Membrane anchoring cytokines, engineered immune cells and uses thereof

Abstract

The present disclosure relates to a nucleic acid molecule, polypeptide, protein, cell or system comprising a cytokine, an optional targeting domain (e.g., a chimeric antigen receptor), and an anchoring structure attachable to the surface of a cell (e.g., an engineered immune cell), the invention also relates to a preparation method of the nucleic acid molecule, the polypeptide, the protein, the cell or the system and the use of the nucleic acid molecule, the polypeptide, the protein, the cell or the system for treating diseases or conditions.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to international patent application PCT / CN2023 / 071969, filed on January 12, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0003] Lymphocyte clearance is currently part of chimeric antigen receptor (CAR)-T cell therapy, which may involve administering cyclophosphamide or fludarabine to the patient before CAR-T cell infusion. This step prevents autologous lymphocytes from attacking the infused CAR-T cells and prolongs the effects of immunotherapy. However, the process of clearing large amounts of autologous lymphocytes complicates the medical procedure, increases the patient's medical burden, and raises the risk of post-treatment infection.

[0004] Some researchers have attempted to combine different cytokines to help stimulate the proliferation of immune cells in the body and improve their survival rate. However, a drawback of this approach is that it may also stimulate the proliferation of other unwanted lymphocytes and increase the risk of cytokine release syndrome (CRS).

[0005] There is a need in this field for novel reagents that can improve the proliferation and survival of specific cells in a targeted manner. Summary of the Invention

[0006] In some aspects, this document discloses a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide, said polypeptide comprising a signal peptide, a cytokine peptide, and a non-peptide anchoring signal; wherein said cytokine peptide comprises at least a portion thereof or a variant thereof selected from any one of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α.

[0007] In some aspects, this document discloses an immune cell comprising an exogenous nucleic acid sequence encoding a polypeptide, said polypeptide comprising cytokine peptides and non-peptide anchoring signals; wherein said cytokine peptides comprise at least a portion or variants of any one of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19 or IL-1α.

[0008] In some aspects, disclosed herein is an immune cell comprising an exogenous nucleic acid sequence encoding a polypeptide comprising a cytokine peptide and a non-peptide anchor attachment 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 simultaneously encode IL-12p40 and IL-12p35; and the immune cell does not comprise a stimulatory response element (SRE) derived from PDE5. In some cases, the immune cell does not comprise a stimulatory response element (SRE), and the cytokine peptide is IL-12p40.

[0009] In some aspects, disclosed herein is 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 any one selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36y, IL-23p19, or IL-la, or a variant thereof. In some cases, the protein is processed from a polypeptide comprising the cytokine peptide and a non-peptide anchor attachment signal, and during protein processing, the non-peptide anchor attachment signal is replaced by the non-peptide anchor. In some cases, the polypeptide is encoded by an exogenous nucleic acid sequence. In some cases, the polypeptide or the protein comprises a signal peptide. In some cases, the signal peptide, cytokine peptide, and non-peptide anchor attachment signal are operably linked in the direction from the N-terminus to the C-terminus of the polypeptide. In some cases, the non-peptide anchor attachment signal comprises a glycolipid attachment signal.

[0010] In some aspects, disclosed herein 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 any one selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36y, IL-23p19, or IL-la, or a variant thereof.

[0011] In some aspects, disclosed herein is a cell comprising a polypeptide comprising a cytokine peptide and a peptide anchor; wherein the cytokine peptide comprises at least a portion of any one selected from the group consisting of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36y, IL-23p19, or IL-la, or a variant thereof.

[0012] In any one or a related aspect of the above, the polypeptide comprises a signal peptide. In some cases, the signal peptide, cytokine peptide, and peptide anchor are operably linked in the direction from the N-terminus to the C-terminus of the polypeptide. In some cases, the peptide anchor comprises a transmembrane peptide sequence. In some cases, the signal peptide comprises a CD4 signal peptide, a CD8a 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 b2M signal peptide, or a combination thereof. In some cases, 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 any one of SEQ ID NOs: 2-7. In some cases, 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 the sequence set forth in any one of SEQ ID NOs: 52-57. In some cases, the signal peptide is a naturally occurring signal peptide of a wild-type cytokine. In some cases, 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-12 p40 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-36y signal peptide, an IL-23p19 signal peptide, or an IL-la signal peptide, or a functional variant thereof. In some cases, 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 any one of SEQ ID NOs: 113 or 114. In some cases, 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 the sequence set forth in any one of SEQ ID NOs: 58 or 59. In some cases, the cytokine peptide comprises at least a portion of IL-2 or a variant thereof. In some cases, the cytokine peptide comprises at least a portion of IL-4 or a variant thereof. In some cases, the cytokine peptide comprises at least a portion of IL-7 or a variant thereof. In some cases, the cytokine peptide comprises at least a portion of IL-9 or a variant thereof. In some cases, the cytokine peptide comprises at least a portion of IL-10 or a variant thereof. In some cases, the cytokine peptide comprises at least a portion of IL-18 or a variant thereof. In some cases, the cytokine peptide comprises at least a portion of IL-23 or a variant thereof. In some cases, the cytokine peptide comprises at least a portion of IL-27 or a variant thereof.In some cases, the cytokine peptide comprises at least a portion of IL-36y or a variant thereof. In some cases, the cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. In some cases, the cytokine peptide comprises at least a portion of IL-la or a variant thereof. In some cases, the variant of the cytokine peptide comprises one or more alterations, substitutions, deletions, additions, or chemical modifications of amino acids, comprises one or more unnatural amino acids, or any combination thereof. In some cases, the 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 any one of SEQ ID NOs: 100-104, 107, or 109-112. In some cases, the 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 any one of SEQ ID NOs: 150-154, 157, or 159-164.

[0013] In any one of the above or related aspects, the polypeptide or protein further comprises a polypeptide linker. In some cases, the polypeptide linker links the cytokine peptide to the peptide anchor. In some cases, the polypeptide linker links the cytokine peptide to the non-peptide anchor attachment signal. In some cases, the polypeptide linker links the peptide anchor to the cleavable linker. In some cases, the polypeptide linker links the non-peptide anchor attachment signal to the cleavable linker.

[0014] In any one of the above or related aspects, the immune cell has increased cytotoxicity relative to a control immune cell; the control immune cell does not comprise an exogenous nucleic acid sequence encoding a polypeptide comprising the cytokine peptide and non-peptide anchor attachment signal, does not comprise a nucleic acid sequence encoding a polypeptide comprising the signal peptide, cytokine peptide, and non-peptide anchor attachment signal, does not comprise a nucleic acid sequence encoding a polypeptide comprising the cytokine peptide and peptide anchor, or does not comprise a protein comprising the cytokine peptide and (i) the non-peptide anchor or (ii) the peptide anchor. In some cases, the increased cytotoxicity of the immune cell is measured by an in vitro cytotoxicity assay described in Examples A-D. In some cases, the increased cytotoxicity of the immune cell is measured by an in vitro or in vivo method. In some cases, the increased cytotoxicity of the immune cell is at least about 5%, 10%, 20%, 30%, 40%, 50%, or more.

[0015] In any one or related aspects described above, the immune cell population has a longer duration of proliferation than a control immune cell population; the control immune cell population does not comprise an exogenous nucleic acid sequence encoding a polypeptide comprising the cytokine peptide and the non-peptide anchor attachment signal, does not comprise a nucleic acid sequence encoding a polypeptide comprising the signal peptide, the cytokine peptide, and the non-peptide anchor attachment signal, does not comprise a nucleic acid sequence encoding a polypeptide comprising the cytokine peptide and the peptide anchor, or does not comprise a protein comprising the cytokine peptide and (i) the non-peptide anchor or (ii) the peptide anchor. In some cases, the proliferation of the immune cell population is measured by the in vitro immune cell proliferation assay described in Examples A-E. In some cases, the proliferation of the immune cell population is measured by in vitro or in vivo means. In some cases, the immune cell population has a duration of proliferation that is at least about 5%, 10%, 20%, 30%, 40%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or more longer than the control immune cell population.

[0016] In some aspects, disclosed herein 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 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 first cytokine peptide is a pro-inflammatory cytokine peptide and the second cytokine peptide is an anti-inflammatory cytokine peptide.

[0017] In some aspects, disclosed herein is a system comprising a first nucleic acid sequence and a second, different 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 first cytokine peptide is a pro-inflammatory cytokine peptide and the second cytokine peptide is an anti-inflammatory cytokine peptide.

[0018] In some aspects, disclosed herein 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; the second protein comprises a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor; and the first cytokine peptide is a pro-inflammatory cytokine peptide and the second cytokine peptide is an anti-inflammatory cytokine peptide. In some cases, the first protein is processed from a first polypeptide comprising the first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) the first peptide anchor, wherein during protein processing, the first non-peptide anchor attachment signal is replaced with the first non-peptide anchor.

[0019] In any one of the above or related aspects, the anti-inflammatory cytokine peptide comprises at least a portion of, or a variant of, any one of IL-4, IL-10, or IL-27, and the pro-inflammatory cytokine peptide comprises at least a portion of, or a variant of, any one of IL-2, IL-7, IL-9, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36y, IL-23p19, or IL-la. In some cases, the anti-inflammatory cytokine peptide comprises at least a portion of, or a variant of, IL-4, and the pro-inflammatory cytokine peptide comprises at least a portion of, or a variant of, any one of IL-2, IL-7, IL-9, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36y, IL-23p19, or IL-la. 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 any one of SEQ ID NOs: 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 the sequence set forth in any one of 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 any one of SEQ ID NOs: 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 the sequence set forth in any one of SEQ ID NOs: 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 the sequence set forth in any one of 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 any one of SEQ ID NOs: 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 the sequence set forth in any one of SEQ ID NOs: 150, 152, 153, 155-158, or 160-162.

[0020] In some aspects, disclosed herein 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 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 first cytokine peptide and the second cytokine peptide each independently comprise at least a portion of any one of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-23, IL-27, IL-36y, IL-23p19, or IL-la, 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 any one of SEQ ID NOs: 100-107 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 any one of SEQ ID NOs: 150-157 or 159-164. In some cases, the second 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 any one of SEQ ID NOs: 100-107 or 109-112. In some cases, 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 the sequence set forth in any one of SEQ ID NOs: 150-157 or 159-164.

[0021] In some aspects, disclosed herein 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; the second protein comprises a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor; and the first cytokine peptide and the second cytokine peptide each independently comprises at least a portion of any one of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-23, IL-27, IL-36y, IL-23p19, or IL-la, 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 any one of SEQ ID NOs: 100-107 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 any one of SEQ ID NOs: 150-157 or 159-164. In some cases, the second 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 any one of SEQ ID NOs: 100-107 or 109-112. In some cases, 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 the sequence set forth in any one of SEQ ID NOs: 150-157 or 159-164.

