figure 35A] Figure 1 shows the proliferation of CD4+ engineered T cells expressing membrane-anchored CAR19-TeIL-4 and CD4+ engineered T cells expressing CAR19 without TeIL-4 upon exposure to tumor cells at an E:T ratio of 1:8 for 12 days. [Figure 35B] Figure 1 shows the proliferation of CD8+ engineered T cells expressing membrane-anchored CAR19-TeIL-4 and CD8+ engineered T cells expressing CAR19 without TeIL-4 upon exposure to tumor cells at an E:T ratio of 1:8 for 12 days. [Figure 36A] Figure 1 shows the proliferation of engineered CD4+ T cells expressing membrane-anchored CAR19-TeIL-4 and CD4+ engineered T cells expressing CAR19 without TeIL-4 when exposed to tumor cells at an E:T ratio of 1:16 for 12 days. [Figure 36B] Figure 1 shows the proliferation of engineered CD8+ engineered T cells expressing membrane-anchored CAR19-TeIL-4 and CD8+ engineered T cells expressing CAR19 without TeIL-4 when exposed to tumor cells at an E:T ratio of 1:16 for 12 days. [Figure 37] Figure 1 shows the sequential cytotoxicity of engineered T cells expressing membrane-anchored CAR19-TeIL-10, engineered T cells expressing soluble CAR19-sIL-10, and engineered T cells expressing CAR19 without TeIL-10 or sIL-10 upon continuous exposure to tumor cells for 45 days. [Figure 38A] Figure 1 shows the continuous proliferation of CD8+ engineered T cells expressing membrane-anchored CAR19-TeIL-10, CD8+ engineered T cells expressing soluble CAR19-sIL-10, and CD8+ engineered T cells expressing CAR19 without TeIL-10 or sIL-10 upon continuous exposure to tumor cells for 51 days. [Figure 38B] Figure 1 shows the continuous proliferation of CD4+ engineered T cells expressing membrane-anchored CAR19-TeIL-10, CD4+ engineered T cells expressing soluble CAR19-sIL-10, and CD8+ engineered T cells expressing CAR19 without TeIL-10 or sIL-10 upon continuous exposure to tumor cells for 51 days. [Figure 39] Figure 1 shows the tumor-killing ability of engineered T cells expressing CAR19-TeIL-4 and CAR19-TeIL-4 + TeIL-15. Fluorescence intensity (total flux p / s) was used to indicate tumor burden. DETAILED DESCRIPTION OF THE INVENTION

[0073] Disclosed herein, in some aspects, are (a) a polypeptide comprising a cytokine peptide and an anchor structure, (b) a nucleic acid encoding the polypeptide, (c) a protein processed from the polypeptide, and (d) a cell (e.g., an immune cell) expressing the polypeptide ((a)-(d) hereinafter collectively referred to as a "single cytokine-anchor material"). In some embodiments, the anchor structure is capable of binding a cytokine to the surface of a cell comprising a nucleic acid molecule encoding the polypeptide. In some embodiments, the cytokine peptide comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. Optionally, the cytokine peptide is at least a portion of IL-12p40. Optionally, the cytokine peptide is at least a portion of IL-12p30. Optionally, the cytokine peptide comprises IL-12p40 and does not comprise IL-12p35. Optionally, immune cells comprising a polypeptide described herein do not comprise a stimulus response element (SRE) derived from PDE5. Optionally, immune cells comprising a polypeptide described herein do not comprise a stimulus response element (SRE). In some embodiments, the polypeptide further comprises a signal peptide. In some embodiments, the anchor structure is a peptide anchor. In some embodiments, the anchor structure is a non-peptide anchor-binding signal that is subsequently replaced by a non-peptide anchor during protein processing. In some embodiments, the nucleic acid molecule further comprises a targeting sequence. In some embodiments, the targeting sequence encodes a chimeric antigen receptor (CAR), a T cell receptor, a B cell receptor, or any combination thereof. In some embodiments, the targeting sequence encodes a CAR comprising a recognition region that targets CD19. In some embodiments, the targeting sequence encodes CAR19 (e.g., as described in Table 5A). In some embodiments, CAR19 and the polypeptide are linked via a cleavable linker.In some embodiments, the signal peptide targets the polypeptide to the surface of a cell. In some embodiments, the peptide anchor comprises a transmembrane peptide sequence. In some embodiments, the non-peptide anchor attachment signal is replaced by a non-peptide anchor, such as a lipid anchor. In some embodiments, the non-peptide anchor attachment signal comprises a glycosylphosphatidylinositol (GPI) attachment signal. In some embodiments, the non-peptide anchor is a GPI anchor. In some embodiments, the peptide anchor is attached to a membrane of a cell. In some embodiments, the non-peptide anchor is attached to a membrane of a cell. In some embodiments, the cleavable linker comprises a P2A peptide, a T2A peptide, an E2A peptide, an F2A peptide, or an IRES peptide. In some embodiments, the nucleic acid molecule is a vector such as a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector. In some embodiments, the cell comprises an immune cell or a tumor cell. In some embodiments, the cell comprises an engineered immune cell, such as a T cell or a natural killer (NK) cell. In some embodiments, the cytotoxicity of the immune cells is increased compared to comparable immune cells that do not contain a nucleic acid sequence encoding the cytokine peptide and anchor structure (e.g., as shown in Example D). In some embodiments, the population of immune cells proliferates for a longer period of time compared to a comparable population of immune cells that do not contain a nucleic acid sequence encoding the cytokine peptide and anchor structure (e.g., as shown in Example E). Optionally, the polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 515-517, 523-526, 607, 618-624, or 667-675. Optionally, the nucleic acid comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 510-514, 518-519, 521-522, 606, 611-617, or 658-666.

[0074] Disclosed herein, in some embodiments, are: (a) a system comprising a first polypeptide and a different second polypeptide, wherein the first polypeptide comprises a first cytokine peptide and a first anchor structure, and the second polypeptide comprises a second cytokine peptide and a second anchor structure; (b) a system comprising a protein comprising the first polypeptide and a protein comprising the second polypeptide; (c) a system comprising a nucleic acid encoding the first polypeptide and a nucleic acid encoding the second polypeptide; (d) nucleic acid molecules encoding the first polypeptide and the second polypeptide; and (e) a cell (e.g., an immune cell) expressing the first polypeptide and the different second polypeptide ((a)-(e) hereinafter collectively referred to as "two cytokine-anchor materials"). In some embodiments, the nucleic acid molecules encoding the first polypeptide and the second polypeptide comprise a first nucleic acid sequence encoding the first polypeptide and a second nucleic acid sequence encoding the second polypeptide. In some embodiments, the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are different. In some embodiments, the first cytokine peptide is a pro-inflammatory cytokine peptide and the second cytokine peptide is an anti-inflammatory cytokine peptide. In some embodiments, the first anchor structure is capable of binding the first cytokine to the surface of a cell containing the nucleic acid molecule. In some embodiments, the second anchor structure is capable of binding the second cytokine to the surface of a cell containing the nucleic acid molecule. In some embodiments, the first cytokine peptide and the second cytokine peptide each independently comprise at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.In other embodiments, each of the first cytokine peptide and the second cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-15, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. In some embodiments, the first polypeptide comprises a first signal peptide. In some embodiments, the second polypeptide comprises a second signal peptide. In some embodiments, the first anchor structure is a peptide anchor. In some embodiments, the first anchor structure is a non-peptide anchor binding signal that is replaced by a non-peptide anchor during protein processing. In some embodiments, the second anchor structure is a peptide anchor. In some embodiments, the second anchor structure is a non-peptide anchor binding signal that is replaced by a non-peptide anchor during processing. In some embodiments, the nucleic acid molecule further comprises a targeting sequence. In some embodiments, the targeting sequence encodes a chimeric antigen receptor (CAR), a T cell receptor, a B cell receptor, or any combination thereof. In some embodiments, the targeting sequence encodes a CAR including a recognition region that targets CD19. In some embodiments, the targeting sequence encodes CAR19 (e.g., as described in Table 5A). In some embodiments, CAR19 and the first polypeptide are linked via a cleavable linker. In some embodiments, CAR19 and the second polypeptide are linked via a cleavable linker. In some embodiments, the first polypeptide and the second polypeptide are linked via a cleavable linker. In some embodiments, the signal peptide targets the polypeptide to the surface of the cell. In some embodiments, the peptide anchor comprises a transmembrane peptide sequence. In some embodiments, the non-peptide anchor binding signal is replaced by a non-peptide anchor, such as a lipid anchor.In some embodiments, the non-peptide anchor attachment signal comprises a glycosylphosphatidylinositol (GPI) attachment signal. In some embodiments, the non-peptide anchor is a GPI anchor. In some embodiments, the peptide anchor is attached to a membrane of a cell. In some embodiments, the non-peptide anchor is attached to a membrane of a cell. In some embodiments, the cleavable linker comprises a P2A peptide, a T2A peptide, an E2A peptide, an F2A peptide, or an IRES peptide. In some embodiments, the nucleic acid molecule is a vector such as a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector. In some embodiments, the cell comprises an immune cell or a tumor cell. In some embodiments, the cell comprises an engineered immune cell such as a T cell or a natural killer (NK) cell. In some embodiments, the cytotoxicity of the immune cell is increased compared to a comparable immune cell that does not contain a nucleic acid sequence encoding the cytokine peptide and the anchor structure (e.g., as shown in Example D). In some embodiments, the population of immune cells proliferates for a longer period of time compared to a comparable population of immune cells that does not contain a nucleic acid sequence encoding the cytokine peptide and the anchor structure (e.g., as shown in Example E). Optionally, the first polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 515-517, 523-526, 607, 618-624, or 667-675. Optionally, the first nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 510-514, 518-519, 521-522, 606, 611-617, or 658-666.Optionally, the second polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 515-517, 523-526, 607, 618-624, or 667-675. Optionally, the second nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 510-514, 518-519, 521-522, 606, 611-617, or 658-666. Optionally, the nucleic acid comprises a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 527-529, 533, 600-602, 625-638, or 676-686. Optionally, the nucleic acid molecule encodes an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 530-532, 534, 603-605, 639-651, or 687-697. Optionally, the anti-inflammatory cytokine peptide comprises at least a portion of IL-4, IL-10, or IL-27, or a variant thereof. Optionally, the proinflammatory cytokine peptide comprises at least a portion of IL-2, IL-7, IL-9, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. Optionally, the anti-inflammatory cytokine peptide comprises at least a portion of IL-4 or a variant thereof, and the proinflammatory cytokine peptide comprises at least a portion of IL-2, IL-7, IL-9, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.In some cases, the anti-inflammatory cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 101, 104, or 109, and the pro-inflammatory cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 100, 102, 103, 105-108, or 110-112. In some cases, the anti-inflammatory cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 151, 163, 154, 164, or 159, and the pro-inflammatory cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NO: 150, 152, 153, 155-158, or 160-162. In some cases, the anti-inflammatory cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 101, and the pro-inflammatory cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 100, 102, 103, 105-108, or 110-112. In some cases, the anti-inflammatory cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 151 or 163, and the pro-inflammatory cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 150, 152, 153, 155-158, or 160-162.Optionally, the first cytokine peptide comprises at least a portion of IL-4 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-2, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. Optionally, the first cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 101, and the second cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 100 or 102-112. Optionally, the first cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 151 or 163, and the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 150, 152-162, or 164. Optionally, the first cytokine peptide comprises at least a portion of IL-4 or a variant thereof, and the second cytokine peptide comprises IL-2, IL-7. , IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. Optionally, the first cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 101, and the second cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 100, 102-107, or 109-112. In some cases, the first cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 151 or 163, and the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 150, 152-157, 159-162, or 164. Optionally, the first cytokine peptide comprises at least a portion of IL-4 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-2, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. Optionally, the first cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 101, and the second cytokine peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 100, 102-105, or 107-112.In some cases, the first cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 151 or 163, and the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 150, 152-155, 157-162, or 164.

[0075]

[0010] In some embodiments, the present invention provides a system comprising: (a) a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide comprises a first cytokine peptide and a first anchor structure, the second polypeptide comprises a second cytokine peptide and a second anchor structure, and the third polypeptide comprises a third cytokine peptide and a third anchor structure, and wherein the first polypeptide, the second polypeptide, and the third polypeptide are different; (b) a protein comprising the first polypeptide, the second polypeptide, and the third polypeptide, wherein the first polypeptide, the second polypeptide, and the third polypeptide are different; Disclosed are: (a) a system comprising a protein containing a cytokine; (c) a system comprising a nucleic acid encoding a first polypeptide, a nucleic acid encoding a second polypeptide, and a nucleic acid encoding a third polypeptide; a system comprising a nucleic acid encoding any combination of the first polypeptide, the second polypeptide, and the third polypeptide; (d) a nucleic acid molecule encoding the first polypeptide, the second polypeptide, and the third polypeptide; and (e) a cell (e.g., an immune cell) expressing the first polypeptide, the second polypeptide, and the third polypeptide ((a)-(e) hereinafter collectively referred to as "three cytokine-anchor materials"). In some embodiments, the nucleic acid molecules encoding the first polypeptide, the second polypeptide, and the third polypeptide comprise a first nucleic acid sequence encoding the first polypeptide, a second nucleic acid sequence encoding the second polypeptide, and a third nucleic acid sequence encoding the third polypeptide. In some embodiments, the first anchor structure is capable of binding the first cytokine to the surface of a cell comprising the nucleic acid molecule. In some embodiments, the second anchor structure is capable of binding the second cytokine to the surface of a cell comprising the nucleic acid molecule. In some embodiments, the third anchor structure is capable of binding the second cytokine to the surface of a cell that contains the nucleic acid molecule.In some embodiments, the first cytokine peptide, the second cytokine peptide, and the third cytokine peptide each independently comprise at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. In some embodiments, the first polypeptide comprises a first signal peptide. In some embodiments, the second polypeptide comprises a second signal peptide. In some embodiments, the third polypeptide comprises a third signal peptide. In some embodiments, the first anchor structure is a peptide anchor. In some embodiments, the first anchor structure is a non-peptide anchor-binding signal that is replaced by a non-peptide anchor during protein processing. In some embodiments, the second anchor structure is a peptide anchor. In some embodiments, the second anchor structure is a non-peptide anchor-binding signal that is replaced by a non-peptide anchor during processing. In some embodiments, the third anchor structure is a peptide anchor. In some embodiments, the third anchor structure is a non-peptide anchor binding signal that is replaced by a non-peptide anchor during processing. In some embodiments, the nucleic acid molecule further comprises a targeting sequence. In some embodiments, the targeting sequence encodes a chimeric antigen receptor (CAR), a T cell receptor, a B cell receptor, or any combination thereof. In some embodiments, the targeting sequence encodes a CAR comprising a recognition region that targets CD19. In some embodiments, the targeting sequence encodes CAR19 (e.g., as described in Table 5A). In some embodiments, CAR19 and the first polypeptide are linked via a cleavable linker. In some embodiments, CAR19 and the second polypeptide are linked via a cleavable linker. In some embodiments, CAR19 and the third polypeptide are linked via a cleavable linker.In some embodiments, the first polypeptide and the second polypeptide are linked via a cleavable linker. In some embodiments, the second polypeptide and the third polypeptide are linked via a cleavable linker. In some embodiments, the first polypeptide and the third polypeptide are linked via a cleavable linker. In some embodiments, the signal peptide targets the polypeptide to the surface of a cell. In some embodiments, the peptide anchor comprises a transmembrane peptide sequence. In some embodiments, the non-peptide anchor attachment signal is replaced by a non-peptide anchor, such as a lipid anchor. In some embodiments, the non-peptide anchor attachment signal comprises a glycosylphosphatidylinositol (GPI) attachment signal. In some embodiments, the non-peptide anchor is a GPI anchor. In some embodiments, the peptide anchor is attached to a membrane of a cell. In some embodiments, the non-peptide anchor is attached to a membrane of a cell. In some embodiments, the cleavable linker comprises a P2A peptide, a T2A peptide, an E2A peptide, an F2A peptide, or an IRES peptide. In some embodiments, the nucleic acid molecule is a vector such as a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector. In some embodiments, the cells comprise immune cells or tumor cells. In some embodiments, the cells comprise engineered immune cells, such as T cells or natural killer (NK) cells. In some embodiments, the cytotoxicity of the immune cells is increased compared to comparable immune cells that do not contain a nucleic acid sequence encoding a cytokine peptide and an anchor structure (e.g., as shown in Example D). In some embodiments, the population of immune cells proliferates for a longer period of time compared to a comparable population of immune cells that do not contain a nucleic acid sequence encoding a cytokine peptide and an anchor structure (e.g., as shown in Example E).Optionally, the first polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 515-517, 523-526, 607, 618-624, or 667-675. Optionally, the first nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 510-514, 518-519, 606, 521-522, 611-617, or 658-666. Optionally, the second polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 515-517, 523-526, 607, 618-624, or 667-675. Optionally, the second nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 510-514, 518-519, 606, 521-522, 611-617, or 658-666. Optionally, the third polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 515-517, 523-526, 607, 618-624, or 667-675. Optionally, the third nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 510-514, 518-519, 606, 521-522, 611-617, or 658-666. Optionally, the nucleic acid molecule comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 652-654 or 698. Optionally, the nucleic acid molecule encodes an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 655-657 or 699.

[0076] Disclosed herein, in some embodiments, are methods, compositions, kits, and vectors relating to any of the single cytokine-anchor materials, two cytokine-anchor materials, and three cytokine-anchor materials disclosed herein (hereinafter collectively referred to as "cytokine-anchor materials" unless otherwise specified).

[0077] Disclosed herein, in some embodiments, are nucleic acid molecules comprising a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 510-514, 518-519, 521-522, 527-529, 533, 600-602, 606, 611-617, 625-638, 652-654, 658-666, 676-686, or 698. Optionally, the nucleic acid molecule comprises a nucleic acid sequence that is at least 80% identical to any of the sequences set forth in SEQ ID NOs: 510-514, 518-519, 521-522, 527-529, 533, 600-602, 606, 611-617, 625-638, 652-654, 658-666, 676-686, or 698. Optionally, the nucleic acid molecule comprises a nucleic acid sequence that is at least 85% identical to any of the sequences set forth in SEQ ID NOs: 510-514, 518-519, 521-522, 527-529, 533, 600-602, 606, 611-617, 625-638, 652-654, 658-666, 676-686, or 698. Optionally, the nucleic acid molecule comprises a nucleic acid sequence that is at least 90% identical to any of the sequences set forth in SEQ ID NOs: 510-514, 518-519, 521-522, 527-529, 533, 600-602, 606, 611-617, 625-638, 652-654, 658-666, 676-686, or 698. Optionally, the nucleic acid molecule comprises a nucleic acid sequence that is at least 95% identical to any of the sequences set forth in SEQ ID NOs: 510-514, 518-519, 521-522, 527-529, 533, 600-602, 606, 611-617, 625-638, 652-654, 658-666, 676-686, or 698. Optionally, the nucleic acid molecule comprises a nucleic acid sequence that is at least 100% identical to any of the sequences set forth in SEQ ID NOs: 510-514, 518-519, 521-522, 527-529, 533, 600-602, 606, 611-617, 625-638, 652-654, 658-666, 676-686, or 698.