[0022] In some aspects, disclosed herein is a system comprising a first nucleic acid sequence and a different 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 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 first and second cytokine peptides each independently comprise at least a portion of any one of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-23, IL-27, IL-36y, IL-23p19, or IL-la, 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 any one of SEQ ID NOs: 100-107 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 any one of SEQ ID NOs: 150-157 or 159-164. In some cases, the second 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 any one of SEQ ID NOs: 100-107 or 109-112. In some cases, 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 the sequence set forth in any one of SEQ ID NOs: 150-157 or 159-164.

[0023] In any one or related aspects of the above, 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 any one of IL-2, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-23, IL-27, IL-36y, IL-23p19, or IL-1a 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 the sequence set forth in any one of 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 any one of SEQ ID NOs: 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 the sequence set forth in any one of SEQ ID NOs: 150, 152-157, 159-162, or 164.

[0024] In some aspects, disclosed herein 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 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 first cytokine peptide and the second cytokine peptide each independently comprise at least a portion of any one of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36y, IL-23p19, or IL-la, 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 any one of SEQ ID NOs: 100-105 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 any one of SEQ ID NOs: 150-155 or 157-164. In some cases, the second 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 any one of SEQ ID NOs: 100-105 or 107-112. In some cases, 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 the sequence set forth in any one of SEQ ID NOs: 150-155 or 157-164.

[0025] In some aspects, disclosed herein is a system comprising a first nucleic acid sequence and a different 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 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 first and second cytokine peptides each independently comprise at least a portion of any one of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36y, IL-23p19, or IL-la, 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 any one of SEQ ID NOs: 100-105 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 any one of SEQ ID NOs: 150-155 or 157-164. In some cases, the second 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 any one of SEQ ID NOs: 100-105 or 107-112. In some cases, 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 the sequence set forth in any one of SEQ ID NOs: 150-155 or 157-164.

[0026] In some aspects, disclosed herein 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; the second protein comprises a second cytokine peptide and (i) a second non-peptide anchor or (ii) a second peptide anchor; and the first cytokine peptide and the second cytokine peptide each independently comprises at least a portion of any one of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36y, IL-23p19, or IL-la, 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 any one of SEQ ID NOs: 100-105 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 any one of SEQ ID NOs: 150-155 or 157-164. In some cases, the second 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 any one of SEQ ID NOs: 100-105 or 107-112. In some cases, 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 the sequence set forth in any one of SEQ ID NOs: 150-155 or 157-164.

[0027] In any of the above or related aspects, 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 any one of IL-2, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36y, IL-23p19, or IL-1a 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 the sequence set forth in any one of 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 any one of SEQ ID NOs: 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 the sequence set forth in any one of SEQ ID NOs: 150, 152-155, 157-162, or 164.

[0028] In any of the above or related aspects, the immune cell has increased cytotoxicity relative to a control immune cell; the control immune cell does not comprise both: (1) a first nucleic acid sequence encoding a first polypeptide comprising the first cytokine peptide and (i) the first non-peptide anchor attachment signal or (ii) the first peptide anchor; and (2) a second nucleic acid sequence encoding a second polypeptide comprising the second cytokine peptide and (i) the second non-peptide anchor attachment signal or (ii) the second peptide anchor; or does not comprise 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) the second non-peptide anchor or (ii) the second peptide anchor. In some cases, the increased cytotoxicity of the immune cell is measured by the in vitro cytotoxicity assay described in Examples A-D. In some cases, the increased cytotoxicity of the immune cell is measured by in vitro or in vivo means. In some cases, the increased cytotoxicity of the immune cell is at least about 5%, 10%, 20%, 30%, 40%, 50%, or more.

[0029] In any one or a related aspect of the above, the immune cell population has a longer duration of proliferation compared to a control immune cell population; the control immune cell population does not comprise a first nucleic acid sequence encoding a first polypeptide comprising the first cytokine peptide and (i) the first non-peptide anchor attachment signal or (ii) the first peptide anchor, and / or does not comprise a second nucleic acid sequence encoding a second polypeptide comprising the second cytokine peptide and (i) the second non-peptide anchor attachment signal or (ii) the second peptide anchor; or does not comprise a first protein comprising the first cytokine peptide and (i) the first non-peptide anchor or (ii) the first peptide anchor, and / or does not comprise a second protein comprising the second cytokine peptide and (i) the second non-peptide anchor or (ii) the second peptide anchor. In some cases, the proliferation of the immune cell population is measured by the in vitro immune cell proliferation assay described in Examples A-E. In some cases, the proliferation of the immune cell population is measured by in vitro or in vivo means. In some cases, the immune cell population has a duration of proliferation that is at least about 5%, 10%, 20%, 30%, 40%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold or more longer than the control immune cell population.

[0030] In any one or a related aspect of the above, the variant of the first cytokine peptide and the second cytokine peptide comprises one or more alterations, substitutions, deletions, additions, or chemical modifications of amino acids, or comprises one or more non-natural amino acids, or any combination thereof.

[0031] In any of the above or related aspects, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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.In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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.

[0032] In some aspects, disclosed herein 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; 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; and the first cytokine peptide, the second cytokine peptide, and the third cytokine peptide each independently comprises at least a portion of any one 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.

[0033] In some aspects, disclosed herein 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; 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; and the first cytokine peptide, the second cytokine peptide, and the third cytokine peptide each independently comprises at least a portion of any one 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-36y, IL-23p19, or IL-la, or a variant thereof; and the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are not the same.

[0034] In some aspects, disclosed herein 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; the third protein comprises a third cytokine peptide and (i) a third non-peptide anchor or (ii) a third peptide anchor; and the first cytokine peptide, the second cytokine peptide, and the third cytokine peptide each independently comprises at least a portion of any one 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-36y, IL-23p19, or IL-la, or a variant thereof.

[0035] the second cytokine peptide, and the third cytokine peptide each independently comprises at least a portion of any one 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-36y, IL-23p19, or IL-la, or a variant thereof.

[0036] In any one or a related aspect of the above, the immune cell has increased cytotoxicity compared to a control immune cell; the control immune cell does not comprise all of: (1) a first nucleic acid sequence encoding a first polypeptide comprising the first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) the first peptide anchor; (2) a second nucleic acid sequence encoding a second polypeptide comprising the second cytokine peptide and (i) the second non-peptide anchor attachment signal or (ii) the second peptide anchor; (3) a third nucleic acid sequence encoding a third polypeptide comprising the third cytokine peptide and (i) the third non-peptide anchor attachment signal or (ii) the third peptide anchor; or does not comprise all of: (1) a first protein comprising the first cytokine peptide and (i) the first non-peptide anchor or (ii) the first peptide anchor; (2) a second protein comprising the second cytokine peptide and (i) the second non-peptide anchor or (ii) the second peptide anchor; (3) a third protein comprising the third cytokine peptide and (i) the third non-peptide anchor or (ii) the third peptide anchor. In some cases, the increased cytotoxicity of the immune cell is measured by the in vitro cytotoxicity assay described in Examples A-D. In some cases, the increased cytotoxicity of the immune cell is measured by in vitro or in vivo methods. In some cases, the increased cytotoxicity of the immune cell is at least about 5%, 10%, 20%, 30%, 40%, 50% or more.

[0037] In any one or a related aspect of the above, the immune cell population has a longer duration of proliferation than a control immune cell population; the control immune cell population does not comprise all of: (1) a first nucleic acid sequence encoding a first polypeptide comprising the first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) the first peptide anchor; (2) a second nucleic acid sequence encoding a second polypeptide comprising the second cytokine peptide and (i) the second non-peptide anchor attachment signal or (ii) the second peptide anchor; (3) a third nucleic acid sequence encoding a third polypeptide comprising the third cytokine peptide and (i) the third non-peptide anchor attachment signal or (ii) the third peptide anchor; or does not comprise all of: (1) a first protein comprising the first cytokine peptide and (i) the first non-peptide anchor or (ii) the first peptide anchor; (2) a second protein comprising the second cytokine peptide and (i) the second non-peptide anchor or (ii) the second peptide anchor; (3) a third protein comprising the third cytokine peptide and (i) the third non-peptide anchor or (ii) the third peptide anchor. In some cases, the proliferation of the immune cell population is measured by the in vitro immune cell proliferation assay described in Examples A-E. In some cases, the proliferation of the immune cell population is measured by in vitro or in vivo means. In some cases, the immune cell population has a duration of proliferation that is at least about 5%, 10%, 20%, 30%, 40%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold or more longer than the control immune cell population.

[0038] In any of the above related aspects, the first protein is processed from a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchor attachment signal or (ii) a first peptide anchor structure, wherein the first non-peptide anchor attachment signal is replaced with the first non-peptide anchor structure during protein processing. In some cases, the first polypeptide is encoded by a first nucleic acid sequence. In any of the above related aspects, the second protein is processed from a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchor attachment signal or (ii) a second peptide anchor structure, wherein the second non-peptide anchor attachment signal is replaced with the second non-peptide anchor structure during protein processing. In some cases, the second polypeptide is encoded by a second nucleic acid sequence. In some cases, the second nucleic acid sequence is under the control of the same promoter as the first nucleic acid sequence. In some cases, the second nucleic acid sequence is under the control of two different promoters from the first nucleic acid sequence. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are operably linked in a 5' to 3' orientation. In some cases, the second nucleic acid sequence and the first nucleic acid sequence are operably linked in a 5' to 3' orientation. In some cases, the second nucleic acid sequence and the first nucleic acid sequence are linked by a nucleic acid sequence encoding a cleavable linker. In some cases, the first protein or the first polypeptide comprises a first signal peptide. In some cases, the first signal peptide, the first cytokine peptide, and (i) the first non-peptide anchor attachment signal or (ii) the first peptide anchor structure are operably linked in a N-terminal to C-terminal orientation of the polypeptide. In some cases, the second protein or the second polypeptide comprises a second signal peptide. In some cases, the second signal peptide, the second cytokine peptide, and (i) the second non-peptide anchor attachment signal or (ii) the second peptide anchor structure are operably linked in a N-terminal to C-terminal orientation of the polypeptide. In some cases, the first non-peptide anchor attachment signal comprises a glycolipid attachment signal. In some cases, the second non-peptide anchor attachment signal comprises a glycolipid attachment signal.