[0078] Disclosed herein, in some embodiments, are polypeptides comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 515-517, 523-526, 530-532, 534, 603-605, 607, 618-624, 639-651, 655-657, 667-675, 687-697, or 699. Optionally, the polypeptide comprises an amino acid sequence that is at least 80% identical to any of the sequences set forth in SEQ ID NOs: 515-517, 523-526, 530-532, 534, 603-605, 607, 618-624, 639-651, 655-657, 667-675, 687-697, or 699. Optionally, the polypeptide comprises an amino acid sequence that is at least 85% identical to any of the sequences set forth in SEQ ID NOs: 515-517, 523-526, 530-532, 534, 603-605, 607, 618-624, 639-651, 655-657, 667-675, 687-697, or 699. Optionally, the polypeptide comprises an amino acid sequence that is at least 90% identical to any of the sequences set forth in SEQ ID NOs: 515-517, 523-526, 530-532, 534, 603-605, 607, 618-624, 639-651, 655-657, 667-675, 687-697, or 699. Optionally, the polypeptide comprises an amino acid sequence that is at least 95% identical to any of the sequences set forth in SEQ ID NOs: 515-517, 523-526, 530-532, 534, 603-605, 607, 618-624, 639-651, 655-657, 667-675, 687-697, or 699. Optionally, the polypeptide comprises an amino acid sequence that is 100% identical to any of the sequences set forth in SEQ ID NOs: 515-517, 523-526, 530-532, 534, 603-605, 607, 618-624, 639-651, 655-657, 667-675, 687-697, or 699.

[0079] In some cases, expressing a specific cytokine or combination of cytokines on the cell membrane includes introducing an exogenous nucleic acid molecule for expressing the specific cytokine or combination and an anchor structure, such that the specific cytokine or combination is expressed at a level higher than its naturally occurring expression level in the host cell or host organism. In other cases, introducing a specific cytokine or combination of cytokines and a tumor-targeting moiety (e.g., CAR) into T cells and expressing them on the cell membrane may include introducing a nucleic acid molecule containing a sequence encoding the specific cytokine or combination of cytokines and the tumor-targeting moiety (e.g., CAR) into immune cells, including T cells, NK cells, monocytes, macrophages, dendritic cells, etc. In some cases, the exogenous nucleic acid sequence encodes IL-12p40. In some cases, the exogenous nucleic acid sequence does not encode both IL-12p40 and IL-12p35. In some cases, the exogenous nucleic acid sequence does not encode a stimulus response element (SRE).

[0080] Without wishing to be bound by any particular theory, the cytokine-anchor materials disclosed herein can provide engineered immune cells with higher anti-tumor activity compared to engineered immune cells (such as CAR-T cells) that express a CAR alone (without expressing an exogenously introduced cytokine or combination of cytokines). When used in immunotherapy, the cytokine-anchor materials disclosed herein can achieve better therapeutic effects. The cytokine-anchor materials disclosed herein enable the expression of a cytokine or combination of cytokines on the cell membrane, thereby regulating the function of immune cells via a vector. When the vectors disclosed herein are introduced into immune cells, the resulting immune cells may have improved immunotherapeutic effects, including improved proliferation and survival rates compared to existing immune cells. By introducing a cytokine or a specific combination of cytokines, such as an interleukin, into immune cells and expressing that cytokine or specific combination of cytokines on the cell membrane together with a tumor-targeting moiety, such as a CAR, anti-tumor activity can be enhanced compared to expressing a tumor-targeting moiety (e.g., a CAR) alone, and in particular, the persistence and proliferation of engineered immune cells can be significantly improved. Delivery of engineered immune cells of cytokine-anchor materials to cancer patients can result in better therapeutic outcomes. In addition, the engineered immune cells of the present disclosure can provide various benefits, such as a reduction in the number of cells administered, given that they enhance antitumor activity without increasing the release of free cytokines, thereby reducing side effects such as cytokine release syndrome (CRS).

[0081] Without wishing to be bound by any particular theory, immobilizing specific cytokines or specific combinations of cytokines on the surface of immune cells can preserve cytokine activity and precisely stimulate immune cells in a targeted manner without inducing the secretion of additional cytokines that could cause severe CRS. Cytokines immobilized on the surface of immune cells according to some embodiments of the present disclosure can include different interleukins, such as IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or variants thereof. Such cytokines immobilized on the cell membrane can improve the ability of immune cells to proliferate and survive.

[0082] Without wishing to be bound by any particular theory, combinations of different cytokines can be immobilized on the surface of immune cells, including combinations of different interleukins, such as IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or variants thereof. Such combinations of cytokines immobilized on the cell membrane can further improve the ability of immune cells to proliferate and survive.

[0083] It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be applied to any aspect unless the content clearly dictates otherwise. Moreover, various embodiments may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art. These and other embodiments of the present disclosure are further described in the detailed description that follows.

[0084] definition In this disclosure, whenever an embodiment is described herein with the word "comprising," other similar embodiments are also provided that are described with the terms "consisting of" and / or "consisting essentially of." All definitions set forth herein should be construed to refer to the definitions used throughout this specification and the appended claims, whether or not specifically referenced.

[0085] Throughout the specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "cells" includes plural samples, including mixtures thereof.

[0086] In the present disclosure, one, some, or all of the characteristics of the various embodiments described herein may be applied to any aspect unless the content clearly indicates otherwise. Furthermore, various embodiments may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art. These and other embodiments of the present disclosure are further described in the detailed description herein.

[0087] Unless otherwise defined throughout the specification and the appended claims, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0088] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0089] The numbering of amino acids in antibody variable domains, CDRs, and framework regions (FRs) follows the definition of Kabat as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise indicated.

[0090] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, in accordance with practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When particular values ​​are described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0091] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified, naturally or by intervention, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. Because the polypeptides described herein are based on antibodies, it is understood that the polypeptides can occur as single chains or associated chains.

[0092] The term "amino acid" refers to natural, unnatural, and synthetic amino acids, including both the D or L optical isomers, and amino acid analogs and peptidomimetics. Amino acids are designated using standard one-letter or three-letter codes.

[0093] A "variant," as applied to a protein, is a protein having sequence homology to a naturally occurring biologically active protein that retains at least some of the therapeutic and / or biological activity of the biologically active protein. For example, a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, or any range between at least 70% and 99%, compared to the reference biologically active protein. As used herein, "variant" can refer to a cytokine that has substantial or significant sequence identity or similarity to a naturally occurring cytokine, such as a naturally occurring cytokine described herein (e.g., IL-2, IL-4, etc.), and that retains one or more biological activities of the naturally occurring cytokine described herein (e.g., IL-2, IL-4, etc.) or an isoform of the naturally occurring cytokine described herein. A variant can include, for example, an alteration, substitution, deletion, addition, or chemical modification of one or more amino acids, one or more unnatural amino acids, or any combination thereof, of a parent peptide, such as a cytokine described herein (e.g., IL-2, IL-4, etc.), and can still retain the ability to specifically bind to its respective receptor, activate downstream targets, and / or induce one or more of the differentiation, proliferation (or death) and activity of cells, e.g., T cells and NK cells, to about the same extent, the same extent, or a greater extent than the parent peptide. Optionally, the variant is a cytokine. With respect to a parent cytokine, a variant can be at least about 80%, about 90%, about 95%, about 99% or more identical in amino acid sequence to the parent cytokine.

[0094] In the context of a polypeptide, a "linear sequence" or "sequence" is the order of amino acids in the polypeptide in the direction from amino terminus to carboxyl terminus, in which residues adjacent to each other in the sequence are contiguous in the primary structure of the polypeptide. A "subsequence" is a linear sequence of a portion of a polypeptide that is known to contain additional residues in one or both directions.

[0095] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps," substitution of one or more analogs of naturally occurring nucleotides, internucleotide modifications such as those with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and those with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralens, etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylators, those with modified linkages (e.g., alpha-anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. Additionally, any of the hydroxyl groups normally present in the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides can further comprise ribose or deoxyribose sugar analogs commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include embodiments in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, or a substituted or unsubstituted alkyl (1-20C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0096] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The heavy and light chain variable regions each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs of each chain are held in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda, MD)), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948). As used herein, CDRs may refer to CDRs defined by either approach or a combination of both approaches.

[0097] A "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0098] A "host cell" includes an individual cell or cell culture that can be or has been a recipient of a vector containing an exogenous polynucleotide. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide of the present disclosure.

[0099] An "individual" or "subject" is a mammal, more preferably a human. Mammals also include farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats.

[0100] As used herein, " vector " refers to the construct that can deliver and preferably express one or more genes or sequences of interest in host cell.Examples of vector include virus vector, naked DNA or RNA expression vector, plasmid, cosmid or phage vector, DNA or RNA expression vector that is associated with cationic condensing agent, DNA or RNA expression vector that is encapsulated in liposome, and certain eukaryotic cells, such as production cells.

[0101] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to produce beneficial or desired results. The therapeutically effective amount may vary depending on one or more of the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the mode of administration, etc., and can be easily determined by one skilled in the art. The term "effective amount" also applies to a dose that provides an image for detection by an appropriate imaging method. The specific dose may vary depending on one or more of the specific agent selected, the administration regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system in which it is delivered. An effective amount of an active agent can be administered in a single dose or multiple doses.

[0102] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any substance that, when combined with an active ingredient, allows the ingredient to retain its biological activity and is non-reactive with the subject's immune system. Examples include any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water, emulsions such as oil / aqueous emulsions, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate-buffered saline or normal (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000).

[0103] Throughout the specification and the appended claims, the methods and systems of the present disclosure described herein may use conventional techniques and descriptions of molecular biology (including recombinant technology), cell biology, biochemistry, microarrays, and sequencing techniques that are within the skill of those skilled in the art, unless otherwise indicated. Such conventional techniques include polymer array synthesis, oligonucleotide hybridization and ligation, oligonucleotide sequencing, and hybridization detection using labels. Specific descriptions of suitable techniques can be obtained by referring to the examples herein. However, it is understood that equivalent conventional procedures can also be used.Such conventional techniques and descriptions are found in Green, et al., Eds., Genome Analysis: A Laboratory Manual Series (Vols. I-IV) (1999); Weiner, et al., Eds., Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler, Eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics: Sequence and Genome Analysis (2004); Sambrook and Russell, Condensed Protocols from Molecular Cloning: A Laboratory Manual (2006); and Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012) (all from Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4th Ed.) W.H. Freeman, N.Y. (1995); Gait, “Oligonucleotide Synthesis: A Practical Approach” IRL Press, London (1984); Nelson and Cox, Lehninger, Principles of Biochemistry, 6. th Ed., W.H. Freeman Pub., New York (2012); R.I. Freshney, Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6 th Ed., Wiley-Blackwell (2010); and Berg et al., Biochemistry, 5 thThe text can be found in standard laboratory manuals such as "The Journal of Experimental Biology," Ed., W.H. Freeman Pub., New York (2002), all of which are incorporated herein by reference in their entirety for all purposes. Before describing the present compositions, research tools, and systems and methods, it should be understood that this disclosure is not limited to the particular systems and methods, compositions, targets, and uses described, which may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is limited only by the appended claims.

[0104] The term "chimeric antigen receptor" or alternatively "CAR" refers in its simplest embodiment to a set of typically two polypeptides that, when present in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and intracellular signal generation. In some embodiments, the CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain of a stimulatory molecule and / or costimulatory molecule. Optionally, the set of polypeptides are contiguous with each other, e.g., in the same polypeptide chain, e.g., comprising a chimeric fusion protein. In some embodiments, the set of polypeptides are not adjacent to each other, e.g., in different polypeptide chains. In some embodiments, the set of polypeptides comprises a dimerization switch that, in the presence of a dimerization molecule, can couple the polypeptides to each other, e.g., couple the antigen-binding domain to the intracellular signaling domain. Optionally, the stimulatory molecule is a zeta chain associated with a T cell receptor complex. Optionally, the cytoplasmic signaling domain further comprises one or more functional signaling domains of at least one costimulatory molecule, as defined below. Optionally, the costimulatory molecule is selected from a costimulatory molecule described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28. Optionally, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0105] "Cytokine release syndrome" and "CRS" refer to an acute systemic inflammatory syndrome characterized by fever and multiple organ dysfunction associated with chimeric antigen receptor (CAR)-T cell therapy, therapeutic antibodies, and haploidentical allogeneic transplantation, as described, for example, in Frey N, Porter D. Cytokine Release Syndrome with Chimeric Antigen Receptor T Cell Therapy. Biol Blood Marrow Transplant 2019;25:e123.

[0106] In this disclosure, "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, this term includes immunoglobulin molecules that specifically bind to an antigen and contain an FcR binding site, which may or may not be functional. As used in this disclosure, this term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, diabodies), Fv fragments, and single-chain (ScFv) mutants that contain the antigen recognition or binding site and have the ability to bind to the antigen. Antigen-binding antibody or immunoglobulin fragments are well known in the art, and such fragments may have a functional or non-functional Fc receptor binding site. Furthermore, as used herein, the term is not limited to only intact polyclonal or monoclonal antibodies, multispecific antibodies, e.g., bispecific or multispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted antibodies, human antibodies, and any other modified immunoglobulin molecule that contains an antigen-binding site, so long as the antibody exhibits the desired biological activity.

[0107] An antibody or CAR that "specifically binds" to an epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates more frequently, rapidly, for a longer duration, and / or with greater affinity with a particular cell, protein, or substance than with another cell, protein, or substance. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, ease, and / or duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to CD19 is an antibody that binds to this epitope with greater affinity, avidity, ease, and / or duration than it binds to other epitopes. As a further example, an antibody (or another moiety) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (but can include) exclusive binding. Generally, although not necessarily always, reference to binding implies preferential binding.

[0108] A "fragment," as applied to a protein or polypeptide, is a truncated form of a native biologically active protein or polypeptide that may or may not retain at least some of its therapeutic and / or biological activity.

[0109] "TeIL," "tethered interleukin," "membrane-anchored cytokine," "membrane-bound cytokine," or "membrane-bound IL" may be used interchangeably to refer to the particular cytokine-anchor structures disclosed herein.

[0110] The term "sIL" refers to a secreted interleukin or cytokine that is neither membrane-anchored nor membrane-bound.

[0111] Sequence identity Sequence identity for cytokine-anchor material or any other amino acid sequence or nucleic acid sequence identified herein is defined as the percentage of amino acid residues (or nucleotides) in a query sequence that are identical to the amino acid residues of a second reference polypeptide sequence or a portion thereof (or a nucleotide or a portion thereof of a second reference nucleic acid sequence), after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity and without considering conservative substitutions as part of the sequence identity.Alignment for the purpose of determining percent amino acid sequence identity or nucleic acid sequence identity can be achieved in a variety of ways within the skill of those in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. Percent identity may be measured over the length of the entire defined polypeptide or nucleic acid sequence, or over a shorter length, for example, over a fragment taken from a larger defined polypeptide or nucleic acid sequence, such as a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues or base pairs or nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein in the tables, figures, or sequence listing can be used to describe the length over which percent identity can be measured. In some embodiments, percent identity is determined over the entire length of a prominent reference sequence, such as the sequences provided herein.For example, sequence comparison between two amino acid sequences (or shorter lengths) of the present disclosure can be performed using the computer program Blastp (protein-protein BLAST) provided online by the National Center for Biotechnology Information (NCBI). The percentage amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can be translated as a given amino acid sequence A having a specific % amino acid sequence identity to a given amino acid sequence B) is calculated by the following formula:

[0112]

number

[0113] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.

[0114] signal peptide In some embodiments, provided herein is a signal peptide present as part of a polypeptide described in the present disclosure, including a polypeptide in a single cytokine-anchor material, a first and second polypeptide in two cytokine-anchor materials, and a first, second, and third polypeptide in three cytokine-anchor materials. Optionally, the signal peptide is removed during protein processing from the polypeptide to form the protein in the single cytokine-anchor material, from the first and second polypeptides to form the first and second proteins, respectively, in the two cytokine-anchor materials, or from the first, second, and third polypeptides to form the first, second, and third proteins, respectively, in the three cytokine-anchor materials.

[0115] The signal peptide can be encoded by a signal peptide nucleic acid sequence, which can be present as part of the nucleic acid sequences described in this disclosure, including the nucleic acid sequence in a single cytokine-anchor material, the first and second nucleic acid sequences in two cytokine-anchor materials, and the first, second, and third nucleic acid sequences in three cytokine-anchor materials.

[0116] A signal peptide can be any suitable peptide capable of directing a polypeptide described herein to a specific compartment of a cell containing such a polypeptide, such as the cell's membrane. In some embodiments, a signal peptide includes a peptide that directs intracellular delivery and localization of the peptide and any linked polypeptide to a specific organelle (such as the endoplasmic reticulum) and / or the cell surface. As used herein, the term "signal peptide" or "signal peptide sequence" refers to a peptide sequence that can be present at the N-terminus of a newly synthesized secreted polypeptide or a newly synthesized transmembrane polypeptide. The signal peptide can translocate the polypeptide through or within the cell's plasma membrane. The signal can then be removed. Specifically, the signal peptide can direct the polypeptide into the cell's secretory pathway.

[0117] Optionally, the signal peptide may be a peptide of any secreted or transmembrane protein that directs delivery of the polypeptides disclosed herein to the cell membrane and cell surface, resulting in accurate localization of the polypeptides of the present disclosure. Optionally, the signal peptide directs the polypeptides of the present disclosure to the cell membrane, where the extracellular portion of the polypeptide is displayed on the cell surface. Optionally, the transmembrane portion spans the plasma membrane, and the active domain is located in the cytoplasm or inside the cell. In some embodiments, the signal peptide is cleaved after passing through the endoplasmic reticulum and is called a cleavable signal peptide. Optionally, the signal peptide may have an amino acid at its end that is recognized and cleaved by a signal peptidase. The signal peptidase can cleave the signal peptide during or after translocation, resulting in a free signal peptide and a mature protein. The free signal peptide can then be digested by a specific protease.

[0118] In some embodiments, the signal peptide is a type I, type II, type III, or type IV transmembrane protein. In some embodiments, the signal peptide comprises an immunoglobulin heavy chain signal peptide.

[0119] Optionally, when another component of the cytokine-anchor material, such as a cytokine and / or CAR, is expressed in a cell (e.g., an engineered immune cell), a signal peptide expressed together with the component in the same polypeptide directs the nascent protein to the endoplasmic reticulum and then to the cell surface, thus allowing the component of the cytokine-anchor material to be expressed on the cell surface. Optionally, the core of the signal peptide consists of a long stretch of hydrophobic amino acids that tend to form an α-helix. Optionally, the signal peptide begins with a short stretch of positively charged amino acids, which helps to ensure the correct topology of the polypeptide during translocation. The signal peptide can be at the N-terminus of the polypeptide. In some embodiments, when a cell expresses a membrane-anchored cytokine and / or CAR, the signal sequence can be excised from the membrane-anchored cytokine and / or CAR.