[0039] In any of the above related aspects, the third protein is processed from a third polypeptide comprising a third cytokine peptide and a third non-peptide anchor attachment signal or a third peptide anchor structure, wherein the third non-peptide anchor attachment signal is replaced with the third non-peptide anchor structure during protein processing. In some cases, the third polypeptide is encoded by a third nucleic acid sequence. In some cases, the second nucleic acid sequence and the third nucleic acid sequence are regulated by the same promoter. In some cases, the second nucleic acid sequence and the third nucleic acid sequence are regulated by two different promoters. In some cases, the first nucleic acid sequence and the third nucleic acid sequence are regulated by the same promoter. In some cases, the first nucleic acid sequence and the third nucleic acid sequence are regulated by two different promoters. In some cases, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are operably linked in a 5' to 3' orientation. In some cases, the first nucleic acid sequence, the third nucleic acid sequence, and the second nucleic acid sequence are operably linked in a 5' to 3' orientation. In some cases, the second nucleic acid sequence, the first nucleic acid sequence, and the third nucleic acid sequence are operably linked in a 5' to 3' orientation. In some cases, the second nucleic acid sequence, the third nucleic acid sequence, and the first nucleic acid sequence are operably linked in a 5' to 3' orientation. In some cases, the third nucleic acid sequence, the second nucleic acid sequence, and the first nucleic acid sequence are operably linked in a 5' to 3' orientation. In some cases, the third nucleic acid sequence, the first nucleic acid sequence, and the second nucleic acid sequence are operably linked in a 5' to 3' orientation. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are linked by a nucleic acid sequence encoding a cleavable linker. In some cases, the second nucleic acid sequence and the third nucleic acid sequence are linked by a nucleic acid sequence encoding a cleavable linker. In some cases, the first nucleic acid sequence and the third nucleic acid sequence are linked by a nucleic acid sequence encoding a cleavable linker.

[0040] In any of the above related aspects, the third protein or the third polypeptide comprises a third signal peptide. In some cases, the third signal peptide, the third cytokine peptide, and (i) the third non-peptide anchor attachment signal or (ii) the third peptide anchor structure are operably linked in a N-terminal to C-terminal orientation of the polypeptide. In some cases, the third non-peptide anchor attachment signal comprises a glycolipid attachment signal.

[0041] In any of the above related aspects, the first signal peptide, the second signal peptide, and the third signal peptide each independently comprises a CD4 signal peptide, a CD8a 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 b2M signal peptide, or a combination thereof. In some cases, the first signal peptide, the second signal peptide, and the third signal peptide each independently 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 any one of SEQ ID NOs: 2-7. In some cases, the first signal peptide, the second signal peptide, and the third signal peptide each independently 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 any one of SEQ ID NOs: 52-57.

[0042] In any of the above related aspects, 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 any one of SEQ ID NOs: 100-112. 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 any one of SEQ ID NOs: 150-164. In some cases, the first cytokine peptide comprises at least a portion of IL-2 or a variant thereof. In some cases, the first cytokine peptide comprises at least a portion of IL-4 or a variant thereof. In some cases, the first cytokine peptide comprises at least a portion of IL-7 or a variant thereof. In some cases, the first cytokine peptide comprises at least a portion of IL-9 or a variant thereof. In some cases, the first cytokine peptide comprises at least a portion of IL-10 or a variant thereof. In some cases, the first cytokine peptide comprises at least a portion of IL-12 or a variant thereof.

[0043] In some cases, the first cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. In some cases, the first cytokine peptide comprises at least a portion of IL-18 or a variant thereof. In some cases, the first cytokine peptide comprises at least a portion of IL-23 or a variant thereof. In some cases, the first cytokine peptide comprises at least a portion of IL-27 or a variant thereof. In some cases, the first cytokine peptide comprises at least a portion of IL-36y or a variant thereof. In some cases, the first 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-la or a variant thereof. In some cases, the first cytokine peptide comprises at least a portion of IL-15 or a variant thereof. In some cases, the first cytokine peptide comprises at least a portion of IL-21 or a variant thereof.

[0044] In any of the above related aspects, the second 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 any one of SEQ ID NOs: 100-112. In some cases, 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 the sequence set forth in any one of SEQ ID NOs: 150-164. In some cases, the second cytokine peptide comprises at least a portion of IL-2 or a variant thereof. In some cases, the second cytokine peptide comprises at least a portion of IL-4 or a variant thereof. In some cases, the second cytokine peptide comprises at least a portion of IL-7 or a variant thereof. In some cases, the second cytokine peptide comprises at least a portion of IL-9 or a variant thereof. In some cases, the second cytokine peptide comprises at least a portion of IL-10 or a variant thereof. In some cases, the second cytokine peptide comprises at least a portion of IL-12 or a variant thereof. In some cases, the second cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. In some cases, the second cytokine peptide comprises at least a portion of IL-18 or a variant thereof. In some cases, the second cytokine peptide comprises at least a portion of IL-23 or a variant thereof. In some cases, the second cytokine peptide comprises at least a portion of IL-27 or a variant thereof. In some cases, the second cytokine peptide comprises at least a portion of IL-36y or a variant thereof. In some cases, the second cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. In some cases, the second cytokine peptide comprises at least a portion of IL-la or a variant thereof. In some cases, the second cytokine peptide comprises at least a portion of IL-15 or a variant thereof. In some cases, the second cytokine peptide comprises at least a portion of IL-21 or a variant thereof.

[0045] In any of the above related aspects, the third 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 any one of SEQ ID NOs: 100-112. In some cases, the third 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 any one of SEQ ID NOs: 150-164. In some cases, the third cytokine peptide comprises at least a portion of IL-2 or a variant thereof. In some cases, the third cytokine peptide comprises at least a portion of IL-4 or a variant thereof. In some cases, the third cytokine peptide comprises at least a portion of IL-7 or a variant thereof. In some cases, the third cytokine peptide comprises at least a portion of IL-9 or a variant thereof. In some cases, the third cytokine peptide comprises at least a portion of IL-10 or a variant thereof. In some cases, the third cytokine peptide comprises at least a portion of IL-12 or a variant thereof. In some cases, the third cytokine peptide comprises at least a portion of IL-12p40 or a variant thereof. In some cases, the third cytokine peptide comprises at least a portion of IL-18 or a variant thereof. In some cases, the third cytokine peptide comprises at least a portion of IL-23 or a variant thereof. In some cases, the third cytokine peptide comprises at least a portion of IL-27 or a variant thereof. In some cases, the third cytokine peptide comprises at least a portion of IL-36y or a variant thereof. In some cases, the third cytokine peptide comprises at least a portion of IL-23p19 or a variant thereof. In some cases, the third cytokine peptide comprises at least a portion of IL-la or a variant thereof. In some cases, the third cytokine peptide comprises at least a portion of IL-15 or a variant thereof. In some cases, the third cytokine peptide comprises at least a portion of IL-21 or a variant thereof. In some cases, 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. In some cases, 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. In some cases, 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.In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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.In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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.In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. 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-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-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-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.

[0046] In any of the foregoing related aspects, the glycolipid attachment signal comprises a glycosylphosphatidylinositol (GPI) attachment signal. In some cases, the GPI attachment signal comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:200. In some cases, the GPI attachment signal is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:250-252.

[0047] In any of the aforementioned related aspects, the first peptide anchoring structure, the second peptide anchoring structure, and the third peptide anchoring structure each independently contain a transmembrane peptide sequence.

[0048] In any of the foregoing related aspects, variants of the first cytokine peptide, the second cytokine peptide, and the third cytokine peptide comprise changes, substitutions, deletions, additions, or chemical modifications of one or more amino acids, or comprise one or more non-natural amino acids, or any combination thereof.

[0049] In any of the foregoing related aspects, the first peptide anchoring structure, the second peptide anchoring structure, and the third peptide anchoring structure each independently comprise a transmembrane peptide sequence. In some cases, the transmembrane peptide sequence comprises a sequence selected from the B7-1 transmembrane amino acid sequence, the B7-2 transmembrane amino acid sequence, the B7-H1 transmembrane amino acid sequence, the B7-H3 transmembrane amino acid sequence, the tumor necrosis factor receptor 2 (TNFR2) transmembrane amino acid sequence, the CD8α transmembrane amino acid sequence, the CD28 transmembrane amino acid sequence, the CD3ζ transmembrane amino acid sequence, the CTLA-4 (CD152) transmembrane amino acid sequence, or the PD-L1 transmembrane amino acid sequence, or any fragment or variant thereof. In some cases, the transmembrane peptide sequence comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:203-204. In some cases, the transmembrane peptide sequence is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:253-255.

[0050] In any of the foregoing related aspects, the nucleic acid molecules, immune cells, systems, or cells described herein further include a targeting sequence encoding a targeting portion.

[0051] In any of the foregoing related aspects, the target sequence is linked to the nucleic acid sequence or the exogenous nucleic acid sequence via a nucleic acid sequence encoding a cleavable adapter.

[0052] In any of the above related aspects, the targeting sequence and the first nucleic acid sequence are regulated by two different promoters. In any of the above related aspects, the targeting sequence and the first nucleic acid sequence are regulated by the same promoter. In some cases, the targeting sequence and the second nucleic acid sequence are regulated by two different promoters. In some cases, the targeting sequence and the second nucleic acid sequence are regulated by the same promoter. In some cases, the targeting sequence and the third nucleic acid sequence are regulated by two different promoters. In some cases, the targeting sequence and the third nucleic acid sequence are regulated by the same promoter. In some cases, the cleavable linker connects a chimeric antigen receptor (CAR) to the first signal peptide, the second signal peptide, and / or the third signal peptide. In some cases, the cleavable linker connects a chimeric antigen receptor (CAR) to the first peptide anchor structure, the second peptide anchor structure, and / or the third peptide anchor structure. In some cases, the cleavable linker connects a chimeric antigen receptor (CAR) to the first non-peptide anchor attachment signal, the second non-peptide anchor attachment signal, and / or the third non-peptide anchor attachment signal.

[0053] In any of the above related aspects, 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. In some cases, the peptide linker connects the first cytokine peptide to the first peptide anchor structure, the second cytokine peptide to the second peptide anchor structure, and / or the third cytokine peptide to the third peptide anchor structure. In some cases, the peptide linker connects the first cytokine peptide to the first non-peptide anchor attachment signal, the second cytokine peptide to the second non-peptide anchor attachment signal, and / or the third cytokine peptide to the third non-peptide anchor attachment signal. In some cases, the peptide linker connects the first peptide anchor structure, the second peptide anchor structure, and / or the third peptide anchor structure to the cleavable linker. In some cases, the targeting sequence and the third nucleic acid sequence are present in the same plasmid within the system. In some cases, the targeting sequence and the third nucleic acid sequence are present in two different plasmids within the system. In some cases, the first nucleic acid sequence and the third nucleic acid sequence are present in the same plasmid within the system. In some cases, the first nucleic acid sequence and the third nucleic acid sequence are present in two different plasmids within the system. In some cases, the second nucleic acid sequence and the third nucleic acid sequence are present in the same plasmid within the system. In some cases, the second nucleic acid sequence and the third nucleic acid sequence are present in two different plasmids within the system. In some cases, the targeting sequence and the third nucleic acid sequence are present in the same plasmid within the cell. In some cases, the targeting sequence and the third nucleic acid sequence are present in two different plasmids within the cell. In some cases, the first nucleic acid sequence and the third nucleic acid sequence are present in the same plasmid within the cell. In some cases, the first nucleic acid sequence and the third nucleic acid sequence are present in two different plasmids within the cell. In some cases, the second nucleic acid sequence and the third nucleic acid sequence are present in the same plasmid within the cell. In some cases, the second nucleic acid sequence and the third nucleic acid sequence are present in two different plasmids within the cell. In some cases, 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.