[0120] Without wishing to be bound by any particular theory, a signal peptide can promote expression of a membrane-anchored cytokine and / or CAR, and the presence of a signal peptide in an expressed membrane-anchored cytokine and / or CAR can contribute to membrane anchoring of the cytokine and / or CAR, but the presence of a signal peptide may be unrelated to the function of the cell membrane-anchored cytokine and / or CAR.

[0121] In some embodiments, the signal peptide is a signal peptide derived from a transmembrane protein. In some embodiments, the signal peptide comprises a CD4 signal peptide, a Cd8α signal peptide, a CD28 signal peptide, a CD33 signal peptide, a CD137 (4-1BB) signal peptide, an IL-2 signal peptide, an IgE signal peptide, an IgG1 signal peptide, a GM-CSF signal peptide, an HLA-A signal peptide, an HLA signal peptide, a TCR signal peptide, or a β2M signal peptide, or a combination thereof. In some embodiments, the signal peptide comprises a CD4 signal peptide, a Cd8α signal peptide, a CD28 signal peptide, a CD33 signal peptide, a CD137 (4-1BB) signal peptide, an IL-2 signal peptide, an IgE signal peptide, an IgG1 signal peptide, a GM-CSF signal peptide, an HLA-A signal peptide, an HLA signal peptide, a TCR signal peptide, or a β2M signal peptide, or a combination thereof. In some embodiments, the signal peptide comprises a CD4 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises a CD8α signal peptide or a variant thereof. In some embodiments, the signal peptide comprises a CD28 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises a CD33 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises a CD137 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IL-2 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IgE signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IgG1 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises a CD4 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises a GM-CSF signal peptide or a variant thereof.In some embodiments, the signal peptide comprises a CD4 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an HLA-A signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an HLA1 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises a TCR signal peptide or a variant thereof. In some embodiments, the signal peptide comprises a β2M signal peptide or a variant thereof.

[0122] In some embodiments, the signal peptide is a naturally occurring signal peptide of a wild-type cytokine. In some embodiments, the signal peptide comprises an IL-2 signal peptide, an IL-4 signal peptide, an IL-7 signal peptide, an IL-9 signal peptide, an IL-10 signal peptide, an IL-12p40 signal peptide, an IL-15 signal peptide, an IL-18 signal peptide, an IL-21 signal peptide, an IL-23 signal peptide, an IL-27 signal peptide, an IL-36γ signal peptide, an IL-23p19 signal peptide, or an IL-1α signal peptide, or a functional variant thereof. In some embodiments, the signal peptide comprises an IL-4 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IL-10 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IL-7 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IL-9 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IL-12p40 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IL-15 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IL-18 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IL-21 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IL-23 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IL-27 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IL-36γ signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IL-23p19 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises an IL-1α signal peptide or a variant thereof.

[0123] In some embodiments, the signal peptide and cytokine peptide are encoded by the same gene. In some embodiments, the signal peptide and cytokine peptide are encoded by different genes. In some embodiments, the signal peptide comprises a naturally occurring signal peptide of IL-4, and the cytokine peptide comprises at least a portion of IL-4 or a variant thereof. In some embodiments, the signal peptide comprises a naturally occurring signal peptide of IL-10, and the cytokine peptide comprises at least a portion of IL-10 or a variant thereof. In some embodiments, the signal peptide comprises a naturally occurring signal peptide of IL-4, and the cytokine peptide comprises at least a portion of IL-10 or a variant thereof. In some embodiments, the signal peptide comprises a naturally occurring signal peptide of IL-10, and the cytokine peptide comprises at least a portion of IL-4 or a variant thereof.

[0124] In some embodiments, the signal peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 2-7 or 113-114. In some embodiments, the signal peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 2. In some embodiments, the signal peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 3. In some embodiments, the signal peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 4. In some embodiments, the signal peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 5. In some embodiments, the signal peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 6. In some embodiments, the signal peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 7. In some embodiments, the signal peptide comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 113. In some embodiments, the signal peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO:114.

[0125] In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 52-59. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 52. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 53. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 54. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 55. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 56. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 57. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 58. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO:59.

[0126] [Table 1A]

[0127] [Table 1B]

[0128] cytokines In some embodiments, provided herein are cytokines or cytokine peptides present as part of the polypeptides described herein, including a polypeptide in a single cytokine-anchor material, a first polypeptide and a second polypeptide in two cytokine-anchor materials, and a first polypeptide, a second polypeptide, and a third polypeptide in three cytokine-anchor materials. The cytokines or cytokine peptides present as part of the polypeptides are operably linked to the anchor structures described herein. The anchor structures can bind the cytokine to the membrane of a cell.

[0129] The cytokine can be encoded by a cytokine nucleic acid sequence, which can be present as part of the nucleic acid sequences described in the present disclosure, including a nucleic acid sequence in a single cytokine-anchor material, a first nucleic acid sequence and a second nucleic acid sequence in two cytokine-anchor materials, and a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence in three cytokine-anchor materials.

[0130] In various embodiments, the term "cytokine" can include a full-length cytokine or interleukin, or a fragment (e.g., truncated form) or variant thereof that substantially retains the biological activity of the corresponding wild-type cytokine or interleukin (e.g., has a biological activity that is at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% of the biological activity of the corresponding wild-type cytokine or interleukin). Cytokines can be secreted by immune cells (e.g., monocytes, macrophages, T cells, B cells, NK cells, etc.). Cytokines can be secreted by certain non-immune cells (e.g., endothelial cells, epithelial cells, fibroblasts, etc.). Cytokines can be produced by monocytes, macrophages, B cells, dendritic cells, TH1 and TH2, mast cells, NK cells, and bone marrow stromal cells. Cytokines can have the ability to regulate multiple cellular and bodily functions, including innate and adaptive immunity, hematopoiesis, cell proliferation, APSC pluripotent cells, and damaged tissue repair and other functions. Cytokines can include interleukins, interferon-α (IFN-α), interferon-β (IFN-β), or tissue necrosis factor (TNF).

[0131] In some embodiments, the cytokines used may be derived from any mammalian species. In some embodiments, the cytokines are derived from species including human, horse, cow, mouse, pig, rabbit, cat, dog, rat, goat, sheep, or non-human primate. In some embodiments, the cytokines are derived from humans. In some embodiments, the cytokines may be mutant forms of their native or wild-type forms.

[0132] In some cases, the cytokine is specifically selected from interleukins (abbreviated as "IL"). Interleukins are a type of cytokine produced by various cells and act on various cells. Interleukins can play a role in signal transduction, activation and regulation of immune cells, mediation of T cell and B cell activation, proliferation and differentiation, and inflammatory responses. Interleukins can be produced by helper CD4+ T lymphocytes. Interleukins can be synthesized by monocytes, macrophages, and endothelial cells. Interleukins can bind to receptors and affect the activation and inhibition of the immune system and cell division.

[0133] In some embodiments, the cytokine comprises an IL from the interleukin-1 family. The interleukin-1 family can include IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-1Ra, IL-36Ra, and IL-38, as well as an anti-inflammatory cytokine (IL-37). Optionally, the cytokine comprises a pro-inflammatory cytokine. The pro-inflammatory cytokine can include IL-2, IL-7, IL-9, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-36γ, IL-23p19, or IL-1α. Optionally, the cytokine comprises an anti-inflammatory cytokine. The anti-inflammatory cytokine can include IL-4, IL-10, or IL-27.

[0134] In some embodiments, the cytokine comprises an IL from the IL-2 family. The IL-2 cytokine family, also known as the common gamma chain family, can include IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The IL-2 family can bind to the common gamma receptor, also known as CD132. The IL-2 family can act as a growth and proliferation factor for progenitor and mature cells.

[0135] In some embodiments, the cytokine comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof. In some embodiments, the cytokine comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-112. In some embodiments, the cytokine is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-162.

[0136] In some embodiments, the cytokine comprises IL-2 or a variant thereof. IL-2 can be produced primarily by CD4+ and CD8+ T cells. IL-2 can also be expressed by dendritic cells and NK cells. IL-2 can bind to IL-2R, which contains three subunits (CD25, CD122, and common γc), all of which are necessary for binding. IL-2 can act as a B cell growth factor in the development of regulatory T (Treg) cells, stimulate antibody synthesis, and promote the proliferation and differentiation of NK cells and T helper cells. In some embodiments, the cytokine comprises human IL-2 or a variant thereof. In some embodiments, the cytokine comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 100. In some embodiments, the cytokine is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO:150.

[0137] In some embodiments, the cytokine comprises IL-4 or a variant thereof. IL-4 can be produced by Th2 cells, basophils, eosinophils, and mast cells. IL-4 can bind to two receptors, IL4-R type I, which includes CD124 (IL-4rα) and CD132, and IL4-R type II, which includes IL-4Rα and IL-13Rα1. IL-4 can play several different roles, including controlling allergic conditions and activating immune responses to extracellular parasites. IL-4 can stimulate the development of Th2 cells. In some embodiments, the cytokine comprises human IL-4 or a variant thereof. In some embodiments, the cytokine comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 101. In some embodiments, the cytokine is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 151. In some embodiments, the cytokine is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 163.

[0138] In some embodiments, the cytokine comprises IL-7 or a variant thereof. IL-7 may be a homeostatic cytokine. IL-7 can be found in T cells, B cell precursors, and bone marrow macrophages. IL-7 can bind to its receptor IL-7R, including the gamma chain fraction and IL-7Rα (CD127). IL-7 may be involved in the survival and proliferation of thymocytes and the development of naive and memory B and T cells, mature T cells, and NK cells. In some embodiments, the cytokine comprises human IL-7 or a variant thereof. In some embodiments, the cytokine comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 102. In some embodiments, the cytokine is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 152. In some embodiments, the human IL-7 or variant thereof comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to a sequence set forth in GenBank Accession Nos. AAC63047.1, EAW87060.1, EAW87061.1, EAW87062.1, ABK41904.1, BAD89408.1, BAD89409.1, BAD89411.1, BAD89412.1, BAD89414.1, BAD89422.1, BAF84227.1, AAH47698.1, ACX53627.1, AAA59156.1, ANQ68335.1.

[0139] In some embodiments, the cytokine comprises IL-9 or a variant thereof. IL-9 can be produced by Th2 cells, eosinophils, or mast cells. IL-9 can bind to its receptor, IL-9R, which comprises CD132 and the IL-9Rα domain. IL-9 can be a potent growth factor for T cells and mast cells. IL-9 can inhibit cytokine production by Th1 cells, IgE production, and mucus secretion by bronchial epithelium. In some embodiments, the cytokine comprises human IL-9 or a variant thereof. In some embodiments, the cytokine comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 103. In some embodiments, the cytokine is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 153.

[0140] In some embodiments, the cytokine comprises IL-10 or a variant thereof. In some embodiments, the cytokine comprises human IL-10 or a variant thereof. In some embodiments, the cytokine comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 104. In some embodiments, the cytokine is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 154. In some embodiments, the cytokine is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 164.

[0141] In some embodiments, the cytokine comprises IL-12 or a variant thereof. IL-12 can comprise the IL-12p35 and IL-12p40 subunits. Coexpression of the IL-12p35 and IL-12p40 subunits can result in the secretion of disulfide-linked bioactive IL-12p70. IL-12 can be produced by various hematopoietic cell types, including antigen-presenting cells, such as dendritic cells and macrophages. IL-12 can bind to its receptor IL-12Rβ1 / IL-12Rβ2, which can be expressed on activated T cells, NK cells, and dendritic cells. Binding of IL-12 to its receptor can activate TYK2 (tyrosine kinase 2), JAK2, and STAT pathways. In some embodiments, the cytokine comprises human IL-12 or a variant thereof. In some embodiments, the human IL-12 or variant thereof comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in GenBank Accession Nos. AAM34792.1, AAG32620.1, AAG32620.1, CCA63965.1, AJQ18452.1, AAD56386.1, AAL05890.1, AAL05891., AAH67502.11, AAH67498.1, AAH67498.1, AAH67500.1, AAH67501.1, AAH74723.1, ABM53138.1, AAA35695.1, AAA59938.1.

[0142] In some embodiments, the cytokine comprises IL-12p40 or a variant thereof. In some embodiments, the cytokine comprises human IL-12p40 or a variant thereof. In some embodiments, the cytokine comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 105. In some embodiments, the cytokine is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 155.

[0143] In some embodiments, the cytokine comprises IL-15 or a variant thereof. IL-15 can bind to its receptor IL-15R, which comprises the CD132 subunit, IL-15Rα, and IL-2Rβ chain. IL-15 can be produced by keratinocytes, skeletal muscle cells, monocytes, and activated CD4+ T cells in response to signals that trigger innate immunity. IL-15 can have a similar structure and some of the same functions as IL-2, such as stimulating T cell activation and NK cell proliferation. IL-15 can also be involved in CD8+ memory cell, NK cell, and NKT cell homeostasis. In some embodiments, the cytokine comprises human IL-15 or a variant thereof. In some embodiments, the cytokine comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 106. In some embodiments, the cytokine is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 156. In some embodiments, human IL-15 or a variant thereof is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 156. In some embodiments, human IL-15 or a variant thereof is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 156. In some embodiments, human IL-15 or a variant thereof is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 156. 2.1, AAB97518.1, BAG53839.1, BAF83308.1, AAU21241.1, CAA71044.1, AAH18149.1, AAB97518.1, CAA63914.1, CAG46777.1, CAG46804.1, AAD15004.1, AAA21551.1, and CAA63913.1, and includes an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in

[0144] In some embodiments, the cytokine comprises IL-18 or a variant thereof. IL-18 can promote TH1 and TH2 cell responses. IL-18, together with IL-2, can induce IL-13 production in T cells and NK cells. IL-18 can enhance NK toxicity by promoting Fas ligand expression in NK cells. IL-18 may be involved in several autoimmune diseases, myocardial infarction, metabolic syndrome, etc. In some embodiments, the cytokine comprises human IL-18 or a variant thereof. In some embodiments, the cytokine comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 107. In some embodiments, the cytokine is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 157.

[0145] In some embodiments, the cytokine comprises IL-21 or a variant thereof. IL-21 can be produced by T cells, NKT cells, and Th17. IL-21 can bind to its receptors, including CD132 and IL-21R. IL-21 can be involved in B cell function. IL-21 can increase the proliferation of CD8+ T cells, NK cells, and NKT cells. In some embodiments, the cytokine comprises human IL-21 or a variant thereof. In some embodiments, the cytokine comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 108. In some embodiments, the cytokine is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 158. In some embodiments, human IL-15 or a variant thereof is selected from the group consisting of GenBank Accession Nos. AAU88182.1, EAX05226.1, CA194500.1, CAJ47524.1, CAL81203.1, CAN87399.1, CAS03522.1, CAV33288.1, CBE74752.1, CB170418.1, CBI85469.1, CB185472.1, CBL and AAH66262.1, CCA63962.1, AAG29348.1, AAH66258.1, AAH66259.1, AAH66260.1, AAH66261.1, AAH66262.1, AAH69124.1, ABG36529.1, and BBA22643.1.

[0146] In some embodiments, the cytokine comprises IL-27 or a variant thereof. In some embodiments, the cytokine comprises human IL-27 or a variant thereof. IL-27 can bind to the ubiquitously expressed gp130 protein and its receptor IL-27R, including WSX-1 / TCCR. The biological effects of IL-27 can be mediated through activation of JAK1, JAK2, TYK2, STAT1, and STAT3. In some embodiments, the cytokine comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 109. In some embodiments, the cytokine is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 159.

[0147] In some embodiments, the cytokine comprises IL-36γ or a variant thereof. In some embodiments, the cytokine comprises human IL-36γ or a variant thereof. In some embodiments, the cytokine comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 110. In some embodiments, the cytokine is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 160.

[0148] In some embodiments, the cytokine comprises IL-23 or a variant thereof. In some embodiments, the cytokine comprises IL-23p19 or a variant thereof. IL-23 can bind to its receptor derived from the combination of IL-12Rβ1 and the unique IL-23 receptor subunit (IL-23R). The biological effects of IL-23 on its target cells may be mediated through activation of TYK2, JAK2, STAT3, and STAT4. In some embodiments, the cytokine comprises human IL-23 or a variant thereof. In some embodiments, the cytokine comprises human IL-23p19 or a variant thereof. In some embodiments, the cytokine comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO:111. In some embodiments, the cytokine is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO:161.

[0149] In some embodiments, the cytokine comprises IL-1α or a variant thereof. In some embodiments, the cytokine comprises human IL-1α or a variant thereof. In some embodiments, the cytokine comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 112. In some embodiments, the cytokine is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 162.

[0150] [Table 2-1]

[0151] [Table 2-2]

[0152] [Table 3-1]

[0153] [Table 3-2]

[0154] [Table 3-3]

[0155] Anchor Structure In some embodiments, provided herein are anchor structures present as part of the polypeptides described in the present disclosure, including a polypeptide in a single cytokine-anchor material, a first polypeptide and a second polypeptide in two cytokine-anchor materials, and a first polypeptide, a second polypeptide, and a third polypeptide in three cytokine-anchor materials. The anchor structure can be any structure that anchors itself and any peptides linked to it to a cell membrane.

[0156] In some embodiments of the present disclosure, the cell membrane anchor structure is a polypeptide cell membrane anchor structure comprising an amino acid sequence. The anchor structure can bind the cytokine to the membrane of a cell. In various embodiments, a cytokine described herein is operably linked to the anchor structure. The anchor structure can include a peptide anchor or a non-peptide anchor. The non-peptide anchor can be bound to the polypeptide via a non-peptide anchor binding signal. The non-peptide anchor binding signal can be part of the polypeptide. Optionally, the non-peptide anchor binding signal is operably linked to a cytokine described herein. Optionally, the non-peptide anchor binding signal is operably linked to a cytokine described herein. Optionally, the peptide anchor is operably linked to a cytokine described herein.

[0157] The peptide anchor provided herein can be encoded by a peptide anchor nucleic acid sequence.The peptide anchor nucleic acid sequence can be present as part of the nucleic acid sequence described in the present disclosure, including the nucleic acid sequence in a single cytokine-anchor material, the first nucleic acid sequence and the second nucleic acid sequence in two cytokine-anchor materials, and the first nucleic acid sequence, the second nucleic acid sequence and the third nucleic acid sequence in three cytokine-anchor materials.

[0158] The non-peptide anchor binding signal can be encoded by a non-peptide anchor binding signal nucleic acid sequence. The non-peptide anchor binding signal nucleic acid sequence can be present as part of the nucleic acid sequences described in the present disclosure, including the nucleic acid sequence in a single cytokine-anchor material, the first and second nucleic acid sequences in two cytokine-anchor materials, and the first, second, and third nucleic acid sequences in three cytokine-anchor materials.