[0054] In any of the foregoing related aspects, the targeting portion comprises a chimeric antigen receptor (CAR), a T-cell receptor (TCR), a B-cell receptor (BCR), or any combination thereof. In some cases, the targeting sequence and the nucleic acid sequence or the exogenous nucleic acid sequence are regulated by different promoters. In some cases, the targeting sequence and the nucleic acid sequence or the exogenous nucleic acid sequence are regulated by the same promoter. In some cases, the targeting sequence encodes a chimeric antigen receptor (CAR). In some cases, the chimeric antigen receptor (CAR) includes a ligand-binding domain that targets CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, CD138, CD171, CD5, CD7, MUC1, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), interleukin-13 receptor α2 (IL13Rα2), disialotyl-ganglioside (GD2), natural killer cell family 2 member D (NKG2D), epidermal growth factor receptor variant III (EGFRvIII), CS1, and chemokine (CC motif) ligand 1 (CCL1). The following proteins are listed: B cell maturation antigen (BCMA), mesothelin, receptor tyrosine kinase-like orphan receptor 1 (ROR1), FMS-like tyrosine kinase 3 (FLT3), fibroblast activation protein (FAP), tumor-associated glycoprotein 72 (TAG72), CD44 variant 6 (CD44v6), epithelial cell adhesion molecule (EPCAM), B7 homolog 3 (B7H3), stem cell factor receptor (KIT), protease serine 21 (PRSS21), vascular endothelial growth factor receptor 2 (VEGR2), Lewis Y antigen (LewisY), CD24, platelet-derived growth factor receptor β (PDGFR-β), stage-specific embryonic antigen 4 (SSEA-4), neural cell adhesion molecule (NCAM), and tight junction protein 18.2 (Claudin18).2) glypican 3 (GPC3), ganglioside GM3 (GM3), T cell immunoglobulin domain and mucin domain 5 (TGS5), high-molecular-weight-melanoma-associated antigen (HMWMAA), tumor endothelial marker 7 receptor (TEM7R), claudin 6 (CLDN6), G protein-coupled receptor class C group 5 member D (GPRC5D), X chromosome open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), placenta-specific 1 (PLAC1), GloboH, NY-BR-1, urokinase-type plasminogen activator receptor 2 (UPK2), hepatocytin A receptor 2 (HAVCR1), beta 3 adrenergic receptor (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex locus K (LY6K), olfactory receptor 51E2 (OR51E2), tumor-associated phosphoprotein (TRAP), Wilms tumor 1 (WT1), NY-ESO-1, LAGE-la, or melanoma antigen gene 1 (MAGE-A1). In some cases, the ligand binding domain targets CD19. In some cases, the targeting sequence encodes a chimeric antigen receptor that targets CD19 (CAR19). In some cases, the chimeric antigen receptor (CAR) 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 any one of SEQ ID NOs: 4, 400-407. In some cases, the chimeric antigen receptor (CAR) 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 any one of SEQ ID NOs: 54, 450-457.

[0055] In any of the above related aspects, the targeting sequence is linked to the first nucleic acid sequence or the second nucleic acid sequence by a nucleic acid sequence encoding a cleavable linker. In some cases, the cleavable linker links a chimeric antigen receptor (CAR) to the first signal peptide and / or the second signal peptide. In some cases, the cleavable linker links a chimeric antigen receptor (CAR) to the first peptide anchor structure and / or the second peptide anchor structure. In some cases, the cleavable linker links a chimeric antigen receptor (CAR) to the first non-peptide anchor attachment signal and / or the second non-peptide anchor attachment signal.

[0056] In any of the above related aspects, the cleavable linker comprises a peptide selected from a P2A peptide, a T2A peptide, an E2A peptide, an F2A peptide, or an IRES peptide. In some cases, 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 the sequence set forth in any one of SEQ ID NOS: 350-355. In some cases, the cleavable linker connects a chimeric antigen receptor (CAR) to the signal peptide. In some cases, the cleavable linker connects a chimeric antigen receptor (CAR) to the peptide anchoring structure. In some cases, the cleavable linker connects a chimeric antigen receptor (CAR) to the non-peptide anchoring attachment signal. In some cases, the cleavable linker 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 any one of SEQ ID NOS: 300-303.

[0057] In any of the above related aspects, the first polypeptide, the first protein, the second polypeptide, and the second protein each independently further comprise a peptide linker. In some cases, the peptide linker connects the first cytokine peptide to the first peptide anchoring structure and / or the second cytokine peptide to the second peptide anchoring structure. In some cases, the peptide linker connects the first cytokine peptide to the first non-peptide anchoring attachment signal and / or the second cytokine peptide to the second non-peptide anchoring attachment signal. In some cases, the peptide linker connects the first peptide anchoring structure and / or the second peptide anchoring structure to the cleavable linker. In some cases, the peptide linker connects the first non-peptide anchoring attachment signal and / or the second non-peptide anchoring attachment signal to the cleavable linker.

[0058] In any of the above related aspects, the peptide linker comprises a peptide selected from a GS linker, a Lrl linker, or a Lr8 linker. In some cases, the peptide linker 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 any one of SEQ ID NOS: 500, 501, 504, 506, 507, or comprises a sequence selected from LE, AS, GSG, or EF. In some cases, the peptide linker 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 ggctccggc, ggaagcgga, gagttc, SEQ ID NO: 509, or SEQ ID NO: 520.

[0059] In any of the above related aspects, the nucleic acid molecule is RNA. In any of the above related aspects, the nucleic acid molecule is DNA. In some cases, the nucleic acid molecule is a linear RNA. In some cases, the nucleic acid molecule is a circular RNA. In some cases, the nucleic acid molecule is a vector. In some cases, the vector is a viral vector. In some cases, the vector is a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

[0060] In any of the above related aspects, the targeting sequence is present in the same plasmid as the first nucleic acid sequence within the system. In some cases, the targeting sequence is present in two different plasmids as the first nucleic acid sequence within the system. In some cases, the targeting sequence is present in the same plasmid as the second nucleic acid sequence within the system. In some cases, the targeting sequence is present in two different plasmids as the second nucleic acid sequence within the system. In some cases, the first nucleic acid sequence is present in the same plasmid as the second nucleic acid sequence within the system. In some cases, the first nucleic acid sequence is present in two different plasmids as the second nucleic acid sequence within the system.

[0061] In any of the above related aspects, the cell is a bacterial cell, a yeast cell, or an insect cell. In some cases, the cell is an immune cell or a tumor cell. In some cases, the immune cell is an engineered immune cell. In some cases, the immune cell is a T cell. In some cases, the immune cell is a tumor infiltrating lymphocyte (TIL). In some cases, the engineered immune cell is a natural killer (NK) cell. In some cases, the targeting sequence is present in the same plasmid as the nucleic acid sequence within the immune cell. In some cases, the targeting sequence is present in two different plasmids as the nucleic acid sequence within the immune cell. In some cases, the targeting sequence is present in the genome of the immune cell.

[0062] In any of the above related aspects, the targeting sequence is present in the same plasmid as the first nucleic acid sequence within the cell. In some cases, the targeting sequence is present in two different plasmids as the first nucleic acid sequence within the cell. In some cases, the targeting sequence is present in the same plasmid as the second nucleic acid sequence within the cell. In some cases, the targeting sequence is present in two different plasmids as the second nucleic acid sequence within the cell. In some cases, the first nucleic acid sequence is present in the same plasmid as the second nucleic acid sequence within the cell. In some cases, the first nucleic acid sequence is present in two different plasmids as the second nucleic acid sequence within the cell. In some cases, the targeting sequence, the first nucleic acid sequence, and the second nucleic acid sequence are present in the genome of the cell.

[0063] Disclosed herein, in some aspects, is a nucleic acid molecule comprising a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in any one of 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, 698.

[0064] Disclosed herein, in some aspects, is a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 515-517, 523-526, 530-532, 534, 603-605, 607, 618-624, 639-651, 655-657, 667-675, 687-697, 699.

[0065] Disclosed herein, in some aspects, is a nucleic acid molecule comprising a nucleic acid sequence that encodes a polypeptide described herein.

[0066] Disclosed herein, in some aspects, is a cell comprising a nucleic acid molecule described herein.

[0067] Disclosed herein, in some aspects, is a pharmaceutical composition comprising an immune cell described herein, a cell described herein, or a pharmaceutical composition described herein, and a pharmaceutically acceptable excipient or carrier.

[0068] Disclosed herein, in some aspects, is a kit comprising: (a) an immune cell described herein, a cell described herein, or a pharmaceutical composition described herein; and (b) informational material comprising instructions for administering a dose of the immune cell, the cell, or a dosage form of the pharmaceutical composition to a subject.

[0069] Disclosed herein, in some aspects, is a method of treating a subject in need thereof, comprising administering to the subject an immune cell described herein, a cell described herein, or a pharmaceutical composition described herein. In some cases, the immune cell or the cell is allogeneic with respect to the subject. In some cases, the immune cell or the cell is autologous with respect to the subject. In some cases, the method further comprises obtaining a population of immune cells, and engineering the population of immune cells or progeny thereof to make the engineered immune cell. In some cases, the immune cell, the cell, or the pharmaceutical composition is used to treat a cancer in the subject. In some cases, the cancer comprises a solid tumor. In some cases, the cancer comprises a leukemia. In some cases, the cancer comprises a melanoma. In some cases, the cancer comprises a lymphoma. In some cases, the cancer comprises a cancer selected from the group consisting of an adrenal cancer, a bladder cancer, a bone cancer, a brain tumor, a breast cancer, a cervical cancer, a colorectal cancer, an esophageal cancer, a fallopian tube cancer, a gastrointestinal cancer, a glioma, a glioblastoma, a head and neck cancer, a hematopoietic malignancy, a leukemia, a liver cancer, a lung cancer, a lymphoma, a myeloma, a nasal cancer, a nasopharyngeal cancer, an oral cancer, an oropharyngeal cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a sarcoma, a stomach cancer, a squamous cell lung cancer, a testicular cancer, a thyroid cancer, a uterine cancer, or any combination thereof. In some cases, the subject has not received a lymphodepleting treatment prior to administration.

[0070] Disclosed herein, in some aspects, is a method of treating a subject in need thereof, comprising administering to the subject an immune cell described herein, a cell described herein, or a pharmaceutical composition described herein. In some cases, the immune cell or the cell is allogeneic with respect to the subject. In some cases, the immune cell or the cell is autologous with respect to the subject. In some cases, the method further comprises obtaining a population of immune cells, and engineering the population of immune cells or progeny thereof to make the engineered immune cell. In some cases, the immune cell, the cell, or the pharmaceutical composition is used to treat a cancer in the subject. In some cases, the cancer comprises a solid tumor. In some cases, the cancer comprises a leukemia. In some cases, the cancer comprises a melanoma. In some cases, the cancer comprises a lymphoma. In some cases, the cancer comprises a cancer selected from the group consisting of an adrenal cancer, a bladder cancer, a bone cancer, a brain tumor, a breast cancer, a cervical cancer, a colorectal cancer, an esophageal cancer, a fallopian tube cancer, a gastrointestinal cancer, a glioma, a glioblastoma, a head and neck cancer, a hematopoietic malignancy, a leukemia, a liver cancer, a lung cancer, a lymphoma, a myeloma, a nasal cancer, a nasopharyngeal cancer, an oral cancer, an oropharyngeal cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a sarcoma, a stomach cancer, a squamous cell lung cancer, a testicular cancer, a thyroid cancer, a uterine cancer, or any combination thereof.

[0071] Disclosed herein, in some aspects, is a method of making an engineered immune cell, comprising introducing a nucleic acid molecule described herein or a system described herein into an immune cell.

[0072] Disclosed herein in some aspects is a method of making an immune cell described herein or a cell described herein.

[0073] Disclosed herein in some aspects is a method of making a pharmaceutical composition comprising mixing an immune cell described herein or a cell described herein with a pharmaceutically acceptable excipient or carrier.

[0074] Incorporation by Reference

[0075] 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 DESCRIPTION OF DRAWINGS

[0076] The novel features of the 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, which sets forth illustrative

[0077] Figure 1A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-2 and CAR19 engineered T cells not expressing TeIL-2 is shown at an effector cell to target cell ratio (E:T ratio) of 1 : 10 for 24 hours.

[0078] Figure 1B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-2 and CAR19 CD8+ engineered T cells not expressing TeIL-2 is shown in co-culture with tumor cells for 12 days.

[0079] Figure 2A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-7 and CAR19 engineered T cells not expressing TeIL-7 is shown at an effector cell to target cell ratio (E:T ratio) of 1 : 10 for 24 hours.

[0080] Figure 2B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-7 and CAR19 CD8+ engineered T cells not expressing TeIL-7 is shown in co-culture with tumor cells for 12 days.

[0081] Figure 3ACytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-9, and CAR19 engineered T cells not expressing TeIL-9, is shown under conditions of an E:T ratio of 1 :9 for 24 hours.

[0082] Figure 3B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-9, and CAR19 CD8+ engineered T cells not expressing TeIL-9, is shown under conditions of co-culture with tumor cells for 6 days.

[0083] Figure 4A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-12p40, and CAR19 engineered T cells not expressing TeIL-12p40, is shown under conditions of an E:T ratio of 1 :9 for 24 hours.

[0084] Figure 4B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-12p40, and CAR19 CD8+ engineered T cells not expressing TeIL-12p40, is shown under conditions of co-culture with tumor cells for 9 days.

[0085] Figure 5A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-15, and CAR19 engineered T cells not expressing TeIL-15, is shown under conditions of an E:T ratio of 1 :9 for 24 hours.

[0086] Figure 5B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-15, and CAR19 CD8+ engineered T cells not expressing TeIL-15, is shown under conditions of co-culture with tumor cells for 9 days.

[0087] Figure 6A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-18, and CAR19 engineered T cells not expressing TeIL-18, is shown under conditions of an E:T ratio of 1 :9 for 24 hours.

[0088] Figure 6B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-18, and CAR19 CD8+ engineered T cells not expressing TeIL-18, is shown under conditions of co-culture with tumor cells for 6 days.

[0089] Figure 7A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored CAR19-TeIL-21, and CAR19 engineered T cells not expressing TeIL-21, is shown under conditions of an E:T ratio of 1 :9 for 24 hours.

[0090] Figure 7B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-21, and CAR19 CD8+ engineered T cells not expressing TeIL-21, is shown under conditions of co-culture with tumor cells for 6 days.

[0091] Figure 8A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-36y, and CAR19 engineered T cells not expressing TeIL-36y, is shown under conditions of an E:T ratio of 1 :9 for 24 hours.

[0092] Figure 8B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-36y, and CAR19 CD8+ engineered T cells not expressing TeIL-36y, is shown under conditions of co-culture with tumor cells for 6 days.

[0093] Figure 9A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-12p40 and TeIL-7, and CAR19 engineered T cells not expressing TeIL-12p40 and TeIL-7, is shown under conditions of an E:T ratio of 1 :9 for 24 hours.

[0094] Figure 9B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-12p40 and TeIL-7, and CAR19 CD8+ engineered T cells not expressing TeIL-12p40 and TeIL-7, is shown under conditions of co-culture with tumor cells for 9 days.

[0095] Figure 10A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-12p40 and TeIL-15, and CAR19 engineered T cells not expressing TeIL-12p40 and TeIL-15, is shown under conditions of an E:T ratio of 1 :9 for 24 hours.

[0096] Figure 10BProliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-12p40 and TeIL-15, and CAR19 CD8+ engineered T cells not expressing TeIL-12p40 and TeIL-15, under conditions of 9 days of co-culture with tumor cells is shown.

[0097] Figure 11A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-12p40 and TeIL-21, and CAR19 engineered T cells not expressing TeIL-12p40 and TeIL-21, under conditions of E:T ratio of 1 :18, for 72 hours is shown.

[0098] Figure 11B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-12p40 and TeIL-21, and CAR19 CD8+ engineered T cells not expressing TeIL-12p40 and TeIL-21, under conditions of 9 days of co-culture with tumor cells is shown.

[0099] Figure 12A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-15 and TeIL-2, and CAR19 engineered T cells not expressing TeIL-15 and TeIL-2, under conditions of E:T ratio of 1 :9, for 24 hours is shown.

[0100] Figure 12B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-15 and TeIL-2, and CAR19 CD8+ engineered T cells not expressing TeIL-15 and TeIL-2, under conditions of 9 days of co-culture with tumor cells is shown.

[0101] Figure 13A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-15 and TeIL-7, and CAR19 engineered T cells not expressing TeIL-15 and TeIL-7, under conditions of E:T ratio of 1 :9, for 24 hours is shown.

[0102] Figure 13B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-15 and TeIL-7, and CAR19 CD8+ engineered T cells not expressing TeIL-15 and TeIL-7, under conditions of 6 days of co-culture with tumor cells is shown.

[0103] Figure 14A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-15 and TeIL-12p40, and CAR19 engineered T cells not expressing TeIL-12p40 and TeIL-15, is shown under conditions of an E:T ratio of 1 :18 for 72 hours.

[0104] Figure 14B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-15 and TeIL-12p40, and CAR19 CD8+ engineered T cells not expressing TeIL-12p40 and TeIL-15, is shown under conditions of co-culture with tumor cells for 9 days.

[0105] Figure 15A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-15 and TeIL-21, and CAR19 engineered T cells not expressing TeIL-15 and TeIL-21, is shown under conditions of an E:T ratio of 1 :9 for 24 hours.

[0106] Figure 15B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-15 and TeIL-21, and CAR19 CD8+ engineered T cells not expressing TeIL-15 and TeIL-21, is shown under conditions of co-culture with tumor cells for 9 days.

[0107] Figure 16A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-21 and TeIL-7, and CAR19 engineered T cells not expressing TeIL-21 and TeIL-7, is shown under conditions of an E:T ratio of 1 :9 for 24 hours.

[0108] Figure 16B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-21 and TeIL-7, and CAR19 CD8+ engineered T cells not expressing TeIL-21 and TeIL-7, is shown under conditions of co-culture with tumor cells for 13 days.

[0109] Figure 17ACytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-21 and TeIL-12p40, and CAR19 engineered T cells not expressing TeIL-21 and TeIL-12p40, at an E:T ratio of 1 :9 for 24 hours is shown.

[0110] Figure 17B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-21 and TeIL-12p40, and CAR19 CD8+ engineered T cells not expressing TeIL-21 and TeIL-12p40, in co-culture with tumor cells for 9 days is shown.

[0111] Figure 18A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-21 and TeIL-15, and CAR19 engineered T cells not expressing TeIL-21 and TeIL-15, at an E:T ratio of 1 :9 for 24 hours is shown.

[0112] Figure 18B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-21 and TeIL-15, and CAR19 CD8+ engineered T cells not expressing TeIL-21 and TeIL-15, in co-culture with tumor cells for 13 days is shown.

[0113] Figure 19A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-2, TeIL-7, and TeIL-15, and CAR19 engineered T cells not expressing TeIL-2, TeIL-7, and TeIL-15, at an E:T ratio of 1 :10 for 24 hours is shown.

[0114] Figure 19B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-2, TeIL-7, and TeIL-15, and CAR19 CD8+ engineered T cells not expressing TeIL-2, TeIL-7, and TeIL-15, in co-culture with tumor cells for 12 days is shown.

[0115] Figure 20ACytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-12p40, TeIL-7, and TeIL-21, and CAR19 engineered T cells not expressing TeIL-12p40, TeIL-7, and TeIL-21, at an E:T ratio of 1 :9 for 24 hours is shown.

[0116] Figure 20B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-12p40, TeIL-7, and TeIL-21, and CAR19 CD8+ engineered T cells not expressing TeIL-12p40, TeIL-7, and TeIL-21, in co-culture with tumor cells for 6 days is shown.

[0117] Figure 21A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-12p40, TeIL-15, and TeIL-21, and CAR19 engineered T cells not expressing TeIL-12p40, TeIL-15, and TeIL-21, at an E:T ratio of 1 :18 for 72 hours is shown.

[0118] Figure 21B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-12p40, TeIL-15, and TeIL-21, and CAR19 CD8+ engineered T cells not expressing TeIL-12p40, TeIL-15, and TeIL-21, in co-culture with tumor cells for 9 days is shown.

[0119] Figure 22A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-15, TeIL-7, and TeIL-21, and CAR19 engineered T cells not expressing TeIL-15, TeIL-7, and TeIL-21, at an E:T ratio of 1 :9 for 24 hours is shown.

[0120] Figure 22B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-15, TeIL-7, and TeIL-21, and CAR19 CD8+ engineered T cells not expressing TeIL-15, TeIL-7, and TeIL-21, in co-culture with tumor cells for 6 days is shown.

[0121] Figure 23ACytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-15, TeIL-12p40, and TeIL-21, and CAR19 engineered T cells not expressing TeIL-15, TeIL-12p40, and TeIL-21, at an E:T ratio of 1 :9 for 24 hours is shown.

[0122] Figure 23B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-15, TeIL-12p40, and TeIL-21, and CAR19 CD8+ engineered T cells not expressing TeIL-15, TeIL-12p40, and TeIL-21, under conditions of co-culture with tumor cells for 9 days is shown.

[0123] Figure 24A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-15, TeIL-21, and TeIL-12p40, and CAR19 engineered T cells not expressing TeIL-15, TeIL-21, and TeIL-12p40, at an E:T ratio of 1 :9 for 24 hours is shown.

[0124] Figure 24B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-15, TeIL-21, and TeIL-12p40, and CAR19 CD8+ engineered T cells not expressing TeIL-15, TeIL-21, and TeIL-12p40, under conditions of co-culture with tumor cells for 9 days is shown.