[0159] Non-peptide anchor In some embodiments, the non-peptide anchor does not comprise an amino acid sequence. In some embodiments, the non-peptide anchor can be attached to a protein during protein processing. In some embodiments, the non-peptide anchor can be attached to a protein by replacing the non-peptide anchor attachment signal located at the C-terminus of the protein.

[0160] The non-peptide anchors provided herein can include lipids such as glycolipids. Glycolipids can be lipids with carbohydrates linked by glycosidic bonds. Glycolipids can maintain the stability of cell membranes. Glycolipids can promote cell recognition. Glycolipids can be located on the surface of all eukaryotic cell membranes. Glycolipids can extend from the phospholipid bilayer to the extracellular environment. Glycolipids can include glyceroglycolipids and sphingoglycolipids. Glycolipids can include acetylated or non-acetylated glycerol with at least one fatty acid as a lipid complex. Glycolipids can further include galactolipids and sulfolipids.

[0161] Optionally, the glycolipid comprises a glycosylphosphatidylinositol (GPI). Optionally, the non-peptide anchor comprises a GPI anchor. Optionally, the non-peptide anchor is linked to a non-peptide anchor attachment signal. Optionally, the non-peptide anchor attachment signal comprises a glycolipid attachment signal. Optionally, the non-peptide anchor attachment signal comprises a GPI-attachment signal. Optionally, the C-terminal GPI attachment signal spans 20-30 amino acids starting from the amino acid to which the GPI is attached after the GPI attachment signal is cleaved. Optionally, such amino acids to which the GPI is attached comprise Ser, Asn, Asp, Ala, Gly, Cys, or Thr. The GPI-attachment signal peptide can comprise a stretch of about 10 hydrophilic amino acids. The GPI-attachment signal peptide can comprise a stretch of about 20 hydrophobic amino acids.

[0162] In some embodiments, a non-peptide anchor can be attached to the C-terminus of a polypeptide during post-translational modification, anchoring the cytokine to the cell membrane. In some embodiments, the non-peptide anchor is a glycosylated phosphatidylinositol anchor (GPI anchor). The GPI anchor can include a phosphoethanolamine linker, a core glycan, and a phospholipid tail. The structure of the core glycan can be EtNP-6Manα2-Manα6-(EtNP)2Manα4-GlNα6-myoIno-P-lipid (EtNP, ethanolamine phosphate; Man, mannose; GlcN, glucosamine; Ino, inositol). Optionally, the GPI anchor can be linked to the C-terminus of the polypeptide via an amide bond formed between the C-terminal carboxyl group and the amino group of the terminal EtNP. Optionally, the core glycan can be modified with a side chain selected from, for example, a phosphoethanolamine group, mannose, galactose, sialic acid, or other sugars. Examples of GPI anchors, their synthesis, structure, and function are described in Kinoshita Taroh, 2020 Biosynthesis and biology of mammalian GPI-anchored proteins Open Biol. 10190290190290; Paulick MG et al., Biochemistry. 2008;47(27):6991-7000, all of which are incorporated by reference in their entirety for all purposes.

[0163] The attachment of a GPI anchor can be a post-translational modification of a protein that adds glycosylated phosphatidylinositol, which can allow the protein to be anchored to the extracellular surface of the cell membrane. In some cases, wild-type proteins with GPI anchors do not contain transmembrane or cytoplasmic domains. GPI anchors can be found in different families of proteins, including membrane-bound enzymes, adhesion molecules, and proteins that coat the outer surface of protoparasites such as Trypanosoma brucei.

[0164] GPI anchors can be attached to polypeptides via a GPI-linkage signal located at the C-terminus of the polypeptide. After translocation through the endoplasmic reticulum, the GPI-linkage signal can be cleaved and replaced by a GPI anchor by specific transamidases. Protein modification by the addition of a GPI anchor confers specific properties to the protein because the added lipid moiety allows the protein to insert into the cell membrane, thereby anchoring the protein. In some embodiments, the GPI anchor is selected from the group consisting of rat brain Thy-1, human erythrocyte AChE, hamster brain scrapie prion protein, human urinary CD59, mouse skeletal muscle NCAM, bovine liver 5'-nucleotidase, human placental APase, human CD52, porcine kidney membrane dipeptidase, human kidney membrane dipeptidase, Trypanosoma brucei VSG, Trypanosoma cruzi IG7, T. cruzimucins, Trypanosoma cruzi NETNES, Leishmania miltiorrhiza gp63, Saccharomyces cerevisiae gp125, Aspergillus fumigatus PhoAp, Pyrus commmunis arabinogalactan proteins, Dictyostelium discoideum PsA, Trypanosoma congo L. congolense) VSG, or Torpedo AChE.

[0165] In some embodiments, the GPI attachment signal comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 200. In some embodiments, the GPI attachment signal is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 250-252. In some embodiments, the GPI attachment signal is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NO: 250. In some embodiments, the GPI attachment signal is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NO: 251. In some embodiments, the GPI attachment signal is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NO:252.

[0166] [Table 4A]

[0167] [Table 4B]

[0168] Peptide anchor In some embodiments, the peptide anchor comprises an amino acid sequence derived from at least a portion of a transmembrane domain of any suitable transmembrane protein, hi some embodiments, the peptide anchor comprises a transmembrane peptide sequence.

[0169] In some embodiments, the peptide anchor comprises a portion of a transmembrane peptide sequence. In some embodiments, the transmembrane peptide sequence comprises a B7-1 transmembrane amino acid sequence, a B7-2 transmembrane amino acid sequence, a B7-H1 transmembrane amino acid sequence, a B7-H3 transmembrane amino acid sequence, a tumor necrosis factor receptor 2 (TNFR2) transmembrane amino acid sequence, a CD8α transmembrane amino acid sequence, a CD28 transmembrane amino acid sequence, a CD3ζ transmembrane amino acid sequence, a CTLA-4 (CD152) transmembrane amino acid sequence, or a PD-L1 transmembrane amino acid sequence, or any variant thereof. In some embodiments, the peptide anchor comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO:203. In some embodiments, the peptide anchor is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO:253. In some embodiments, the peptide anchor comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 204. In some embodiments, the peptide anchor is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 254. In some embodiments, the peptide anchor is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 255.

[0170] In some embodiments, the peptide anchor comprises at least a portion of the transmembrane domain of B7-1, B7-2, CD8, and CD28.

[0171] In some embodiments, the peptide anchor comprises at least a portion of the B7-1 transmembrane-intracellular amino acid sequence. In some embodiments, the peptide anchor comprises at least a portion of the B7-2 transmembrane-intracellular amino acid sequence. In some embodiments, the peptide anchor comprises at least a portion of the CD8α transmembrane-intracellular amino acid sequence. In some embodiments, the peptide anchor comprises at least a portion of the B7-1 transmembrane amino acid sequence. In some embodiments, the peptide anchor comprises at least a portion of the B7-2 transmembrane amino acid sequence. In some embodiments, the peptide anchor comprises at least a portion of the CD8α transmembrane amino acid sequence. In some embodiments, the peptide anchor comprises the B7-H1 transmembrane amino acid sequence, or any fragment or variant thereof. In some embodiments, the peptide anchor comprises the B7-H3 transmembrane amino acid sequence, or any fragment or variant thereof. In some embodiments, the peptide anchor comprises the tumor necrosis factor receptor 2 (TNFR2) transmembrane amino acid sequence, or any fragment or variant thereof. In some embodiments, the peptide anchor comprises the CD28 transmembrane amino acid sequence, or any fragment or variant thereof. In some embodiments, the peptide anchor comprises a CD3ζ transmembrane amino acid sequence or any fragment or variant thereof. In some embodiments, the peptide anchor comprises a CTLA-4 (CD152) transmembrane amino acid sequence or any fragment or variant thereof. In some embodiments, the peptide anchor comprises a PD-L1 transmembrane amino acid sequence or any fragment or variant thereof.

[0172] In some embodiments, the peptide anchor is located at the C-terminus of the polypeptide. In some embodiments, the signal peptide described herein, the cytokine peptide described herein, and the peptide anchor are operably linked in the direction from the N-terminus of the polypeptide to the C-terminus of the polypeptide.

[0173] [Table 5A]

[0174] [Table 5B]

[0175] targeting part In some embodiments, provided herein is a targeting moiety present as part of a polypeptide described in the present disclosure, including a polypeptide in a single cytokine-anchor material, a first polypeptide and a second polypeptide in two cytokine-anchor materials, and a first polypeptide, a second polypeptide, and a third polypeptide in three cytokine-anchor materials. The targeting moiety can include a CAR, a T cell receptor (TCR), or a B cell receptor (BCR), or a fragment thereof.

[0176] In some embodiments, the targeting moiety comprises a TCR or a fragment thereof. The TCR can be a recombinant TCR produced by recombinant expression of a gene encoding one or more exogenous TCR alpha, beta, gamma, and / or delta chains. The recombinant TCR can be a chimeric or hybrid TCR composed of amino acid sequences of TCRs from two or more mammalian species. The TCR can be a humanized TCR. The TCR can comprise an alpha chain. The TCR can comprise a beta chain. The TCR can comprise a gamma chain of the TCR. The TCR can comprise a delta chain. Polypeptide chains of TCRs are known in the art.

[0177] The targeting moiety can be connected to the polypeptide described herein via a cleavable linker as described herein.The targeting moiety is encoded by a targeting sequence that can be connected to a nucleic acid sequence via a cleavable linker nucleic acid sequence.Optionally, the targeting moiety recognizes an antigen.Optionally, the targeting moiety binds to an antigen.

[0178] In some cases, the targeting moiety recognizes a tumor-specific antigen. A tumor-specific antigen can be a molecule, including a protein, polypeptide, peptide, lipid, carbohydrate, etc., that is predominantly or overexpressed by tumor cells, such that the antigen can be considered specifically associated with tumors or cancer. A tumor-specific antigen can be expressed by normal cells, non-tumor cells, or non-cancerous cells, but at a lower or less potent level than that expressed by tumor cells. Tumor cells can overexpress tumor-specific antigens or express tumor-specific antigens at a significantly higher level than that expressed by normal non-cancerous cells. A tumor-specific antigen can be expressed by cells in different states of development or maturation. For example, a tumor-specific antigen can be expressed by embryonic or fetal cells that are not normally found in adult subjects. A tumor-specific antigen can be expressed by stem or progenitor cells that are not normally found in adult subjects. In some cases, a tumor-specific antigen can be a mutant antigen that is predominantly or overexpressed by tumor or cancer cells and not expressed or expressed at a significantly lower level by normal non-cancerous cells.

[0179] Examples of tumor-specific antigens include mesothelin, gp100, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, CD138, CD276, CD171, CD5, CD7, MUC1, AFP, CEA, PSCA, PSMA, HER2, EGFR, IL13Ralpha2, GD2, NKG2D, EGFTvIII, CS1, CCL1, BCMA, mesothelin, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ralpha2, PRSS21, VEGR2, LewisY, CD24, PDGFR-beta, SSEA-4, AFP, NCAM, Claudin18.2, and GPC. 3, GM3, TGS5, HMWMAA, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TRAP, WT1, NY-ESO-1, LAGE-la, MAGE -A1, MAGE-A2, BRCA, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, EGFRVIII, VEGFR-2, TRP-1, TRP-2, Tyrosinase, Human Papilloma Virus (HPV) 16 E6, HPV 16 E7, HPV 18 E6, HPV 18 E7, KK-LC-1, NY-BR-, NY-ESO-1 (or CAG-3), SSX-2, SSX-3, SSX-4, SSX-5, SSX-9, SSX-1. In some embodiments, the tumor-specific antigen is CD19.

[0180] In some embodiments, the targeting moiety comprises a chimeric antigen receptor (CAR). The CAR can comprise a ligand binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0181] In some embodiments, the ligand-binding domain recognizes a tumor-specific antigen described herein. In some embodiments, the ligand-binding domain binds to a tumor-specific antigen described herein. In some embodiments, the ligand-binding domain is derived from an antibody or antibody fragment (e.g., a murine, human, or humanized antibody) that recognizes a tumor-specific antigen described herein.

[0182] In some embodiments, the CAR is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, CD5, CD7, MUC1, AFP, CEA, PSCA, PSMA, Her2, EGFR, IL13Ra2, GD2, NKG2D, EGFRvIII, CS1, CCL1, BCMA, mesothelin, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, PRSS21, VEGFR 2, LewisY, CD24, PDGFR-β, SSEA-4, AFP, NCAM, Claudin18.2, GM3, TGS5, HMWMAA, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, or any combination thereof.

[0183] In some embodiments, the ligand binding domain of the CAR is selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, CD5, CD7, MUC1, AFP, CEA, PSCA, PSMA, Her2, EGFR, IL13Ra2, GD2, NKG2D, EGFRvIII, CS1, CCL1, BCMA, mesothelin, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, PRSS21 , VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, AFP, NCAM, Claudin18.2, GM3, TGS5, HMWMAA, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, or any combination thereof.

[0184] In some embodiments, the ligand-binding domain is an scFv fragment. In some embodiments, the ligand-binding domain targets CD19. In some embodiments, the ligand-binding domain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO:401. In some embodiments, the ligand-binding domain comprises an amino acid sequence at least 75% identical to the sequence set forth in SEQ ID NO:401. In some embodiments, the ligand-binding domain comprises an amino acid sequence at least 80% identical to the sequence set forth in SEQ ID NO:401. In some embodiments, the ligand-binding domain comprises an amino acid sequence at least 85% identical to the sequence set forth in SEQ ID NO:401. In some embodiments, the ligand-binding domain comprises an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO:401. In some embodiments, the ligand-binding domain comprises an amino acid sequence at least 95% identical to the sequence set forth in SEQ ID NO:401. In some embodiments, the ligand-binding domain comprises an amino acid sequence at least 98% identical to the sequence set forth in SEQ ID NO:401. In some embodiments, the ligand-binding domain comprises an amino acid sequence at least 99% identical to the sequence set forth in SEQ ID NO:401. In some embodiments, the ligand-binding domain comprises an amino acid sequence 100% identical to the sequence set forth in SEQ ID NO:401. In some embodiments, the ligand-binding domain is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO:451. In some embodiments, the ligand-binding domain is encoded by a nucleic acid sequence at least 75% identical to the sequence set forth in SEQ ID NO:451. In some embodiments, the ligand-binding domain is encoded by a nucleic acid sequence at least 80% identical to the sequence set forth in SEQ ID NO:451. In some embodiments, the ligand-binding domain is encoded by a nucleic acid sequence at least 85% identical to the sequence set forth in SEQ ID NO:451.In some embodiments, the ligand-binding domain is encoded by a nucleic acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 451. In some embodiments, the ligand-binding domain is encoded by a nucleic acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 451. In some embodiments, the ligand-binding domain is encoded by a nucleic acid sequence that is at least 98% identical to the sequence set forth in SEQ ID NO: 451. In some embodiments, the ligand-binding domain is encoded by a nucleic acid sequence that is at least 99% identical to the sequence set forth in SEQ ID NO: 451. In some embodiments, the ligand-binding domain is encoded by a nucleic acid sequence that is 100% identical to the sequence set forth in SEQ ID NO: 451.

[0185] Optionally, the CAR comprises a leader. The leader can be located at the N-terminus of the CAR. The leader can be connected to the ligand-binding domain. In some embodiments, the leader comprises a CD8α leader or a variant thereof. In some embodiments, the leader comprises a β2M signal peptide or a variant thereof. In some embodiments, the leader comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 400. In some embodiments, the leader comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 4. In some embodiments, the leader is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 450. In some embodiments, the leader is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO:54.

[0186] Optionally, the CAR comprises a hinge region. The hinge region can connect the ligand-binding domain and the transmembrane domain. Optionally, the hinge region is derived from a human protein. In some embodiments, the hinge region comprises the hinge region of a human Ig hinge, such as an IgG1 IgG4, IgD, FcγRIIIα, KIR2DS2 hinge, or CD8α hinge. In some embodiments, the hinge region comprises a peptide linker described herein, e.g., a GS linker. In some embodiments, the hinge region comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 402. In some embodiments, the hinge region is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 452.

[0187] In some cases, the CAR comprises a transmembrane domain, which can be the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 zeta subunit, a CD3 epsilon subunit, a CD3 gamma subunit, a CD3 delta subunit, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD123, CD134, CD137, CD154, or any combination thereof. In some embodiments, the transmembrane domain comprises the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 zeta subunit, a CD3 epsilon subunit, a CD3 gamma subunit, a CD3 delta subunit, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD123, CD134, CD137, CD154, or any fragment or variant thereof.

[0188] In some embodiments, the transmembrane domain comprises the transmembrane domain of CD8α or a fragment thereof. In some embodiments, the transmembrane domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 403. In some embodiments, the transmembrane domain is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 453.

[0189] In some cases, the CAR comprises a costimulatory domain. In some cases, the costimulatory domain comprises at least a portion of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD16, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, LFA-1, CD3ε, or any combination thereof. In other cases, the costimulatory domain comprises at least a portion of any other costimulatory domain having an immunoreceptor tyrosine-based activation motif. In some embodiments, the costimulatory domain is the costimulatory domain of CD28. In some embodiments, the costimulatory domain is the costimulatory domain of CD137 (4-1BB). In some embodiments, the costimulatory domain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 404. In some embodiments, the costimulatory domain is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 454.

[0190] In some embodiments, the CAR comprises an intracellular signaling domain. The intracellular signaling domain can comprise at least a portion of the intracellular signaling domain from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, or CD66d. In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ. In some embodiments, the intracellular signaling domain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 405. In some embodiments, the intracellular signaling domain is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 455.

[0191] In some embodiments, the CAR comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NO:4 or 400-407. In some embodiments, the CAR comprises an amino acid sequence that is at least 75% identical to any of the sequences set forth in SEQ ID NO:4 or 400-407. In some embodiments, the CAR comprises an amino acid sequence that is at least 80% identical to any of the sequences set forth in SEQ ID NO:4 or 400-407. In some embodiments, the CAR comprises an amino acid sequence that is at least 85% identical to any of the sequences set forth in SEQ ID NO:4 or 400-407. In some embodiments, the CAR comprises an amino acid sequence that is at least 90% identical to any of the sequences set forth in SEQ ID NO:4 or 400-407. In some embodiments, the CAR comprises an amino acid sequence that is at least 95% identical to any of the sequences set forth in SEQ ID NO:4 or 400-407. In some embodiments, the CAR comprises an amino acid sequence that is at least 98% identical to any of the sequences set forth in SEQ ID NO:4 or 400-407. In some embodiments, the CAR comprises an amino acid sequence at least 99% identical to any of the sequences set forth in SEQ ID NO:4 or 400-407. In some embodiments, the CAR comprises an amino acid sequence 100% identical to any of the sequences set forth in SEQ ID NO:4 or 400-407. In some embodiments, the CAR comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO:406. In some embodiments, the CAR comprises an amino acid sequence at least 80% identical to the sequence set forth in SEQ ID NO:406. In some embodiments, the CAR comprises an amino acid sequence at least 85% identical to the sequence set forth in SEQ ID NO:406. In some embodiments, the CAR comprises an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO:406. In some embodiments, the CAR comprises an amino acid sequence at least 95% identical to the sequence set forth in SEQ ID NO:406.In some embodiments, the CAR comprises an amino acid sequence that is 100% identical to the sequence set forth in SEQ ID NO: 406. In some embodiments, the CAR comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 407.