[0125] Figure 25A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-21, TeIL-7, and TeIL-15, and CAR19 engineered T cells not expressing TeIL-21, TeIL-7, and TeIL-15, at an E:T ratio of 1 :9 for 24 hours is shown.

[0126] Figure 25B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-21, TeIL-7, and TeIL-15, and CAR19 CD8+ engineered T cells not expressing TeIL-21, TeIL-7, and TeIL-15, under conditions of co-culture with tumor cells for 13 days is shown.

[0127] Figure 26ACytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-21, TeIL-12p40, and TeIL-15, and CAR19 engineered T cells not expressing TeIL-21, TeIL-12p40, and TeIL-15, at an E:T ratio of 1:9 for 24 hours is shown.

[0128] Figure 26B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-21, TeIL-12p40, and TeIL-15, and CAR19 CD8+ engineered T cells not expressing TeIL-21, TeIL-12p40, and TeIL-15, in co-culture with tumor cells for 13 days is shown.

[0129] Figure 27A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored IL-12p40-Lr1-Ar1-E2A-IL-15-Lr1-Ar2, and CAR19 engineered T cells not expressing IL-12p40-Lr1-Ar1-E2A-IL-15-Lr1-Ar2, at an E:T ratio of 1:3 for 72 hours is shown.

[0130] Figure 27B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored IL-12p40-Lr1-Ar1-E2A-IL-15-Lr1-Ar2, and CAR19 CD8+ engineered T cells not expressing IL-12p40-Lr1-Ar1-E2A-IL-15-Lr1-Ar2, in co-culture with tumor cells for 12 days is shown.

[0131] Figure 28A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored IL-12p40-Lr1-Ar1-F2A-IL-15-Lr1-Ar2, and CAR19 engineered T cells not expressing IL-12p40-Lr1-Ar1-F2A-IL-15-Lr1-Ar2, at an E:T ratio of 1:3 for 72 hours is shown.

[0132] Figure 28BProliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored IL-12p40-Lrl-Arl-F2A-IL-15-Lrl-Ar2 and CAR19 CD8+ engineered T cells not expressing IL-12p40-Lrl-Arl-F2A-IL-15-Lrl-Ar2 under conditions of co-culture with tumor cells for 12 days is shown.

[0133] Figure 29A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored IL-12p40-Lrl-Arl-P2A-IL-15-Lrl-Ar2 and CAR19 engineered T cells not expressing IL-12p40-Lrl-Arl-P2A-IL-15-Lrl-Ar2 under conditions of E:T ratio of 1 :3 for 72 hours is shown.

[0134] Figure 29B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored IL-12p40-Lrl-Arl-P2A-IL-15-Lrl-Ar2 and CAR19 CD8+ engineered T cells not expressing IL-12p40-Lrl-Arl-P2A-IL-15-Lrl-Ar2 under conditions of co-culture with tumor cells for 12 days is shown.

[0135] Figure 30A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored IL-12p40-Lrl-Arl-T2A-IL-15-Lrl-Ar2 and CAR19 engineered T cells not expressing IL-12p40-Lrl-Arl-T2A-IL-15-Lrl-Ar2 under conditions of E:T ratio of 1 :3 for 72 hours is shown.

[0136] Figure 30B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored IL-12p40-Lrl-Arl-T2A-IL-15-Lrl-Ar2 and CAR19 CD8+ engineered T cells not expressing IL-12p40-Lrl-Arl-T2A-IL-15-Lrl-Ar2 under conditions of co-culture with tumor cells for 12 days is shown.

[0137] Figure 31ACytotoxicity of engineered T cells expressing CAR19 + membrane-anchored IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar1 and CAR19 engineered T cells not expressing IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar1 at an E:T ratio of 1 :3 for 72 hours is shown.

[0138] Figure 31B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar1 and CAR19 CD8+ engineered T cells not expressing IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar1 in co-culture with tumor cells for 12 days is shown.

[0139] Figure 32A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar2 and CAR19 engineered T cells not expressing IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar2 at an E:T ratio of 1 :3 for 72 hours is shown.

[0140] Figure 32B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar2 and CAR19 CD8+ engineered T cells not expressing IL-12p40-Lr1-Ar2-T2A-IL-15-Lr1-Ar2 in co-culture with tumor cells for 12 days is shown.

[0141] Figure 33A Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored IL-12p40-Lr8-Ar1-E2A-IL-15-Lr1-Ar2 and CAR19 engineered T cells not expressing IL-12p40-Lr8-Ar1-E2A-IL-15-Lr1-Ar2 at an E:T ratio of 1 :3 for 72 hours (both in co-culture with tumor cells for 12 days and 9 days, respectively) is shown.

[0142] Figure 33BProliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored IL-12p40-Lr8-Ar1-E2A-IL-15-Lr1-Ar2 and CAR19 CD8+ engineered T cells not expressing IL-12p40-Lr8-Ar1-E2A-IL-15-Lr1-Ar2 are shown under conditions of co-culture with tumor cells for 12 days.

[0143] Figure 34 Cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-4 and CAR19 engineered T cells not expressing TeIL-4 are shown under conditions of E:T ratio of 1 :8 and 1 :16 for 72 hours.

[0144] Figure 35A Proliferation of CD4+ engineered T cells expressing CAR19 + membrane-anchored TeIL-4 and CAR19 CD4+ engineered T cells not expressing TeIL-4 are shown under conditions of E:T ratio of 1 :8 and co-culture with tumor cells for 12 days.

[0145] Figure 35B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-4 and CAR19 CD8+ engineered T cells not expressing TeIL-4 are shown under conditions of E:T ratio of 1 :8 and co-culture with tumor cells for 12 days.

[0146] Figure 36A Proliferation of CD4+ engineered T cells expressing CAR19 + membrane-anchored TeIL-4 and CAR19 CD4+ engineered T cells not expressing TeIL-4 are shown under conditions of E:T ratio of 1 :16 and co-culture with tumor cells for 12 days.

[0147] Figure 36B Proliferation of CD8+ engineered T cells expressing CAR19 + membrane-anchored TeIL-4 and CAR19 CD8+ engineered T cells not expressing TeIL-4 are shown under conditions of E:T ratio of 1 :16 and co-culture with tumor cells for 12 days.

[0148] Figure 37 Sustained cytotoxicity of engineered T cells expressing CAR19 + membrane-anchored TeIL-10, engineered T cells expressing CAR19 + soluble sIL-10, and CAR19 engineered T cells not expressing TeIL-10 and sIL-10 are shown under conditions of continuous co-culture with tumor cells for 45 days.

[0149] Figure 38A Proliferation of CD8+ engineered T cells expressing CAR19+ membrane-anchored TeIL-10, CD8+ engineered T cells expressing CAR19+ soluble sIL-10, and CAR19 CD8+ engineered T cells not expressing TeIL-10 and sIL-10 is shown under conditions of continuous co-culture with tumor cells for 51 days.

[0150] Figure 38B Proliferation of CD4+ engineered T cells expressing CAR19+ membrane-anchored TeIL-10, CD4+ engineered T cells expressing CAR19+ soluble sIL-10, and CAR19 CD8+ engineered T cells not expressing TeIL-10 and sIL-10 is shown under conditions of continuous co-culture with tumor cells for 51 days.

[0151] Figure 39 Tumor killing ability of engineered T cells expressing CAR19+ membrane-anchored TeIL-4 and engineered T cells expressing CAR19+ membrane-anchored TeIL-4 and TeIL-15 is shown. Fluorescence intensity (total flux p / s) indicates tumor burden. DETAILED DESCRIPTION

[0152] The present disclosure discloses, in some aspects: (a) a polypeptide comprising a cytokine peptide and an anchoring 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 (hereinafter (a)-(d) collectively referred to as “single cytokine-anchoring substances”). In some embodiments, the anchoring structure is capable of attaching the cytokine to a cell surface comprising a nucleic acid molecule encoding the polypeptide. In some embodiments, the cytokine peptide comprises at least a portion of any one selected from the group consisting 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. In some cases, the cytokine peptide is at least a portion of IL-12p40. In some cases, the cytokine peptide is at least a portion of IL-12p30. In some cases, the cytokine peptide comprises IL-12p40 and does not comprise IL-12p35. In some cases, an immune cell comprising a polypeptide described herein does not comprise a stimulation response element (SRE) derived from phosphodiesterase 5 (PDE5). In some cases, an immune cell comprising a polypeptide described herein does not comprise a stimulation response element (SRE). In some embodiments, the polypeptide further comprises a signal peptide. In some embodiments, the anchoring structure is a peptide anchoring structure. In some embodiments, the anchoring structure is a non-peptide anchoring attachment signal that is replaced with a non-peptide anchoring structure 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 (TCR), a B cell receptor (BCR), or any combination thereof. In some embodiments, the targeting sequence encodes a CAR comprising a recognition region targeting CD19 (CAR19). In some embodiments, the targeting sequence encodes a CAR19 (e.g., as described in Table 5A). In some embodiments, the CAR19 is linked to the polypeptide by a cleavable linker. In some embodiments, the signal peptide directs the polypeptide to the cell surface. In some embodiments, the peptide anchoring structure comprises a transmembrane peptide sequence. In some embodiments, the non-peptide anchoring attachment signal is replaced with a non-peptide anchoring structure (e.g., a lipid anchoring structure). In some embodiments, the non-peptide anchoring attachment signal comprises a glycosylphosphatidylinositol (GPI) attachment signal. In some embodiments, the non-peptide anchoring structure is a GPI anchoring structure. In some embodiments, the peptide anchoring structure is attached to the cell membrane. In some embodiments, the non-peptide anchoring structure is attached to the cell membrane. In some embodiments, the cleavable linker comprises a P2A peptide, a T2A peptide, a E2A peptide, a F2A peptide, or an IRES peptide. In some embodiments, the nucleic acid molecule is a vector, e.g., 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 improved relative to a reference immune cell that does not comprise a nucleic acid sequence encoding the cytokine peptide and the anchor structure (e.g., as shown in Example D). In some embodiments, the duration of proliferation of the population of immune cells is prolonged relative to a population of reference immune cells that does not comprise a nucleic acid sequence encoding the cytokine peptide and the anchor structure (e.g., as shown in Example E). In some cases, the polypeptide 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 any one of SEQ ID NOs: 515-517, 523-526, 607, 618-624, or 667-675. In some cases, the nucleic acid comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 510-514, 518-519, 521-522, 606, 611-617, or 658-666.

[0153] The present disclosure discloses, in some aspects: (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) a nucleic acid molecule encoding the first polypeptide and the second polypeptide, (e) a cell (e.g., an immune cell) expressing the first polypeptide and the different second polypeptide (hereinafter (a)-(e) collectively referred to as “dual cytokine-anchor agents”). In some embodiments, the nucleic acid molecule encoding the first polypeptide and the second polypeptide comprises 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 is different from the nucleic acid encoding the second polypeptide. 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 attaching the first cytokine to a cell surface comprising the nucleic acid molecule. In some embodiments, the second anchor structure is capable of attaching the second cytokine to a cell surface comprising the nucleic acid molecule. In some embodiments, the first cytokine peptide and the second cytokine peptide each independently comprises at least a portion of any one of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36y, IL-23p19, or IL-1a, or a variant thereof. In other embodiments, the first cytokine peptide and the second cytokine peptide each independently comprises at least a portion of any one of IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-15, IL-23, IL-27, IL-36y, IL-23p19, or IL-1a, 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 structure. In some embodiments, the first anchor structure is a non-peptide anchor attachment signal that is replaced with a non-peptide anchor structure during protein processing. In some embodiments, the second anchor structure is a peptide anchor structure. In some embodiments, the second anchor structure is a non-peptide anchor attachment signal that is replaced with a non-peptide anchor structure 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 (TCR), a B cell receptor (BCR), or any combination thereof.In some embodiments, the targeting sequence encodes a CAR comprising a recognition region that targets CD19 (CAR19). In some embodiments, the targeting sequence encodes a CAR19 (e.g., as described in Table 5A). In some embodiments, the CAR19 is linked to the first polypeptide by a cleavable linker. In some embodiments, the CAR19 is linked to the second polypeptide by a cleavable linker. In some embodiments, the first polypeptide is linked to the second polypeptide by a cleavable linker. In some embodiments, the signal peptide directs the polypeptide to the cell surface. In some embodiments, the peptide anchoring structure comprises a transmembrane peptide sequence. In some embodiments, the non-peptide anchoring attachment signal is replaced with a non-peptide anchoring structure (e.g., a lipid anchoring structure). In some embodiments, the non-peptide anchoring attachment signal comprises a glycosyl phosphatidylinositol (GPI) attachment signal. In some embodiments, the non-peptide anchoring structure is a GPI anchoring structure. In some embodiments, the peptide anchoring structure is attached to the cell membrane. In some embodiments, the non-peptide anchoring structure is attached to the cell membrane. 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, e.g., 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, e.g., a T cell or a natural killer (NK) cell. In some embodiments, the cytotoxicity of the immune cell is improved relative to a reference immune cell that does not comprise a nucleic acid sequence encoding the cytokine peptide and the anchoring structure (e.g., as shown in Example D). In some embodiments, the duration of proliferation of the immune cell population is prolonged relative to a reference immune cell population that does not comprise a nucleic acid sequence encoding the cytokine peptide and the anchoring structure (e.g., as shown in Example E). In some cases, the first polypeptide 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 any one of SEQ ID NOs: 515-517, 523-526, 607, 618-624, or 667-675. In some cases, the first nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 510-514, 518-519, 521-522, 606, 611-617, or 658-666.In some cases, the second polypeptide 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 any one of SEQ ID NOs: 515-517, 523-526, 607, 618-624, or 667-675. In some cases, the second nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 510-514, 518-519, 521-522, 606, 611-617, or 658-666. In some cases, the nucleic acid molecule comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 527-529, 533, 600-602, 625-638, or 676-686. In some cases, the nucleic acid molecule encodes an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 530-532, 534, 603-605, 639-651, or 687-697. In some cases, the anti-inflammatory cytokine peptide comprises at least a portion of any one of IL-4, IL-10, or IL-27, or a variant thereof. In some cases, the pro-inflammatory cytokine peptide comprises at least a portion of any one of IL-2, IL-7, IL-9, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36y, IL-23p19, or IL-la, or a variant thereof. In some cases, 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 any one of IL-2, IL-7, IL-9, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36y, IL-23p19, or IL-la, 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 any one of SEQ ID NOs: 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 the sequence set forth in any one of 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 any one of SEQ ID NOs: 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 the sequence set forth in any one of SEQ ID NOs: 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 the sequence set forth in any one of 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 any one of SEQ ID NOs: 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 the sequence set forth in any one of SEQ ID NOs: 150, 152, 153, 155-158, or 160-162. In some cases, 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 any one of IL-2, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36y, IL-23p19, or IL-la 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 the sequence set forth in any one of SEQ ID NOs: 100 or 102-112.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 any one of SEQ ID NOs: 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 the sequence set forth in any one of SEQ ID NOs: 150, 152-162, or 164. In some cases, 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 any one of IL-2, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36y, IL-23p19, or IL-1a 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 the sequence set forth in any one of 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 any one of SEQ ID NOs: 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 the sequence set forth in any one of SEQ ID NOs: 150, 152-157, 159-162, or 164. In some cases, 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 any one of IL-2, IL-7, IL-9, IL-10, IL-12, IL-12p40, IL-18, IL-21, IL-23, IL-27, IL-36y, IL-23p19, or IL-1a 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 the sequence set forth in any one of 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 any one of SEQ ID NOs: 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 the sequence set forth in any one of SEQ ID NOs: 150, 152-155, 157-162, or 164.

[0154] The present disclosure discloses, in some aspects: (a) a system comprising 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, the third polypeptide comprises a third cytokine peptide and a third anchor structure, and the first polypeptide, the second polypeptide, and the third polypeptide are not the same), (b) a system comprising a protein comprising the first polypeptide, a protein comprising the second polypeptide, and a protein comprising the third polypeptide, (c) a system comprising a nucleic acid encoding the first polypeptide, a nucleic acid encoding the second polypeptide, a nucleic acid encoding the third polypeptide, and 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, (e) a cell (e.g., an immune cell) expressing the first polypeptide, the second polypeptide, and the third polypeptide (hereinafter (a)-(e) collectively referred to as “triple cytokine-anchor systems”). In some embodiments, the nucleic acid molecule encoding the first polypeptide, the second polypeptide, and the third polypeptide comprises 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 attaching the first cytokine to a cell surface of a cell comprising the nucleic acid molecule. In some embodiments, the second anchor structure is capable of attaching the second cytokine to a cell surface of a cell comprising the nucleic acid molecule. In some embodiments, the third anchor structure is capable of attaching the second cytokine to a cell surface of a cell comprising the nucleic acid molecule. In some embodiments, the first cytokine peptide, the second cytokine peptide, and the third cytokine peptide each independently comprises at least a portion of any one 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-36y, IL-23p19, or IL-la, 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 structure. In some embodiments, the first anchor structure is a non-peptide anchor attachment signal that is replaced with a non-peptide anchor structure during protein processing. In some embodiments, the second anchor structure is a peptide anchor structure. In some embodiments, the second anchor structure is a non-peptide anchor attachment signal that is replaced with a non-peptide anchor structure during processing. In some embodiments, the third anchor structure is a peptide anchor structure. In some embodiments, the third anchor structure is a non-peptide anchor attachment signal that is replaced with a non-peptide anchor structure 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 (TCR), a B cell receptor (BCR), or any combination thereof. In some embodiments, the targeting sequence encodes a CAR comprising a recognition region that targets CD19 (CAR19). In some embodiments, the targeting sequence encodes a CAR19 (e.g., as described in Table 5A). In some embodiments, the CAR19 is linked to the first polypeptide by a cleavable linker. In some embodiments, the CAR19 is linked to the second polypeptide by a cleavable linker. In some embodiments, the CAR19 is linked to the third polypeptide by a cleavable linker. In some embodiments, the first polypeptide is linked to the second polypeptide by a cleavable linker. In some embodiments, the second polypeptide is linked to the third polypeptide by a cleavable linker. In some embodiments, the first polypeptide is linked to the third polypeptide by a cleavable linker. In some embodiments, the signal peptide directs the polypeptide to the cell surface. In some embodiments, the peptide anchoring structure comprises a transmembrane peptide sequence. In some embodiments, the non-peptide anchoring attachment signal is replaced with a non-peptide anchoring structure (e.g., a lipid anchoring structure). In some embodiments, the non-peptide anchoring attachment signal comprises a glycosyl phosphatidylinositol (GPI) attachment signal. In some embodiments, the non-peptide anchoring structure is a GPI anchoring structure. In some embodiments, the peptide anchoring structure is attached to the cell membrane. In some embodiments, the non-peptide anchoring structure is attached to the cell membrane. 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, e.g., 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, e.g., a T cell or a natural killer (NK) cell. In some embodiments, the cytotoxicity of the immune cell is improved relative to a reference immune cell that does not comprise a nucleic acid sequence encoding the cytokine peptide and the anchoring structure (e.g., as shown in Example D). In some embodiments, the duration of proliferation of the immune cell population is prolonged relative to a reference immune cell population that does not comprise a nucleic acid sequence encoding the cytokine peptide and the anchoring structure (e.g., as shown in Example E). In some cases, the first polypeptide 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 any one of SEQ ID NOs: 515-517, 523-526, 607, 618-624, or 667-675.In some cases, the first nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 510-514, 518-519, 606, 521-522, 611-617, or 658-666. In some cases, the second polypeptide 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 any one of SEQ ID NOs: 515-517, 523-526, 607, 618-624, or 667-675. In some cases, the second nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 510-514, 518-519, 606, 521-522, 611-617, or 658-666. In some cases, the third polypeptide 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 any one of SEQ ID NOs: 515-517, 523-526, 607, 618-624, or 667-675. In some cases, the third nucleic acid sequence comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 510-514, 518-519, 606, 521-522, 611-617, or 658-666. In some cases, the nucleic acid molecule comprises a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 652-654, or 698. In some cases, the nucleic acid molecule encodes an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 655-657, or 699.

[0155] The present disclosure discloses, in some aspects, methods, compositions, kits, and vectors related to the single cytokine-anchored species, the double cytokine-anchored species, and the triple cytokine-anchored species described herein (hereinafter collectively referred to as “cytokine-anchored species” unless otherwise specified).

[0156] The present disclosure discloses, in some aspects, a nucleic acid molecule comprising a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in any one of 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. In some cases, the nucleic acid molecule comprises a nucleic acid sequence that is at least 80% identical to the sequence set forth in any one of 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. In some cases, the nucleic acid molecule comprises a nucleic acid sequence that is at least 85% identical to the sequence set forth in any one of 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. In some cases, the nucleic acid molecule comprises a nucleic acid sequence that is at least 90% identical to the sequence set forth in any one of 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. In some cases, the nucleic acid molecule comprises a nucleic acid sequence that is at least 95% identical to the sequence set forth in any one of 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. In some cases, the nucleic acid molecule comprises a nucleic acid sequence that is 100% identical to the sequence set forth in any one of 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.

[0157] In some aspects, the disclosure provides a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in any one of 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. In some cases, the polypeptide comprises an amino acid sequence that is at least 80% identical to the sequence set forth in any one of 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. In some cases, the polypeptide comprises an amino acid sequence that is at least 85% identical to the sequence set forth in any one of 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. In some cases, the polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence set forth in any one of 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. In some cases, the polypeptide comprises an amino acid sequence that is at least 95% identical to the sequence set forth in any one of 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. In some cases, the polypeptide comprises an amino acid sequence that is 100% identical to the sequence set forth in any one of 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.