[0192] In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 54 or 450-457. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 75% identical to any of the sequences set forth in SEQ ID NOs: 54 or 450-457. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 80% identical to any of the sequences set forth in SEQ ID NOs: 54 or 450-457. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 85% identical to any of the sequences set forth in SEQ ID NOs: 54 or 450-457. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 90% identical to any of the sequences set forth in SEQ ID NOs: 54 or 450-457. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 95% identical to any of the sequences set forth in SEQ ID NOs: 54 or 450-457. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 98% identical to any of the sequences set forth in SEQ ID NOs: 54 or 450-457. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 99% identical to any of the sequences set forth in SEQ ID NOs: 54 or 450-457. In some embodiments, the CAR is encoded by a nucleic acid sequence that is 100% identical to any of the sequences set forth in SEQ ID NOs: 54 or 450-457. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 456. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 456. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 85% identical to the sequence set forth in SEQ ID NO: 456. In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 456.In some embodiments, the CAR is encoded by a nucleic acid sequence that is at least 95% identical to the sequence set forth in SEQ ID NO: 456. In some embodiments, the CAR is encoded by a nucleic acid sequence that is 100% identical to the sequence set forth in SEQ ID NO: 456. In some embodiments, the CAR is encoded by a nucleic acid sequence that is 100% identical to the sequence set forth in SEQ ID NO: 457.

[0193] [Table 6A]

[0194] [Table 7-1]

[0195] [Table 7-2]

[0196] Cleavable linker sequence In some embodiments, provided herein are cleavable linkers capable of connecting one or more components described in the present disclosure, including a targeting moiety and a polypeptide in a single cytokine-anchor material, a targeting moiety, a first polypeptide, and a second polypeptide in two cytokine-anchor materials, and a targeting moiety, a first polypeptide, a second polypeptide, and a third polypeptide in three cytokine-anchor materials.

[0197] In some embodiments, a cleavable linker connects a targeting moiety to a signal peptide of a polypeptide. In some embodiments, a cleavable linker connects a targeting moiety to a peptide anchor of a polypeptide. In some embodiments, a cleavable linker connects a targeting moiety to a non-peptide anchor binding signal. In some embodiments, a cleavable linker connects a first polypeptide to a second polypeptide. In some embodiments, a cleavable linker connects a first polypeptide to a third polypeptide. In some embodiments, a cleavable linker connects a third polypeptide to a second polypeptide.

[0198] The cleavable linker can be encoded by a cleavable linker nucleic acid sequence that can connect two or more nucleic acid sequences described in the present disclosure, including a targeting sequence and a nucleic acid sequence in a single cytokine-anchor material, a targeting sequence, a first nucleic acid sequence, and a second nucleic acid sequence in two cytokine-anchor materials, and a targeting sequence, a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence in three cytokine-anchor materials.

[0199] In some embodiments, the second nucleic acid sequence and the first nucleic acid sequence are linked via a cleavable linker nucleic acid sequence. In some embodiments, the third nucleic acid sequence and the first nucleic acid sequence are linked via a cleavable linker nucleic acid sequence. In some embodiments, the second nucleic acid sequence and the third nucleic acid sequence are linked via a cleavable linker nucleic acid sequence. In some embodiments, the targeting sequence and the first nucleic acid sequence are linked via a cleavable linker nucleic acid sequence. In some embodiments, the targeting sequence and the second nucleic acid sequence are linked via a cleavable linker nucleic acid sequence. In some embodiments, the targeting sequence and the third nucleic acid sequence are linked via a cleavable linker nucleic acid sequence.

[0200] In some embodiments, the cleavable linker sequence between a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding a second polypeptide can be cleaved, thereby generating two polypeptides: a first polypeptide comprising a first cytokine and a first anchor structure, and a second polypeptide comprising a second cytokine and a second anchor structure. In some embodiments, the cleavable linker sequence between a second nucleic acid sequence encoding a second polypeptide and a third nucleic acid sequence encoding a third polypeptide can be cleaved, thereby generating two polypeptides: a second polypeptide comprising a second cytokine and a second anchor structure, and a third polypeptide comprising a third cytokine and a second anchor structure. In some embodiments, the cleavable linker sequence between a first nucleic acid sequence encoding a first polypeptide and a third nucleic acid sequence encoding a third polypeptide can be cleaved, thereby generating two polypeptides: a first polypeptide comprising a first cytokine and a first anchor structure, and a third polypeptide comprising a third cytokine and a second anchor structure.

[0201] In some embodiments, the cleavable linker sequence between the first nucleic acid sequence encoding the first polypeptide and the targeting sequence encoding the targeting moiety can be cleaved, thereby generating two polypeptides: a first polypeptide comprising a first cytokine and a first anchor structure, and a targeting moiety comprising a CAR, e.g., CAR19 (e.g., having the sequence set forth in SEQ ID NO: 406). In some embodiments, the cleavable linker sequence between the second nucleic acid sequence encoding the second polypeptide and the targeting sequence encoding the targeting moiety can be cleaved, thereby generating two polypeptides: a second polypeptide comprising a second cytokine and a second anchor structure, and a targeting moiety comprising a CAR, e.g., CAR19. In some embodiments, the cleavable linker sequence between the third nucleic acid sequence encoding the third polypeptide and the targeting sequence encoding the targeting moiety can be cleaved, thereby generating two polypeptides: a third polypeptide comprising a third cytokine and a third anchor structure, and a targeting moiety comprising a CAR, e.g., CAR19.

[0202] The cleavable linker can be any cleavable linker that connects two peptides.

[0203] In some embodiments, the length of the cleavable linker is not limited and can include about 20 to about 30 amino acid residues, for example, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 amino acid residues.

[0204] Optionally, the cleavable linker comprises an IRES element, such as an IRES element derived from encephalomyocarditis virus (EMCV). Optionally, the cleavable linker comprises a self-cleaving 2A peptide. The 2A peptide can be a viral oligopeptide that mediates polypeptide cleavage during translation in eukaryotic cells. The term "2A" refers to a specific region of the viral genome.

[0205] Without wishing to be bound by any particular theory, the mechanism of 2A-mediated autocleavage may be ribosomal "skipping" of glycyl-prolyl peptide bond formation at the C-terminus of the 2A peptide, rather than true proteolytic cleavage.

[0206] In some embodiments, the cleavable linker comprises a 2A self-cleaving peptide or a 2A-like peptide derived from foot-and-mouth disease virus or cardiovirus. In some embodiments, the cleavable linker comprises a 2A peptide sequence, including the amino acid sequence of porcine Czech virus-1 2A (P2A), the amino acid sequence of equine rhinitis A virus (E2A), the amino acid sequence of beta-tetrasomy virus 2A (T2A), or the amino acid sequence of foot-and-mouth disease virus (F2A). In some embodiments, the cleavable linker comprises a P2A peptide sequence. In some embodiments, the cleavable linker comprises a T2A peptide sequence. In some embodiments, the cleavable linker comprises an E2A peptide sequence. In some embodiments, the cleavable linker comprises an F2A peptide sequence. In some embodiments, the cleavable linker comprises an IRES peptide. In some embodiments, the cleavable linker comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 300-303. In some embodiments, the cleavable linker comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 300. In some embodiments, the cleavable linker comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 301. In some embodiments, the cleavable linker comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 302. In some embodiments, the cleavable linker comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO:303.

[0207] In some embodiments, the cleavable linker is encoded by a cleavable linker nucleic acid sequence comprising a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 350-355. In some embodiments, the cleavable linker is encoded by a cleavable linker nucleic acid sequence comprising a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 350. In some embodiments, the cleavable linker is encoded by a cleavable linker nucleic acid sequence comprising a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 351. In some embodiments, the cleavable linker is encoded by a cleavable linker nucleic acid sequence comprising a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 352. In some embodiments, the cleavable linker is encoded by a cleavable linker nucleic acid sequence comprising a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 353. In some embodiments, the cleavable linker is encoded by a cleavable linker nucleic acid sequence comprising a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 354. In some embodiments, the cleavable linker is encoded by a cleavable linker nucleic acid sequence that comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO:355.

[0208] In other cases, the cleavable linker comprises a furin cleavage site or a tobacco etch virus cleavage site. Furin cleavage sites and sequences are described in Klimstra et al., J Virol. 1999 Aug; 73(8):6299-6306, U.S. Patent No. 8,871,906, all of which are incorporated by reference in their entirety for all purposes.

[0209] [Table 8A]

[0210] [Table 8B]

[0211] Peptide Linker In some embodiments, provided herein are peptide linkers capable of connecting one or more components described in the present disclosure, including anchor structures for a cytokine and a polypeptide in a single cytokine-anchor material, anchor structures for a cytokine, a first polypeptide, and a second polypeptide in two cytokine-anchor materials, and anchor structures for a cytokine, a first polypeptide, a second polypeptide, and a third polypeptide in three cytokine-anchor materials.

[0212] In some embodiments, a peptide linker connects the cytokine to the anchor structure. In some embodiments, a peptide linker connects the cytokine to the peptide anchor. In some embodiments, a peptide linker connects the cytokine to a non-peptide anchor binding signal. In some embodiments, a peptide linker connects the peptide anchor to a cleavable linker. In some embodiments, a peptide linker connects the non-peptide anchor binding signal to a cleavable linker.

[0213] The peptide linker provided herein is not particularly limited. Optionally, the peptide linker in the polypeptide provided herein is a flexible peptide linker. Optionally, the peptide linker in the polypeptide provided herein is a non-flexible peptide linker. Optionally, the peptide linker in the polypeptide provided herein is not a self-cleavable linker. The peptide linker can be any suitable linker sequence that connects the anchor structure to any other component. The peptide linker can be any suitable linker sequence that connects the anchor structure to a cytokine. The peptide linker can be any suitable linker sequence that connects the anchor structure to a cleavable linker and further connects to another polypeptide or targeting moiety.

[0214] In some embodiments, the peptide linker is not particularly limited in length. In some embodiments, the peptide linker contains about 2 to about 10 amino acid residues. In some embodiments, the peptide linker contains about 10 to about 65 amino acid residues, about 18 to about 61 amino acid residues, or about 25 to about 50 amino acid residues. In some embodiments, the peptide linker comprises about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, or about 65 amino acid residues.

[0215] In some embodiments, the peptide linker comprises a GS linker, an Lr1 linker, or an Lr8 linker.

[0216] In some embodiments, the peptide linker comprises glycine and serine residues. In some embodiments, the peptide linker comprises one or more repeats of G4S or G3S, e.g., about 3 to about 15 or about 5 to about 12 repeats of G4S and G3S. In some embodiments, the peptide linker comprises about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 repeats of G4S. In some embodiments, the peptide linker comprises about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 repeats of G3S.

[0217] In some embodiments, the peptide linker is a glycine polymer (G) n In some embodiments, the peptide linker comprises a glycine-serine polymer (G 1-5 S 1-5 )n, where n is 1, 2, 3, 4, or 5. In some embodiments, the peptide linker comprises a glycine-alanine polymer. In some embodiments, the peptide linker comprises an alanine-serine polymer. In some embodiments, the peptide linker comprises GGG. In some embodiments, the peptide linker comprises DGGGS. In some embodiments, the peptide linker comprises TGEKP. In some embodiments, the peptide linker comprises (GGGGS) n (wherein n=1, 2, 3, 4, or 5). In some embodiments, the peptide linker comprises EGKSSGSGSESKVD. In some embodiments, the peptide linker comprises KESGSVSSEQLAQFRSLD. In some embodiments, the peptide linker comprises GGRRGGGS. In some embodiments, the peptide linker comprises LQRDGERP. In some embodiments, the peptide linker comprises LRQKDGGGSERP. In some embodiments, the peptide linker comprises LRQKD(GGGS)2ERP.

[0218] In some embodiments, the peptide linker comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO:500, 501, 504, 506, or 507, or any of the sequences of LE, AS, GSG, or EF. In some embodiments, the peptide linker comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO:500. In some embodiments, the peptide linker comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO:501. In some embodiments, the peptide linker comprises the sequence of LE. In some embodiments, the peptide linker comprises the sequence of AS. In some embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO:504. In some embodiments, the peptide linker comprises the sequence of GSG. In some embodiments, the peptide linker comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 506. In some embodiments, the peptide linker comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 507. In some embodiments, the peptide linker comprises the sequence of EF. In some embodiments, the peptide linker is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence of gagttc. In some embodiments, the peptide linker is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO:509.In some embodiments, the peptide linker is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence set forth in SEQ ID NO: 520. In some embodiments, the peptide linker is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence ggctccggc. In some embodiments, the peptide linker is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence ggaagcgga.

[0219] Other examples of peptide linkers are described in Liu et al., PNAS, pp. 5525-5530 (1997); Pomerantz et al., Proc. Natl. Acad. Sci. USA Vol. 92, pp. 9752-9756, October 1995; Kim et al., PNAS, Vol. 93, pp. 1156-1160 (1996); Chaudhary et al. 1990, Proc. Natl. Acad. Sci. USA, 87:1066-1070; Bird et al., 1988, Science, No. 242: pp. 423-426, all of which are incorporated herein by reference in their entirety for all purposes.

[0220] [Table 9A]

[0221] [Table 9B]

[0222] Nucleic Acid Molecules, Vectors, and Systems According to an aspect of the present disclosure, provided herein is a nucleic acid molecule encoding a polypeptide described in this disclosure.

[0223] From the primary amino acid sequence of the polypeptide(s) encoding the cytokine-anchor protein constructs provided herein, or any component thereof, one skilled in the art can determine the appropriate nucleotide sequence(s) encoding the polypeptide(s), and, if necessary, codon-optimized (see, e.g., Mauro and Chappell. Trends Mol Med. 20(11):604-613, 2014).

[0224] The nucleic acid molecule(s) encoding the polypeptides in the cytokine-anchor material, such as in some embodiments of the present disclosure, may be or be part of a vector (e.g., a plasmid, cosmid, or viral vector, or an artificial chromosome, etc.), which may include other functional regions (elements), such as one or more promoters, one or more origins of replication, one or more selectable marker(s), and one or more other elements typically found in expression vectors. Cloning and expression of nucleic acids encoding proteins, including CARs and cytokines, is well established and within the skill of one of ordinary skill in the art.

[0225] In some embodiments, the nucleic acid molecules of the cytokine-anchor material are more than 80% pure, eg, more than 90%, more than 95%, more than 97%, and more than 99% pure.

[0226] In some embodiments, vectors containing one or more of the nucleic acid sequences described in this disclosure are provided herein. The vector may be a transfer vector, which refers to a composition containing an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. "Transfer vector" includes self-replicating plasmids or viruses. This term should be interpreted to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral transfer vectors include adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like. Vectors can include expression vectors, which refer to vectors containing a recombinant polynucleotide comprising an expression control sequence operably linked to the nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0227] In some embodiments, the nucleic acid molecules described herein are vectors. In some embodiments, the vectors are viral vectors. In some embodiments, the vectors are retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors.

[0228] In some embodiments, the vector is a retroviral vector.Retroviral vector generally refers to an RNA virus that can reverse transcribe the complementary strand of DNA in infected cells, use this single strand of DNA as a template to synthesize a second strand of DNA, and then integrate it into the cell genome in DNA.Retroviral vectors can use host cell enzymes to transcribe and replicate RNA, synthesize proteins, repackage the virus, and release it from cells to become infectious virus.The transduction efficiency of retroviruses can be high, and retroviral vectors can effectively improve the transfection rate of genes.

[0229] In some embodiments, the vector is a lentiviral vector. Lentiviral vector refers to a gene therapy vector developed based on HIV-1 (human immunodeficiency virus type 1). Lentiviral vectors can infect both dividing and non-dividing cells. They can effectively infect almost all mammalian cells, including neuronal cells, hepatocytes, etc., with high infection efficiency. Lentiviruses can efficiently integrate foreign genes into host chromosomes to achieve sustained expression.

[0230] In some embodiments, the vector is a transposon plasmid.Transposon plasmid generally refers to the basic unit that exists on chromosomal DNA and can autonomously replicate and replace.Transposon plasmid can "jump" from one position to another in genome through a series of processes such as excision and reintegration.

[0231] In some embodiments, the vector is an expression vector. In some embodiments, the expression vector comprises a nucleic acid sequence encoding a targeting moiety (e.g., a CAR). In some embodiments, the expression vector comprises a nucleic acid sequence encoding a cytokine and an anchor structure. In some embodiments, the expression vector comprises a nucleic acid sequence encoding a CAR, a cytokine, and an anchor structure. In some embodiments, the expression vector comprises a second nucleic acid sequence encoding a second cytokine and a second anchor structure. In some embodiments, the expression vector comprises a second nucleic acid sequence encoding a second CAR, a second cytokine, and a second anchor structure. In some embodiments, the expression vector comprises a third nucleic acid sequence encoding a third cytokine and a third anchor structure. In some embodiments, the expression vector comprises a third nucleic acid sequence encoding a third CAR, a third cytokine, and a third anchor structure. In some embodiments, nucleic acid sequences within the expression vector can be positioned upstream or downstream. Specifically, the nucleic acid encoding the cytokine and the anchor structure can be positioned upstream or downstream of the nucleic acid encoding the CAR molecule. The nucleic acid encoding the CAR and the nucleic acid encoding the cytokine can be linked via a cleavable linker nucleic acid sequence encoding a 2A peptide or an IRES.

[0232] In some embodiments, the nucleic acid molecules and / or vectors of the present disclosure are introduced into host cells. For eukaryotic cells, suitable techniques include, for example, calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia, or baculovirus for insect cells. In some cases, the introduction of nucleic acids into host cells, particularly eukaryotic cells, uses a virus- or plasmid-based system. In some cases, the plasmid system is maintained episomally. In other cases, the plasmid system is integrated into the host cell or into an artificial chromosome. In certain embodiments, integration is by random integration of one or more copies at a single or multiple loci. In some embodiments, integration is by targeted integration of one or more copies at a single or multiple loci. For bacterial cells, suitable techniques include, for example, calcium chloride transformation, electroporation, and transfection using bacteriophage.

[0233] In some embodiments, the nucleic acid sequences are placed on the same vector. In some embodiments, two or more nucleic acid sequences are encoded by a single nucleic acid molecule as a single polypeptide chain in the same frame. Optionally, the targeting moiety (e.g., CAR) and the cytokine-anchor structure can be separated by one or more peptide cleavage sites (e.g., a self-cleavage site or a substrate for an intracellular protease). Optionally, the targeting sequence is under the control of a promoter different from that of the nucleic acid sequence or the exogenous nucleic acid sequence. Optionally, the targeting sequence is under the control of the same promoter as that of the nucleic acid sequence or the exogenous nucleic acid sequence. Optionally, the targeting sequence and the first nucleic acid sequence are under the control of the same promoter. Optionally, the targeting sequence and the first nucleic acid sequence are under the control of two different promoters. Optionally, the targeting sequence and the second nucleic acid sequence are under the control of the same promoter. Optionally, the targeting sequence and the second nucleic acid sequence are under the control of two different promoters. Optionally, the targeting sequence and the third nucleic acid sequence are under the control of the same promoter. Optionally, the targeting sequence and the third nucleic acid sequence are under the control of two different promoters. Optionally, the second nucleic acid sequence and the first nucleic acid sequence are under the control of the same promoter.Optionally, the second nucleic acid sequence and the first nucleic acid sequence are under the control of two different promoters.Optionally, the second nucleic acid sequence and the third nucleic acid sequence are under the control of the same promoter.Optionally, the second nucleic acid sequence and the third nucleic acid sequence are under the control of two different promoters.Optionally, the third nucleic acid sequence and the first nucleic acid sequence are under the control of the same promoter.Optionally, the third nucleic acid sequence and the first nucleic acid sequence are under the control of two different promoters.

[0234] Optionally, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are operably linked in a 5' to 3' direction. Optionally, the first nucleic acid sequence, the third nucleic acid sequence, and the second nucleic acid sequence are operably linked in a 5' to 3' direction. Optionally, the second nucleic acid sequence, the first nucleic acid sequence, and the third nucleic acid sequence are operably linked in a 5' to 3' direction. Optionally, the second nucleic acid sequence, the third nucleic acid sequence, and the first nucleic acid sequence are operably linked in a 5' to 3' direction. Optionally, the third nucleic acid sequence, the second nucleic acid sequence, and the first nucleic acid sequence are operably linked in a 5' to 3' direction. Optionally, the third nucleic acid sequence, the first nucleic acid sequence, and the second nucleic acid sequence are operably linked in a 5' to 3' direction.

[0235] In some cases, the targeting sequence, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are operably linked in a 5' to 3' direction. In some cases, the targeting sequence, the first nucleic acid sequence, the third nucleic acid sequence, and the second nucleic acid sequence are operably linked in a 5' to 3' direction. In some cases, the targeting sequence, the second nucleic acid sequence, the first nucleic acid sequence, and the third nucleic acid sequence are operably linked in a 5' to 3' direction. In some cases, the targeting sequence, the second nucleic acid sequence, the third nucleic acid sequence, and the first nucleic acid sequence are operably linked in a 5' to 3' direction. In some cases, the targeting sequence, the third nucleic acid sequence, the second nucleic acid sequence, and the first nucleic acid sequence are operably linked in a 5' to 3' direction. In some cases, the targeting sequence, the third nucleic acid sequence, the first nucleic acid sequence, and the second nucleic acid sequence are operably linked in a 5' to 3' direction.

[0236] In some cases, the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, and the targeting sequence are operably linked in a 5' to 3' direction. In some cases, the first nucleic acid sequence, the third nucleic acid sequence, the second nucleic acid sequence, and the targeting sequence are operably linked in a 5' to 3' direction. In some cases, the second nucleic acid sequence, the first nucleic acid sequence, the third nucleic acid sequence, and the targeting sequence are operably linked in a 5' to 3' direction. In some cases, the second nucleic acid sequence, the third nucleic acid sequence, the first nucleic acid sequence, and the targeting sequence are operably linked in a 5' to 3' direction. In some cases, the third nucleic acid sequence, the second nucleic acid sequence, the first nucleic acid sequence, and the targeting sequence are operably linked in a 5' to 3' direction. In some cases, the third nucleic acid sequence, the first nucleic acid sequence, the second nucleic acid sequence, and the targeting sequence are operably linked in a 5' to 3' direction.

[0237] In other cases, the nucleic acid sequences are located on separate vectors.

[0238] In some embodiments, the nucleic acid of the present disclosure is integrated into the genome (e.g., chromosome) of the host cell. In certain embodiments, integration is facilitated by including a sequence that facilitates recombination with the genome, according to standard techniques. In some embodiments, the targeting sequence and the nucleic acid sequence are present in the genome of the immune cell. In some embodiments, the targeting sequence, the first nucleic acid sequence, and the second nucleic acid sequence are present in the genome of the cell. In some embodiments, the targeting sequence, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are present in the genome of the cell.

[0239] According to aspects of the present disclosure, a system relating to the cytokine-anchor material described herein is provided herein. The system can include the nucleic acid molecule or vector described herein. In some embodiments, the system includes one nucleic acid molecule or one plasmid encoding a targeting moiety and one or more cytokines, each linked to an anchor structure. In some embodiments, a system having two nucleic acid molecules, e.g., a dual-plasmid system, is provided, where the first plasmid contains a targeting sequence encoding a CAR and the second plasmid contains a nucleic acid sequence encoding a cytokine and an anchor structure. For example, the first and second nucleic acid molecules are co-delivered to a host cell, such as an engineered immune cell. In some embodiments, a system having two or more nucleic acid molecules is provided, where the two or more plasmids independently contain at least one targeting sequence encoding a CAR or at least two or more nucleic acid sequences encoding a cytokine and an anchor structure. For example, three or more nucleic acid molecules are co-delivered to a host cell, such as an engineered immune cell. Optionally, the first plasmid contains a targeting sequence, the second plasmid contains a first nucleic acid sequence encoding a first cytokine and a first anchor structure, with / without a targeting sequence, and the third plasmid contains a second nucleic acid sequence encoding a second cytokine and a second anchor structure, with / without a targeting sequence. Optionally, the first plasmid contains a first nucleic acid sequence encoding a first cytokine and a first anchor structure, with / without a targeting sequence, the second plasmid contains a second nucleic acid sequence encoding a second cytokine and a second anchor structure, with / without a targeting sequence, and the third plasmid contains a third nucleic acid sequence encoding a third cytokine and a third anchor structure, with / without a targeting sequence.Optionally, the first plasmid contains a first nucleic acid sequence encoding a first cytokine and a first anchor structure and a second nucleic acid sequence encoding a second cytokine and a second anchor structure, with / without a targeting sequence, the second plasmid contains the targeting sequence, and the third plasmid contains a third nucleic acid sequence containing a third cytokine and a third anchor structure, with / without a targeting sequence.

[0240] In some embodiments, the targeting sequence and the first nucleic acid sequence are present on the same plasmid within the system. In some embodiments, the targeting sequence and the first nucleic acid sequence are present on two different plasmids within the system. In some embodiments, the targeting sequence and the second nucleic acid sequence are present on the same plasmid within the system. In some embodiments, the targeting sequence and the second nucleic acid sequence are present on two different plasmids within the system. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on the same plasmid within the system. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on two different plasmids within the system. In some embodiments, the targeting sequence and the first nucleic acid sequence are present on the same plasmid within the system. In some embodiments, the targeting sequence and the first nucleic acid sequence are present on two different plasmids within the system. In some embodiments, the targeting sequence and the second nucleic acid sequence are present on the same plasmid within the system. In some embodiments, the targeting sequence and the second nucleic acid sequence are present on two different plasmids within the system. In some embodiments, the targeting sequence and the third nucleic acid sequence are present on the same plasmid within the system. In some embodiments, the targeting sequence and the third nucleic acid sequence are present on two different plasmids within the system. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on the same plasmid within the system. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on two different plasmids within the system. In some embodiments, the first nucleic acid sequence and the third nucleic acid sequence are present on the same plasmid within the system. In some embodiments, the first nucleic acid sequence and the third nucleic acid sequence are present on two different plasmids within the system. In some embodiments, the second nucleic acid sequence and the third nucleic acid sequence are present on the same plasmid within the system. In some embodiments, the second nucleic acid sequence and the third nucleic acid sequence are present on two different plasmids within the system.

[0241] The system can include proteins or polypeptides encoded by the nucleic acid sequences or nucleic acid molecules in the system described herein.

[0242] host cell A further aspect of the present disclosure provides a host cell containing the nucleic acid molecule, vector, or system disclosed herein. In some embodiments, such a host cell is in vitro. In some embodiments, such a host cell is in culture.

[0243] Optionally, the host cell is derived from any species, such as bacteria or yeast. In other cases, the host cell is a mammalian cell, such as a human cell or a rodent cell, for example, an engineered T cell or an engineered NK cell. Optionally, the host cell can be any T cell, for example, a cultured T cell, a primary T cell, a T cell from a cultured T cell line (e.g., Jurkat, SupT1, etc.), or a T cell obtained from a mammal. Optionally, the T cell is obtained from a mammal. Optionally, the T cell is obtained from many sources, including blood, bone marrow, lymph node, thymus, or other tissues or body fluids. The T cell can be enriched or purified. Optionally, the T cell is a human T cell. Optionally, the T cell is a T cell isolated from a human. The T cells can be any type of T cell and can be at any stage of development, including CD4+ / CD8+ double positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD4+ T cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc.

[0244] Optionally, the host cell is treated to cause or allow expression of the cytokine-anchor material protein from the nucleic acid molecule, e.g., by culturing the host cell under conditions for expression of the encoding nucleic acid sequence. In some embodiments, purification of the expressed product is achieved by methods known to those skilled in the art. For example, the expression vector can be introduced into the host cell by physical, chemical, or biological means.

[0245] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acid molecules are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). In some cases, the method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0246] The biological method for introducing the polynucleotide of the present disclosure into host cells includes the use of DNA and RNA vectors.Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, for example, human cells.Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated virus, etc.See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0247] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). Other cutting-edge methods for targeted delivery of nucleic acids are available, such as the delivery of polynucleotides using targeted nanoparticles or other suitable submicron-sized delivery systems.

[0248] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid molecule can be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained with or complexed to micelles, or otherwise associated with lipids. The lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or in a "folded" structure. They are simply dispersed in the solution, sometimes forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include the lipid droplets that occur naturally in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0249] Suitable lipids for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma (St. Louis, MO), dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY), cholesterol ("Choi") can be obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term that includes a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as vesicular structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions with structures in solution that differ from the typical vesicular structure are also encompassed. For example, lipids may adopt a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0250] Regardless of the method used to introduce the nucleic acid molecules, vectors, or systems described herein into host cells, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR, and "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot), or by the assays described herein to identify agents within the scope of the present disclosure.

[0251] The present disclosure provides a vector comprising a targeting sequence encoding a CAR and a nucleic acid sequence encoding a cytokine and an anchor structure.Optionally, the vector can directly transduce cells, such as T cells or NK cells.Optionally, the vector can express the CAR construct in mammalian T cells or NK cells.In one embodiment, the mammalian T cells are human T cells.

[0252] In some embodiments, the nucleic acid molecule of the present disclosure, including a vector nucleic acid comprising a nucleic acid sequence encoding a CAR or cytokine-anchor polypeptide of the present disclosure, is present in an isolated host cell. Optionally, the host cell is part of a clonal population of host cells. As used herein, reference to a host cell also encompasses a clonal population of cells. A clonal population is one that has been propagated from a single parent host cell. Optionally, the host cell is derived from any suitable organism. In some embodiments, the host cell is, for example, a bacterium, yeast, fungus, or mammalian cell. In some embodiments, the host cell is an immune cell or a tumor cell. In some embodiments, the host cell is an engineered immune cell. In some embodiments, the host cell is a T cell. In some embodiments, the host cell is a tumor-infiltrating lymphocyte (TIL). In some embodiments, the engineered immune cell is a natural killer (NK) cell.

[0253] In some embodiments, the targeting sequence and the first nucleic acid sequence are present on the same plasmid inside the cell. In some embodiments, the targeting sequence and the first nucleic acid sequence are present on two different plasmids inside the cell. In some embodiments, the targeting sequence and the second nucleic acid sequence are present on the same plasmid inside the cell. In some embodiments, the targeting sequence and the second nucleic acid sequence are present on two different plasmids inside the cell. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on the same plasmid inside the cell. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on two different plasmids inside the cell. In some embodiments, the targeting sequence and the first nucleic acid sequence are present on the same plasmid inside the cell. In some embodiments, the targeting sequence and the first nucleic acid sequence are present on two different plasmids inside the cell. In some embodiments, the targeting sequence and the second nucleic acid sequence are present on the same plasmid inside the cell. In some embodiments, the targeting sequence and the second nucleic acid sequence are present on two different plasmids inside the cell. In some embodiments, the targeting sequence and the third nucleic acid sequence are present on the same plasmid inside the cell. In some embodiments, the targeting sequence and the third nucleic acid sequence are present on two different plasmids inside the cell. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on the same plasmid inside the cell. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on two different plasmids inside the cell. In some embodiments, the first nucleic acid sequence and the third nucleic acid sequence are present on the same plasmid inside the cell. In some embodiments, the first nucleic acid sequence and the third nucleic acid sequence are present on two different plasmids inside the cell. In some embodiments, the second nucleic acid sequence and the third nucleic acid sequence are present on the same plasmid inside the cell. In some embodiments, the second nucleic acid sequence and the third nucleic acid sequence are present on two different plasmids inside the cell.

[0254] Cell Source Prior to expansion and genetic or other modification, a source of cells, such as T cells or natural killer (NK) cells, can be obtained directly or indirectly from a subject. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In the case of the present disclosure, immune effector cells, such as T cells, can be obtained from a blood unit collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation. In some cases, cells from an individual's circulating blood are obtained by apheresis. The apheresis product may contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Optionally, cells collected by apheresis can be washed to remove the plasma fraction and, optionally, placed in an appropriate buffer or medium for subsequent processing. Optionally, cells are washed with phosphate-buffered saline (PBS). In other cases, the wash solution lacks calcium, may lack magnesium, or may lack many, if not all, divalent cations. An initial activation step in the absence of calcium can result in expanded activation. As those skilled in the art will readily appreciate, washing steps can be accomplished by methods known to those skilled in the art, for example, by using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, cells can be resuspended in various biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffers. Alternatively, undesirable components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0255] Optionally, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation.

[0256] The method can include selecting a particular subpopulation of immune effector cells, e.g., T cells, that are CD25+ depleted cells, e.g., a T regulatory cell-depleted population, e.g., using negative selection techniques. Preferably, the population of T regulatory depleted cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells.

[0257] In some embodiments, T regulatory cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody or fragment thereof, or the CD25-binding ligand IL-2. In some embodiments, the anti-CD25 antibody or fragment thereof, or the CD25-binding ligand, is conjugated to or otherwise coated on a substrate, e.g., a bead. In some embodiments, the anti-CD25 antibody or fragment thereof is conjugated to a substrate described herein.

[0258] In some embodiments, T regulatory cells, eg, CD25+ T cells, are removed from the population using a CD25 depletion reagent from Militenyi™.

[0259] In some embodiments, T regulatory cells, e.g., CD25+ cells, are removed from the population using a CliniMAC system with a depletion tubing set, e.g., tubing 162-01. In some embodiments, the CliniMAC system is run in a depletion setting, e.g., DEPLETION2.1.

[0260] The methods described herein can include more than one selection step, for example, more than one depletion step. Enrichment of a T cell population by negative selection can be achieved, for example, by using a combination of antibodies against surface markers specific to the negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail can include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0261] Without wishing to be bound by a particular theory, providing a subject with immune cells having a CAR and cytokine-anchor as described herein reduces the level of negative regulators of immune cells in the subject prior to apheresis or during the manufacture of the CAR-expressing cell product, which can also reduce the risk of relapse in the subject (e.g., unwanted immune cells, e.g., T REG This can reduce the risk of recurrence in a subject while reducing or eliminating the need for a treatment (reducing the number of cells). In some cases, providing a subject with immune cells having a CAR and cytokine-anchor as described herein can reduce the risk of recurrence in a subject without the process of eliminating specific immune cells in the subject (i.e., without the process of lymphodepletion). REG General methods for depleting cells are known in the art. REG Methods for reducing cells include cyclophosphamide, anti-GITR antibodies (anti-GITR antibodies described herein), CD25 depletion, and combinations thereof.

[0262] In some embodiments, the manufacturing method includes administering a T cell to a subject prior to production of the CAR-expressing cells. REG In some embodiments, the production method does not involve reducing (e.g., depleting) the number of T cells prior to producing the CAR-expressing cells.REG For example, a manufacturing method can include contacting a sample, e.g., an apheresis sample, with an anti-GITR antibody and / or an anti-CD25 antibody (or fragment thereof, or a CD25-binding ligand), thereby reducing (e.g., partially depleting) the CAR-expressing cells (e.g., T cells, NK cells) prior to manufacturing the CAR-expressing cell (e.g., T cell, NK cell) product. REG The method includes a step of partially depleting the cells.

[0263] In some embodiments, the subject is treated with T REG Although not pre-treated with one or more cell-reducing therapies, the subject still has a reduced risk of relapse to CAR-expressing cell treatment due to co-expression of a CAR and cytokine-anchor. In some embodiments, the subject is treated with T REG The subject may have been previously treated with one or more therapies that partially deplete the cells, thereby further reducing the subject's risk of relapse to CAR-expressing cell treatment.

[0264] In some embodiments, the subject is not pre-treated with cyclophosphamide prior to collection of cells for CAR-expressing cell product manufacturing, yet the co-expression of a CAR and a cytokine-anchor still reduces the subject's risk of relapse to CAR-expressing cell treatment. In some embodiments, the subject is pre-treated with cyclophosphamide prior to collection of cells for CAR-expressing cell product manufacturing, thereby further reducing the subject's risk of relapse to CAR-expressing cell treatment due to the co-expression of a CAR and a cytokine-anchor. In some embodiments, the subject is not pre-treated with an anti-GITR antibody prior to collection of cells for CAR-expressing cell product manufacturing, yet the co-expression of a CAR and a cytokine-anchor still reduces the subject's risk of relapse to CAR-expressing cell treatment. In some embodiments, the subject is pre-treated with an anti-GITR antibody prior to collection of cells for CAR-expressing cell product manufacturing, thereby further reducing the subject's risk of relapse to CAR-expressing cell treatment due to the co-expression of a CAR and a cytokine-anchor.

[0265] In some embodiments, other cell populations can be optionally removed, such as those that adversely affect the proliferation and / or function of CAR-T cells, e.g., cells expressing CD14, CD11b, CD33, CD15, or other markers potentially expressed by immunosuppressive cells.

[0266] The method can further include removing cells expressing tumor antigens, such as tumor antigens that do not contain CD25, such as CD30, CD38, CD123, CD20, CD14, or CD11b, from the population, thereby providing a population of T regulatory depleted cells, such as CD25+ depleted cells, and tumor antigen depleted cells suitable for expressing a CAR, such as CAR19, and a cytokine anchor. In some embodiments, tumor antigen-expressing cells are removed simultaneously with T regulatory, such as CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an anti-tumor antigen antibody or fragment thereof can be bound to the same substrate, such as beads, that can be used to remove cells, or an anti-CD25 antibody or fragment thereof, or an anti-tumor antigen antibody or fragment thereof, can be bound to separate beads, and a mixture thereof can be used to remove cells. In some embodiments, the removal of T regulatory cells, such as CD25+ cells, and the removal of tumor antigen-expressing cells are sequential, for example, can be performed in either order.

[0267] Also provided are methods that include removing cells expressing a checkpoint inhibitor, e.g., a checkpoint inhibitor described herein, e.g., one or more of PD1+ cells, LAG3+ cells, and TIM3+ cells, from the population, thereby providing a population of T regulatory-depleted cells, e.g., CD25+-depleted cells, and checkpoint inhibitor-depleted cells, e.g., PD1+, LAG3+, and / or TIM3+-depleted cells. Exemplary checkpoint inhibitors include B7-H1, B&-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLA, and LAIR1. In some embodiments, checkpoint inhibitor-expressing cells are removed simultaneously with T regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an anti-check point inhibitor antibody or fragment thereof can be attached to the same bead that can be used to remove cells, or an anti-CD25 antibody or fragment thereof, or an anti-check point inhibitor antibody or fragment thereof, can be attached to separate beads, a mixture of which can be used to remove cells. In some embodiments, the removal of T regulatory cells, e.g., CD25+ cells, and the removal of check point inhibitor-expressing cells are sequential, e.g., can occur in either order.

[0268] The methods described herein can include a positive selection step. For example, T cells can be isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3x28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a time sufficient for positive selection of the desired T cells. In one embodiment, the time is approximately 30 minutes. In a further embodiment, the time ranges from 30 minutes to 36 hours or more, and all integer values ​​therebetween. In a further embodiment, the time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time is 10 to 24 hours. In one embodiment, the incubation time is 24 hours. In any situation where T cells are scarce compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals, longer incubation times can be used to isolate T cells. Furthermore, longer incubation times can increase the efficiency of CD8+ T cell capture. Thus, by simply shortening or lengthening the time that T cells are allowed to bind to the CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells (as further described herein), subpopulations of T cells can be preferentially selected for or eliminated at the beginning of culture or at other times during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, subpopulations of T cells can be preferentially selected for or eliminated at the beginning of culture or at other desired times.

[0269] In some embodiments, T cell populations can be selected that express one or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other suitable molecules, e.g., other cytokines. Methods for screening cell expression can be defined, for example, by the methods described in U.S. Patent Application Publication No. US20220056116A1, which is incorporated herein by reference in its entirety.

[0270] For the isolation of a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In some embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed (e.g., increase the concentration of cells) to ensure maximum contact between the cells and beads. For example, a concentration of about 10 billion cells / ml, 9 billion / ml, 8 billion / ml, 7 billion / ml, 6 billion / ml, or 5 billion / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In one embodiment, a cell concentration from 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml is used. In a further embodiment, a concentration of 1.25 or 1.5 billion cells / ml can be used.

[0271] The use of high concentrations can increase cell yield, cell activation, and cell proliferation. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells, or from samples containing many tumor cells (e.g., leukemia blood, tumor tissue, etc.). Such cell populations may have therapeutic value and are desirable to obtain. For example, the use of high cell concentrations allows for more efficient selection of CD8+ T cells, which typically have weaker CD28 expression.

[0272] In some embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and a surface (e.g., particles such as beads), interactions between the particles and the cells can be minimized. This selects for cells that express high amounts of the desired antigen bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are captured more efficiently than CD8+ T cells at dilute concentrations. In some embodiments, the concentration of cells used is 5 x 10^6 / ml. In other embodiments, the concentration used is between about 1 x 10^5 / ml and 1 x 10^6 / ml, and any integer value therebetween.

[0273] In some embodiments, cells are incubated on a rotator at various speeds for various times at either 2-10° C. or room temperature.

[0274] The stimulated T cells can also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing step removes granulocytes and, to some extent, monocytes from the cell population, thereby providing a more uniform product. After the washing step, which removes plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or culture medium containing 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, followed by freezing the cells to -80°C at a rate of 10 / min and storing them in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as uncontrolled freezing at -20°C immediately or in liquid nitrogen, can be used.

[0275] In some embodiments, cryopreserved cells are thawed and washed as described herein and allowed to stand at room temperature for 1 hour before activation using the methods of the present disclosure.

[0276] In the context of the present disclosure, collection of a blood sample or apheresis product from a subject at a time before expanded cells as described herein may be needed is also contemplated. Thus, a source of expanded cells can be collected at any time needed, and desired cells, such as T cells, can be isolated and frozen for later use in immune effector cell therapy for any number of diseases or conditions that would benefit from immune effector cell therapy, such as those described herein. In one embodiment, a blood sample or apheresis is generally collected from a healthy subject. Optionally, a blood sample or apheresis is collected from a generally healthy subject who is at risk of developing a disease but has not yet developed the disease, and the desired cells are isolated and frozen for later use. Optionally, T cells can be expanded, frozen, and used at a later time. Optionally, a sample is collected from a patient shortly after diagnosis of a particular disease described herein, but before any treatment. In further embodiments, the cells are isolated from a blood sample or apheresis from the subject prior to any number of relevant treatment modalities, including treatment with drugs such as natalizumab, efalizumab, antivirals, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.

[0277] In further embodiments of the present disclosure, T cells are obtained from a patient immediately after treatment that leaves the subject with functional T cells. In some cases, following certain cancer treatments, particularly treatment with drugs that damage the immune system, the quality of the T cells obtained may be optimal or improved for their ability to expand ex vivo immediately after treatment during the period when the patient is typically recovering from the treatment. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo expansion. Thus, within the context of the present disclosure, it is contemplated to harvest blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery period. Furthermore, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create conditions in the subject that favor the repopulation, recirculation, regeneration, and / or proliferation of specific cell types, particularly during a defined time frame following treatment. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0278] In some embodiments, immune effector cells expressing a CAR molecule, e.g., CAR19 co-expressed with a cytokine-anchor, can be obtained from a subject that has received a low, immune-enhancing dose of an mTOR inhibitor. In some embodiments, a population of immune effector cells, e.g., T cells or NK cells, that express or are engineered to express a CAR, e.g., CAR19 co-expressed with a cytokine-anchor, can be treated ex vivo by contact with an amount of an mTOR inhibitor that increases the number of PD1-negative immune effector cells, e.g., T cells, or increases the ratio of PD1-negative immune effector cells, e.g., T cells / NK cells / PD1-positive immune effector cells, e.g., T cells or NK cells.

[0279] In some embodiments, the T cell population is deficient in diglycerol kinase (DGK).DGK-deficient cells include cells that do not express DGK RNA or protein, or that have reduced or inhibited DGK activity.DGK-deficient cells can be generated by genetic approaches, for example, by administering RNA interference agents, such as siRNA, shRNA, miRNA, to reduce or prevent DGK expression.Alternatively, DGK-deficient cells can be generated by treatment with DGK inhibitors as described herein.

[0280] In some embodiments, the T cell population is Ikaros-deficient.Ikaros-deficient cells include cells that do not express Ikaros RNA or protein, or that have reduced or inhibited Ikaros activity, and Ikaros-deficient cells can be generated by genetic approaches, for example, by administering RNA interference agents, such as siRNA, shRNA, miRNA, to reduce or prevent Ikaros expression.Alternatively, Ikaros-deficient cells can be generated by treatment with Ikaros inhibitors, such as lenalidomide.

[0281] In some embodiments, the T cell population is DGK-deficient and Ikaros-deficient, e.g., does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK-deficient and Ikaros-deficient cells can be produced by any of the methods described herein.

[0282] In some embodiments, the NK cells are obtained from a subject. In another embodiment, the NK cells are an NK cell line, e.g., the NK-92 cell line (Conkwest).

[0283] Pharmaceutical Composition Provided herein are pharmaceutical compositions comprising nucleic acid molecules, cells, immune cells, or systems related to the cytokine-anchor disclosure.In this regard, the present disclosure provides pharmaceutical compositions comprising any of the cytokine-anchor materials described herein and a pharmaceutically acceptable excipient or carrier.In some embodiments, the pharmaceutical composition comprises the cells described herein, for example, engineered immune cells that express a CAR and cytokine-anchor, and a pharmaceutically acceptable excipient or carrier.

[0284] In some embodiments, the carrier is a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be any of those conventionally used for administering cells. Pharmaceutically acceptable carriers are well known to those skilled in the art. The pharmaceutically acceptable carrier can be one that does not have adverse side effects or toxicity under the conditions of use.

[0285] The carrier can be determined in part by the particular method used to administer a particular cytokine-anchor material. Various suitable formulations of the pharmaceutical compositions of the present disclosure exist. Suitable formulations can include any of the formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, or intraperitoneal administration. More than one route can be used to administer the IL-21 / 15 materials of the present invention, and in certain instances, a particular route can provide a more rapid and effective response than another route.

[0286] In some embodiments, one or more CAR-expressing cells disclosed herein can be administered or delivered to a subject via a biopolymer scaffold, e.g., a biopolymer implant. The biopolymer scaffold can support or enhance the delivery, proliferation, and / or dispersion of the CAR-expressing cells described herein. The biopolymer scaffold comprises a biodegradable polymer that is biocompatible (e.g., does not substantially induce an inflammatory or immune response) and / or can be natural or synthetic.

[0287] Examples of suitable biopolymers include agar, agarose, alginate, alginate / calcium phosphate cement (CPC), β-galactosidase (β-GAL), (1,2,3,4,6-pentaacetyl α-D-galactose), cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) (PHBHHx), poly(lactide), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), polyethylene oxide (PEO), poly(lactic-co-glycolic acid) (PLGA), polypropylene oxide (PPO), polyvinyl alcohol) (PVA), silk, soy protein, and soy protein isolate, in any concentration and in any ratio, alone or in combination with any other polymer composition. The biopolymer can be enhanced or modified with adhesion or migration promoting molecules, e.g., collagen mimetic peptides that bind to collagen receptors on lymphocytes, and / or stimulatory molecules to enhance the delivery, proliferation, or function of the delivered cells, e.g., anti-cancer activity. The biopolymer scaffold can be an injectable, e.g., gel or semi-solid, or solid composition.

[0288] In some embodiments, cells expressing a CAR and a cytokine anchor described herein can be seeded onto a biopolymer scaffold before delivery to a subject. In embodiments, the biopolymer scaffold further comprises one or more additional therapeutic agents described herein (e.g., another CAR-expressing cell, an antibody, or a small molecule), or an agent that enhances the activity of the CAR-expressing cell, for example, an agent incorporated into or conjugated to the biopolymer of the scaffold. In embodiments, the biopolymer scaffold is, for example, injected into a tumor or surgically implanted into a tumor or tumor proximity sufficient to mediate an anti-tumor effect. Further examples of biopolymer compositions and methods for their delivery are described in Stephan et al., Nature Biotechnology, 2015, 33:97-101 and U.S. Patent Application Publication No. US20210015863A1, each of which is incorporated herein by reference in its entirety.

[0289] In some embodiments, the cells are administered by injection, e.g., intravenously. Suitable pharmaceutically acceptable carriers for cells for injection may include any isotonic carrier, such as, for example, saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or lactated Ringer's. In some embodiments, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0290] In some embodiments, pharmaceutical compositions comprise a cell or a plurality of cells expressing CAR and cytokine-anchor as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.Such compositions can comprise buffer solutions, such as neutral buffered saline, phosphate buffered saline, carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol, proteins, polypeptides or amino acids, such as glycine, antioxidants, chelating agents, such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives.In one embodiment, the compositions of the present disclosure are formulated for intravenous administration.

[0291] The pharmaceutical composition of the present disclosure can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration are determined by factors such as the patient's condition and the type and severity of the patient's disease, but the appropriate dosage can be determined by clinical trials.

[0292] In some embodiments, the pharmaceutical composition is substantially free of, e.g., completely free of, detectable levels of contaminants selected from the group consisting of endotoxin, mycoplasma, replication-competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture medium components, vector packaging cells or plasmid components, bacteria, and fungi. In some embodiments, the bacteria is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenzae, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes group A.

[0293] kit In some aspects, provided herein are kits comprising a unit dose containing a nucleic acid molecule, vector, system, cell, or pharmaceutical composition of cytokine-anchor material and instructions for use. In some embodiments, the kit comprises a nucleic acid molecule defined in the cytokine-anchor, e.g., at least one CAR19, and / or cells (e.g., engineered immune cells) expressing at least one cytokine linked to an anchor structure, and informational material containing instructions for administering a dosage of the immune cell, cell, or pharmaceutical composition to a subject.

[0294] The kit may further include one or more unit doses containing one or more additional reagents, such as immunosuppressant reagents, or one or more additional therapeutic agents, such as antibodies. The kit typically includes a label indicating the intended use of the contents of the kit. The term label includes any written or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0295] Kits of the present disclosure may further include diagnostic and / or other therapeutic agents. Optionally, the kit includes a cell or pharmaceutical composition of the present disclosure and a diagnostic agent that can be used in a diagnostic method for diagnosing the condition or presence of a disease, condition, or disorder in a subject.

[0296] Methods of Treatment, Administration, and Use In another aspect, provided herein are methods of using the cytokine-anchor materials disclosed herein to treat a subject, and methods of administering the same to a subject. The subject may have a particular disease or condition requiring treatment provided by the present disclosure. The disease or condition may include, for example, reducing or ameliorating a hyperproliferative disease or disorder, such as cancer. The disease or condition may include a solid tumor, a soft tissue tumor, or a metastatic lesion. As used herein, the term "cancer" is meant to include any type of cancerous growth or oncogenic process, metastatic tissue, or malignantly transformed cell, tissue, or organ, regardless of histopathological type or stage of invasiveness.

[0297] Examples of solid tumors include malignant tumors of various organ systems, such as sarcomas, adenocarcinomas, and carcinomas, such as those affecting the liver, lung, breast, lymphatic system, gastrointestinal (e.g., colon), genitourinary tract (e.g., kidney, urothelial cells), prostate, and pharynx. Adenocarcinomas include malignant tumors such as most colon cancers, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, and esophageal cancer. In some embodiments, the cancer is melanoma, e.g., advanced melanoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the present invention. Examples of other cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, cancer of the fallopian tubes, cancer of the endometrium, cancer of the cervix, cancer of the vagina, carcinoma of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cance...

Claims

1. A nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, (a) a first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) a first peptide anchor; (b) the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor attachment signal or (ii) a second peptide anchor; A nucleic acid molecule wherein each of the first cytokine peptide and the second cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

2. A nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, (a) a first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) a first peptide anchor; (b) the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor attachment signal or (ii) a second peptide anchor; A nucleic acid molecule wherein each of the first cytokine peptide and the second cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

3. A system comprising a first nucleic acid sequence and a second nucleic acid sequence different from the first nucleic acid sequence, (a) a first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) a first peptide anchor; (b) the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor attachment signal or (ii) a second peptide anchor; The system, wherein the first cytokine peptide and the second cytokine peptide each independently comprise at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

4. A system comprising a first nucleic acid sequence and a second nucleic acid sequence different from the first nucleic acid sequence, (a) a first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) a first peptide anchor; (b) the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor attachment signal or (ii) a second peptide anchor; wherein the first cytokine peptide and the second cytokine peptide each independently comprise at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

5. A nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, (a) a first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) a first peptide anchor; (b) the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor attachment signal or (ii) a second peptide anchor; The nucleic acid molecule, wherein the first cytokine peptide is a pro-inflammatory cytokine peptide and the second cytokine peptide is an anti-inflammatory cytokine peptide.

6. A system comprising a first nucleic acid sequence and a second nucleic acid sequence different from the first nucleic acid sequence, (a) a first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) a first peptide anchor; (b) the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor attachment signal or (ii) a second peptide anchor; The first cytokine peptide is a pro-inflammatory cytokine peptide and the second cytokine peptide is an anti-inflammatory cytokine peptide.

7. A cell comprising a first protein and a second protein, the first protein comprises a first cytokine peptide and (i) a first non-peptide anchor or (ii) a first peptide anchor; the second protein comprises a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor; The cells, wherein each of the first cytokine peptide and the second cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

8. A cell comprising a first protein and a second protein, the first protein comprises a first cytokine peptide and (i) a first non-peptide anchor or (ii) a first peptide anchor; the second protein comprises a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor; The cells, wherein each of the first cytokine peptide and the second cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

9. A cell comprising a first protein and a second protein, the first protein comprises a first cytokine peptide and (i) a first non-peptide anchor or (ii) a first peptide anchor; the second protein comprises a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor; The cell, wherein the first cytokine peptide is a pro-inflammatory cytokine peptide and the second cytokine peptide is an anti-inflammatory cytokine peptide.

10. 10. The cell of any one of claims 7 to 9, wherein the first protein is processed from a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor binding signal or (ii) a first peptide anchor, wherein the first non-peptide anchor binding signal is replaced by the first non-peptide anchor during protein processing, and the first polypeptide is encoded by a first nucleic acid sequence.

11. The cell of claim 10, wherein the second protein is processed from a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor binding signal or (ii) a second peptide anchor, wherein the second non-peptide anchor binding signal is replaced by the second non-peptide anchor during protein processing, and the second polypeptide is encoded by a second nucleic acid sequence.

12. 12. The nucleic acid molecule of claim 1, 2, or 5, the system of claim 3, 4, or 6, or the cell of claim 11, wherein (a) the first nucleic acid sequence and the second nucleic acid sequence are operably linked in the 5' to 3' direction, or (b) the second nucleic acid sequence and the first nucleic acid sequence are operably linked in the 5' to 3' direction.

13. 12. The nucleic acid molecule of claim 1, 2, or 5, the system of claim 3, 4, or 6, or the cell of claim 11, wherein the second nucleic acid sequence and the first nucleic acid sequence are linked via a nucleic acid sequence encoding a cleavable linker, and optionally the cleavable linker comprises a P2A peptide, a T2A peptide, an E2A peptide, an F2A peptide, or an IRES peptide.

14. (a) the first protein or first polypeptide comprises a first signal peptide, and optionally the first signal peptide, the first cytokine peptide, and (i) the first non-peptide anchor attachment signal or (ii) the first peptide anchor are operably linked in a direction from the N-terminus of the polypeptide to the C-terminus of the polypeptide; and / or (b) the second protein or second polypeptide comprises a second signal peptide, and optionally the second signal peptide, the second cytokine peptide, and (i) the second non-peptide anchor binding signal or (ii) the second peptide anchor are operably linked in the direction from the N-terminus of the polypeptide to the C-terminus of the polypeptide.

15. 15. The nucleic acid molecule, system, or cell of claim 14, wherein each of the first signal peptide and the second signal peptide independently comprises a CD4 signal peptide, a CD8α signal peptide, a CD28 signal peptide, a CD33 signal peptide, a CD137 (4-1BB) signal peptide, an IL-2 signal peptide, an IgE signal peptide, an IgG1 signal peptide, a GM-CSF signal peptide, an HLA-A signal peptide, an HLA signal peptide, a TCR signal peptide, or a β2M signal peptide, or a combination thereof.

16. 15. The nucleic acid molecule, system, or cell of claim 14, wherein each of the first and second signal peptides independently comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 2-7.

17. 15. The nucleic acid molecule, system, or cell of claim 14, wherein each of the first and second signal peptides is independently encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 52-57.

18. (a) the first non-peptide anchor attachment signal comprises a glycolipid attachment signal; and / or (b) the second non-peptide anchor binding signal comprises a glycolipid binding signal, the nucleic acid molecule of any one of claims 1, 2, or 5, the system of any one of claims 3, 4, or 6, or the cell of claim 11.

19. 19. The nucleic acid molecule, system, or cell of claim 18, wherein the glycolipid linkage signal comprises a GPI linkage signal.

20. 20. The nucleic acid molecule, system, or cell of claim 19, wherein the GPI-linked signal comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO:

200.

21. 20. The nucleic acid molecule, system, or cell of claim 19, wherein the GPI-linked signal is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 250-252.

22. The nucleic acid molecule of any one of claims 1, 2 or 5, the system of any one of claims 3, 4 or 6, or the cell of any one of claims 7 to 9, wherein the first peptide anchor and / or the second peptide anchor comprises a transmembrane peptide sequence.

23. 23. The nucleic acid molecule, system, or cell of claim 22, wherein the transmembrane peptide sequence comprises a B7-1 transmembrane amino acid sequence, a B7-2 transmembrane amino acid sequence, a B7-H1 transmembrane amino acid sequence, a B7-H3 transmembrane amino acid sequence, a tumor necrosis factor receptor 2 (TNFR2) transmembrane amino acid sequence, a CD8α transmembrane amino acid sequence, a CD28 transmembrane amino acid sequence, a CD3ζ transmembrane amino acid sequence, a CTLA-4 (CD152) transmembrane amino acid sequence, or a PD-L1 transmembrane amino acid sequence, or any fragment or variant thereof.

24. 23. The nucleic acid molecule, system, or cell of claim 22, wherein the transmembrane peptide sequence comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 203-204.

25. 23. The nucleic acid molecule, system, or cell of claim 22, wherein the transmembrane peptide sequence is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 253-255.

26. 10. The nucleic acid molecule of claim 5, the system of claim 6, or the cell of claim 9, wherein the anti-inflammatory cytokine peptide comprises at least a portion of IL-4, IL-10, or IL-27, or a variant thereof, and the pro-inflammatory cytokine peptide comprises at least a portion of IL-2, IL-7, IL-9, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

27. 10. The nucleic acid molecule of claim 5, the system of claim 6, or the cell of claim 9, wherein the anti-inflammatory cytokine peptide comprises at least a portion of IL-4 or a variant thereof, and the pro-inflammatory cytokine peptide comprises at least a portion of IL-2, IL-7, IL-9, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

28. 10. The nucleic acid molecule of claim 5, the system of claim 6, or the cell of claim 9, wherein the anti-inflammatory cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to a sequence set forth in SEQ ID NO: 101, 104, or 109, and the pro-inflammatory cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NO: 100, 102, 103, 105-108, or 110-112.

29. 10. The nucleic acid molecule of claim 5, the system of claim 6, or the cell of claim 9, wherein the anti-inflammatory cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 151, 163, 154, 164, or 159, and the pro-inflammatory cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NO: 150, 152, 153, 155-158, or 160-162.

30. 10. The nucleic acid molecule of claim 5, the system of claim 6, or the cell of claim 9, wherein the anti-inflammatory cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 101, and the pro-inflammatory cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 100, 102, 103, 105-108, or 110-112.

31. 10. The nucleic acid molecule of claim 5, the system of claim 6, or the cell of claim 9, wherein the anti-inflammatory cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 151 or 163, and the pro-inflammatory cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 150, 152, 153, 155-158, or 160-162.

32. 10. The nucleic acid molecule of claim 1 or 5, the system of claim 3 or 6, or the cell of claim 7 or 9, wherein the first cytokine peptide comprises at least a portion of IL-4 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-2, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

33. 10. The nucleic acid molecule of claim 1 or 5, the system of claim 3 or 6, or the cell of claim 7 or 9, wherein the first cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 101, and the second cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 100, 102-107, or 109-112.

34. 10. The nucleic acid molecule of claim 1 or 5, the system of claim 3 or 6, or the cell of claim 7 or 9, wherein the first cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 151 or 163, and the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 150, 152-157, 159-162, or 164.

35. 10. The nucleic acid molecule of claim 2 or 5, the system of any one of claims 4 or 6, or the cell of claim 8 or 9, wherein the first cytokine peptide comprises at least a portion of IL-4 or a variant thereof, and the second cytokine peptide comprises at least a portion of IL-2, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

36. 10. The nucleic acid molecule of claim 2 or 5, the system of any one of claims 4 or 6, or the cell of claim 8 or 9, wherein the first cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 101, and the second cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 100, 102-105, or 107-112.

37. 10. The nucleic acid molecule of claim 2 or 5, the system of any one of claims 4 or 6, or the cell of claim 8 or 9, wherein the first cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 151 or 163, and the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the sequences set forth in SEQ ID NO: 150, 152-155, 157-162, or 164.

38. 10. The nucleic acid molecule of claim 1 or 5, the system of claim 3 or 6, or the cell of claim 7 or 9, wherein the first cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-107 or 109-112.

39. 10. The nucleic acid molecule of any one of claims 1, 2 or 5, the system of any one of claims 3, 4 or 6, or the cell of any one of claims 7 to 9, wherein the first cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-157 or 159-164.

40. 10. The nucleic acid molecule of claim 1 or 5, the system of claim 3 or 6, or the cell of claim 7 or 9, wherein the first cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-105 or 107-112.

41. 10. The nucleic acid molecule of claim 2 or 5, the system of any one of claims 4 or 6, or the cell of claim 8 or 9, wherein the first cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-155 or 157-164.

42. 10. The nucleic acid molecule of claim 1 or 5, the system of claim 3 or 6, or the cell of claim 7 or 9, wherein the second cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-107 or 109-112.

43. 10. The nucleic acid molecule of claim 1 or 5, the system of claim 3 or 6, or the cell of claim 7 or 9, wherein the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-157 or 159-164.

44. 10. The nucleic acid molecule of claim 1 or 5, the system of claim 3 or 6, or the cell of claim 7 or 9, wherein the second cytokine peptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 100-105 or 107-112.

45. 10. The nucleic acid molecule of claim 1 or 5, the system of claim 3 or 6, or the cell of claim 7 or 9, wherein the second cytokine peptide is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 150-155 or 157-164.

46. 10. The nucleic acid molecule of any one of claims 1, 2 or 5, the system of any one of claims 3, 4 or 6, or the cell of any one of claims 7 to 9, wherein the nucleic acid molecule, system or cell further comprises a targeting sequence encoding a targeting moiety, wherein the targeting moiety comprises a chimeric antigen receptor, a T cell receptor, a B cell receptor, or any combination thereof.

47. 47. The nucleic acid molecule, system, or cell of claim 46, wherein the targeting sequence encodes a chimeric antigen receptor (CAR).

48. The chimeric antigen receptor (CAR) comprises a ligand-binding domain, and the ligand-binding domain may be any of CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, CD138, CD171, CD5, CD7, MUC1, AFP, CEA, PSCA, PSMA, HER2, EGFR, IL13Ralpha2, GD2, NKG2D, EGFTvIII, CS1, CCL1, BCMA, mesothelin, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ralpha2, and PRS.

48. The nucleic acid molecule, system, or cell of claim 47, which targets S21, VEGR2, LewisY, CD24, PDGFR-beta, SSEA-4, AFP, NCAM, Claudin18.2, GPC3, GM3, TGS5, HMWMAA, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TRAP, WT1, NY-ESO-1, LAGE-1a, or MAGE-A1.

49. 49. The nucleic acid molecule, system, or cell of claim 48, wherein the ligand binding domain targets CD19.

50. 47. The nucleic acid molecule, system, or cell of claim 46, wherein the targeting sequence encodes CAR19.

51. 51. The nucleic acid molecule, system, or cell of any one of claims 47 to 50, wherein the CAR comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NO: 4 or 400-407.

52. 51. The nucleic acid molecule, system, or cell of any one of claims 47 to 50, wherein the CAR is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 54 or 450-457.

53. 47. The nucleic acid molecule, system, or cell of claim 46, wherein the targeting sequence is linked to the first nucleic acid sequence or the second nucleic acid sequence via a nucleic acid sequence encoding a cleavable linker, and optionally the cleavable linker comprises a P2A peptide, a T2A peptide, an E2A peptide, an F2A peptide, or an IRES peptide.

54. 54. The nucleic acid molecule, system, or cell of claim 53, wherein the cleavable linker is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 350-355.

55. The cleavable linker may be 54. The nucleic acid molecule, system, or cell of claim 53, wherein (a) the CAR is connected to a first signal peptide and / or a second signal peptide, (b) the CAR is connected to a first peptide anchor and / or a second peptide anchor, and / or (c) the CAR is connected to a first non-peptide anchor binding signal and / or a second non-peptide anchor binding signal.

56. 54. The nucleic acid molecule, system, or cell of claim 53, wherein the cleavable linker comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 300-303.

57. 10. The nucleic acid molecule of any one of claims 1, 2, or 5, the system of any one of claims 3, 4, or 6, or the cell of any one of claims 7 to 9, wherein each of the first polypeptide, the first protein, the second polypeptide, and the second protein independently further comprises a peptide linker.

58. (a) a peptide linker connects a first cytokine peptide to a first peptide anchor and / or a second cytokine peptide to a second peptide anchor; (b) a peptide linker connects the first cytokine peptide to the first non-peptide anchor binding signal and / or connects the second cytokine peptide to the second non-peptide anchor binding signal; (c) a peptide linker connects the first peptide anchor and / or the second peptide anchor to the cleavable linker, and / or 58. The nucleic acid molecule, system, or cell of claim 57, wherein (d) a peptide linker connects the first non-peptide anchor binding signal and / or the second non-peptide anchor binding signal to the cleavable linker.

59. 58. The nucleic acid molecule, system, or cell of claim 57, wherein the peptide linker comprises a GS linker, an Lr1 linker, or an Lr8 linker.

60. 58. The nucleic acid molecule, system, or cell of claim 57, wherein the peptide linker comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 500, 501, 504, 506, or 507, or the sequences LE, AS, GSG, or EF.

61. 58. The nucleic acid molecule, system, or cell of claim 57, wherein the peptide linker is encoded by a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to the sequence ggctccggc, ggaagcgga, gagttc, the sequence of SEQ ID NO: 509, or the sequence of SEQ ID NO:

520.

62. A nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising a signal peptide, a cytokine peptide, and a non-peptide anchor binding signal, wherein the cytokine peptide comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

63. 1. An immune cell comprising an exogenous nucleic acid sequence encoding a polypeptide comprising a cytokine peptide and a non-peptide anchor binding signal, wherein the cytokine peptide comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

64. An immune cell comprising an exogenous nucleic acid sequence encoding a polypeptide comprising a cytokine peptide and a non-peptide anchor binding signal, wherein the cytokine peptide is at least a portion of IL-12p40 or at least a portion of IL-12p35, and the exogenous nucleic acid sequence does not encode both IL-12p40 and IL-12p35, and the immune cell does not contain a PDE5-derived stimulus response element (SRE).

65. An immune cell comprising a protein comprising a cytokine peptide and a non-peptide anchor, wherein the cytokine peptide comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

66. A nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising a cytokine peptide and a peptide anchor, wherein the cytokine peptide comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

67. A cell comprising a polypeptide comprising a cytokine peptide and a peptide anchor, wherein the cytokine peptide comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

68. A nucleic acid molecule comprising a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence, (a) a first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) a first peptide anchor; (b) the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor attachment signal or (ii) a second peptide anchor; (c) the third nucleic acid sequence encodes a third polypeptide comprising a third cytokine peptide and (i) a third non-peptide anchor attachment signal or (ii) a third peptide anchor; A nucleic acid molecule wherein each of the first cytokine peptide, the second cytokine peptide, and the third cytokine peptide independently comprises at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

69. A system comprising a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence, (a) a first nucleic acid sequence encodes a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) a first peptide anchor; (b) the second nucleic acid sequence encodes a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor attachment signal or (ii) a second peptide anchor; (c) the third nucleic acid sequence encodes a third polypeptide comprising a third cytokine peptide and (i) a third non-peptide anchor attachment signal or (ii) a third peptide anchor; The system wherein the first cytokine peptide, the second cytokine peptide, and the third cytokine peptide each independently comprise at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof, and wherein the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are different.

70. A cell comprising a first protein, a second protein, and a third protein, (a) the first protein comprises a first cytokine peptide and (i) a first non-peptide anchor or (ii) a first peptide anchor; (b) the second protein comprises a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor; (c) the third protein comprises a third cytokine peptide and (i) a third non-peptide anchor or (ii) a third peptide anchor; The cells, wherein the first cytokine peptide, the second cytokine peptide, and the third cytokine peptide each independently comprise at least a portion of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or a variant thereof.

71. A nucleic acid molecule comprising a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs: 510-514, 518-519, 521-522, 527-529, 533, 600-602, 606, 611-617, 625-638, 652-654, 658-666, 676-686, or 698.

72. A polypeptide comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of the sequences set forth in SEQ ID NOs:515-517, 523-526, 530-532, 534, 603-605, 607, 618-624, 639-651, 655-657, 667-675, 687-697, or 699.

73. 72. A nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide of claim 71.

74. 74. The nucleic acid molecule of any one of claims 1, 2, 5, 62, 66, 68, 71, or 73, wherein the nucleic acid molecule is RNA, DNA, linear RNA, circular RNA, or a vector.

75. 75. The nucleic acid molecule of claim 74, wherein the vector is a viral vector, optionally the vector is a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

76. A cell comprising the nucleic acid molecule of claim 1.

77. A cell comprising the nucleic acid molecule of claim 2.

78. A cell comprising the nucleic acid molecule of claim 5.

79. A cell comprising the nucleic acid molecule of claim 62.

80. A cell comprising the nucleic acid molecule of claim 66.

81. A cell comprising the nucleic acid molecule of claim 68.

82. A cell comprising the nucleic acid molecule of claim 71.

83. A cell comprising the nucleic acid molecule of claim 73.

84. 84. The cell of any one of claims 7-9, 67, 70, or 76-83, wherein the cell is a bacterial cell, a yeast cell, or an insect cell.

85. The cell of any one of claims 7 to 9, 67, 70, or 76 to 83, wherein the cell is an immune cell or a tumor cell.

86. 84. The cell of any one of claims 7-9, 67, 70, or 76-83, wherein the cell is an engineered immune cell, and optionally the cell is a T cell or a tumor infiltrating lymphocyte (TIL).

87. 87. The cell of claim 86, wherein the engineered immune cell is a natural killer (NK) cell.

88. Cytotoxicity of immune cells (a) does not comprise both: (1) a first nucleic acid sequence encoding a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) a first peptide anchor; and (2) a second nucleic acid sequence encoding a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor attachment signal or (ii) a second peptide anchor; or (b) the immune cell of any one of claims 63-65 or the immune cell of claim 85 is increased compared to a comparable immune cell that does not comprise both (1) a first protein comprising a first cytokine peptide and either (i) a first non-peptide anchor or (ii) a first peptide anchor, and (2) a second protein comprising a second cytokine peptide and either (i) a second non-peptide anchor or (ii) a second peptide anchor.

89. 89. The cell of claim 88, wherein the increase in immune cell cytotoxicity is measured by the in vitro cytotoxicity assay described in Examples A-D.

90. 89. The cell of claim 88, wherein the increase in immune cell cytotoxicity is measured in vitro or in vivo.

91. 89. The cell of claim 88, wherein the increase in cytotoxicity of the immune cell is at least about 5%, 10%, 20%, 30%, 40%, 50% or more.

92. A population of immune cells 86. The immune cell of any one of claims 63-65 or claim 85, wherein the immune cell proliferates for a longer period of time compared to a comparable population of immune cells that (a) do not comprise a first nucleic acid sequence encoding a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor binding signal or (ii) a first peptide anchor, and / or do not comprise a second nucleic acid sequence encoding a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor binding signal or (ii) a second peptide anchor, or (b) do not comprise a first protein comprising a first cytokine peptide and (i) the first non-peptide anchor or (ii) the first peptide anchor and / or do not comprise a second protein comprising a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor.

93. 93. The cell of claim 92, wherein the expansion of the population of immune cells is measured by the in vitro immune cell proliferation assay described in Examples A-E.

94. The cell of claim 92, wherein the expansion of the immune cell population is measured in vitro or in vivo.

95. 93. The cell of claim 92, wherein the expansion of the population of immune cells is sustained for a period of at least about 5%, 10%, 20%, 30%, 40%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold or more than the expansion of a comparable population of immune cells.

96. 83. A pharmaceutical composition comprising an immune cell according to any one of claims 63 to 65, or a cell according to any one of claims 7 to 9, 67, 70, or 76 to 83, and a pharmaceutically acceptable excipient or carrier.

97. (a) the pharmaceutical composition of claim 96; (b) informational material containing instructions for administering a dosage of the immune cells, cells, or pharmaceutical composition formulation to a subject.

98. 83. Use of an immune cell according to any one of claims 63 to 65, or a cell according to any one of claims 7 to 9, 67, 70, or 76 to 83 in the manufacture of a medicament for treating a subject.

99. 99. The use of claim 98, wherein the immune cell or cells treat cancer in a subject, optionally wherein the cancer comprises a solid tumor or melanoma, or adrenal gland cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, fallopian tube cancer, gastrointestinal cancer, glioma, glioblastoma, head and neck cancer, hematopoietic malignancies, leukemia, liver cancer, lung cancer, lymphoma, myeloma, nasal cancer, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, gastric cancer, squamous cell lung cancer, testicular cancer, thyroid cancer, uterine cancer, or any combination thereof.

100. A method for producing an engineered immune cell, comprising the step of introducing a nucleic acid molecule described in any one of claims 1, 2, 5, 62, 66, 68, 71, or 73, or a system described in any one of claims 3, 4, 6, or 69, into an immune cell.

101. 82. A method for producing an immune cell according to any one of claims 63 to 65, or a cell according to any one of claims 7 to 9, 67, 70, or 76 to 83.

102. 10. A method for making a pharmaceutical composition, said method comprising combining an immune cell according to any one of claims 63-65, or a cell according to any one of claims 7-9, 67, 70, or 76-83, with a pharmaceutically acceptable excipient or carrier.

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