[0158] In some cases, expressing a particular cytokine or a combination of cytokines on the surface of a cell, including introducing an exogenous nucleic acid molecule for expressing the particular cytokine or the combination thereof and an anchoring structure, such that the expression level of the particular cytokine or the combination thereof is higher than its natural expression level in a host cell or a host organism. In other cases, introducing a particular cytokine or a combination of cytokines and a tumor targeting moiety (e.g., a CAR) into a T cell and expressing on the surface thereof, can include introducing a nucleic acid molecule comprising a sequence encoding the particular cytokine or the combination of cytokines and the tumor targeting moiety (e.g., a CAR) into an immune cell (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).

[0159] Without being limited by a particular theory, the cytokine-anchoring substance described herein can provide engineered immune cells with higher anti-tumor activity than engineered immune cells (e.g., CAR-T cells, etc.) expressing a CAR alone (without expressing an exogenously introduced cytokine or a combination of cytokines). The cytokine-anchoring substance described herein can achieve a more optimal therapeutic effect when applied in immunotherapy. The cytokine-anchoring substance described herein can enable expression of a cytokine or a combination of cytokines on the cell membrane through a vector, thereby regulating the function of an immune cell. When the vector of the present disclosure is introduced into an immune cell, the resulting immune cell can have a more optimal immunotherapeutic effect, including higher proliferation and survival capacity than existing immune cells. By introducing a cytokine (e.g., an interleukin) or a particular combination of cytokines into an immune cell and co-expressing the cytokine or the particular combination of cytokines with a tumor targeting moiety (e.g., a CAR) on the cell surface, the anti-tumor activity can be enhanced compared to expressing the tumor targeting moiety (e.g., a CAR) alone, and in particular, the persistence and proliferation capacity of the engineered immune cell can be significantly improved. Delivering the engineered immune cell of the cytokine-anchoring substance to a cancer patient in the body can result in a more optimal therapeutic effect. Furthermore, the engineered immune cell of the present disclosure can reduce the number of cells administered due to its enhanced anti-tumor activity, and does not increase the release of free cytokines, thereby reducing the risk of side effects such as cytokine release syndrome (CRS).

[0160] Without being bound by a particular theory, anchoring a particular cytokine or a combination of particular cytokines at the surface of an immune cell can preserve the activity of the cytokine(s) and precisely stimulate the immune cell in a targeted manner without triggering the secretion of additional cytokines that can lead to severe CRS. According to some embodiments of the disclosure, the cytokine anchored at the surface of an immune cell can include a different interleukin, such as any one or a variant thereof selected from 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-36y, IL-23p19, or IL-la. Such a cytokine anchored on the cell membrane can improve the proliferative and survival capacity of the immune cell.

[0161] Without being bound by a particular theory, a combination of different cytokines can be anchored at the surface of an immune cell, including a combination of different interleukins, such as any one or a variant thereof selected from 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-36y, IL-23p19, or IL-la. Such a combination of cytokines anchored on the cell membrane can further improve the proliferative and survival capacity of the immune cell.

[0162] It should be understood that one, some or all of the features of the various embodiments described herein can be applied to any aspect unless the content clearly dictates otherwise. Moreover, the various embodiments can be combined to form other embodiments of the disclosure. These and other aspects of the disclosure will become apparent to those skilled in the art from the following detailed description.

[0163] Definitions

[0164] In the present disclosure, wherever aspects are described with “comprising,” similar aspects described with “consisting of” and / or “consisting essentially of” are also provided. All definitions, whether explicitly recited or not, should be construed in the broadest possible sense unless otherwise indicated.

[0165] Throughout the specification and claims, the singular “a” and “the” include plural referents unless the context clearly dictates otherwise. For example, the term “a cell” includes multiple cells, including mixtures thereof.

[0166] In this disclosure, unless the content clearly dictates otherwise, one, some or all of the features of the embodiments described herein can be applied to any aspect. Furthermore, the embodiments can be combined to form other embodiments of the disclosure. These and other aspects of the disclosure will be apparent from the detailed description that follows.

[0167] Throughout the specification and the appended claims, unless otherwise specified, all technical and scientific terms used herein have a meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, the Merck Manual of Diagnosis and Therapy (17th Ed., 1999), The Pharmacological Basis of Therapeutics (Gilman et al., 8th Ed., 1990), Goodman and Gilman’s The Pharmacological Basis of Therapeutics (Hardman et al., 9th Ed., 1995), and Harrison’s Principles of Internal Medicine (Kasper et al., 17th Ed., 2005) provide one of ordinary skill with a general dictionary of many of the terms used in this disclosure.

[0168] References herein to amino acids can use the commonly accepted three letter symbols or the one-letter codes recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides can be represented by their commonly accepted single-letter codes.

[0169] Unless otherwise indicated, the numbering of amino acids in the variable domains, complementarity determining regions (CDRs), and framework regions (FRs) of antibodies follows the Kabat definition set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. (1991), Public Health Service, National Institutes of Health, Bethesda, MD.

[0170] The term “about” or “approximately” means within an acceptable range of error for the particular value as determined by one of ordinary skill in the art to which the disclosure pertains, which will depend in part on how the value is measured or determined (i.e., limitations of the measurement system). For example, “about” can mean within one or more standard deviations, per art practice. Alternatively, “about” can mean ranges approximately 20% above and below a given value, approximately 10% above and below a given value, approximately 5% above and below a given value, or approximately 1% above and below a given value. Alternatively, especially 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. When a particular value is described in the specification and claims, unless otherwise stated the term “about” meaning within an acceptable range of error for the particular value should be presumed, unless otherwise indicated.

[0171] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acids. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified by natural processes, such as post-translational modifications, or by human intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other 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. It is understood that, because the polypeptides described herein are based on antibodies, the polypeptides can exist as single chains or as associated chains.

[0172] The term "amino acid" refers to both naturally occurring and unnatural, synthetic amino acids, including optical isomers (e.g., D or L forms) and amino acid analogs and peptidomimetics. Amino acids are referred to by either their standard three letter or one letter codes.

[0173] "Variant," when applied to a protein, refers to a protein that has sequence homology with a naturally occurring biologically active protein, and retains at least a portion of the therapeutic activity and / or biological activity of the biologically active protein. For example, a variant protein can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, or any range of identity between at least 70% and 99%, as compared to a reference biologically active protein. "Variant," as used herein, can refer to a cytokine that has substantial or significant sequence identity or similarity to a naturally occurring cytokine (e.g., a naturally occurring cytokine described herein, such as IL-2, IL-4, etc.), and retains one or more biological activities of the naturally occurring cytokine described herein (e.g., IL-2, IL-4, etc.), or one or more biological activities of an isoform of the naturally occurring cytokin. A variant can include, for example, one or more amino acid alterations, substitutions, deletions, additions, or chemical modifications to a parent peptide (e.g., a cytokine described herein, such as IL-2, IL-4, etc.), or the inclusion of one or more unnatural amino acids, or any combination thereof, but still retains the ability to specifically bind to a corresponding receptor, activate a downstream target, and / or induce one or more of differentiation, proliferation (or death), and activity of a cell (e.g., T cells and NK cells), to a similar, identical, or greater extent as the parent peptide. In some cases, the variant is a cytokine. The amino acid sequence of the variant can be at least about 80%, about 90%, about 95%, about 99%, or more identical to the parent cytokine, relative to the parent cytokine.

[0174] In the context of a polypeptide, "linear sequence" or "sequence" refers to the order of amino acids in a polypeptide from the amino terminus (N-terminus) to the carboxy terminus (C-terminus), where adjacent residues in the sequence are contiguous in the primary structure of the polypeptide. "Partial sequence" refers to the linear sequence of a portion of a polypeptide, which is known to contain additional residues in one or both directions.

[0175] "polynucleotide" or "nucleic acid," used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides (such as methylated nucleotides and the like) and / or analogs. If present, modifications to the nucleotide structure can be in the sugar moiety, the phosphate moiety, the nucleobase moiety or any combination thereof. The modifications can be done before or after assembly of the polymer. The nucleic acid sequence can be interrupted by non-nucleotide components. A polynucleotide can also be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, but are not limited to, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, but not limited to, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, amino -phosphoramidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing chiral linkages (e.g.,

[0176] The "variable region" of an antibody refers to the light chain variable region of an antibody or the heavy chain variable region of an antibody, either alone or in combination. The variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs, also known as hypervariable regions). The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs of the other chain, contribute to the formation of the antigen binding site of antibodies. There are at least two methods for determining CDRs: (1) the method based on inter-species sequence variability (i.e., the method proposed by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed. (1991), National Institute of Health, Bethesda, MD); (2) the method based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Mol. Biol. 273:927-948). CDRs as used herein can refer to CDRs defined by either method or a combination of both methods.

[0177] The "constant region" of an antibody refers to the light chain constant region of an antibody or the heavy chain constant region of an antibody, either alone or in combination.

[0178] A "host cell" includes an individual cell or cell culture that can be or has been a recipient for a vector containing a foreign polynucleotide. Host cells include progeny of the original host cell that have a non-identical genotype. Host cells include cells that have been transfected in vivo with a polynucleotide of the disclosure.

[0179] 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.

[0180] A "vector" as used herein refers to a construct that is capable of delivering and, preferably, expressing one or more genes or sequences of interest in a host cell. Examples of vectors include viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or bacteriophage vectors, DNA or RNA expression vectors bound to cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (e.g., producer cells).

[0181] The term "effective amount" or "therapeutically effective amount" refers to the amount of an agent that is sufficient to effect a beneficial or desired result. The therapeutically effective amount can vary depending on one or more of the following factors: the subject and disease condition to be treated, the subject's body mass and age, the severity of the disease condition, the mode of administration, and the like, which can be readily determined by one of ordinary skill in the art. The term "effective amount" also applies to the dosage that enables an image to be detected by an appropriate imaging method. The specific dosage can vary depending on one or more of the following factors: the particular agent selected, the dosing 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 placed. The effective amount of the active agent can be achieved by a single administration or by multiple administrations.

[0182] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or wetting agents, and the like, with which the active ingredient is administered, which are pharmaceutically acceptable in that they do not leave a residue, and which do not interact with the subject's immune system after the combination. Examples include any standard pharmaceutical carrier such as a phosphate buffered saline solution, water, oil / water emulsion such as water-in-oil or oil-in-water emulsion, and various types of wetting agents. The preferred diluent for aerosol or parenteral administration is phosphate buffered saline or normal saline (0.9%). The compositions comprising such carriers are formulated by well-known conventional methods (e.g., see Remington The Science of Pharmacy, 18thEdition, A. Gennaro, Ed., Mack Publishing Company, Easton, PA, 1990; and Remington The Science and Practice of Pharmacy, 20thEdition, Mack Publishing Company, 2000).

[0183] Throughout this specification and the annexed claims, unless otherwise specified, the methods and systems described in the present disclosure employ conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, microarray and sequencing technologies well known to those of ordinary skill in the art. Such conventional techniques include polymer array synthesis, hybridization and ligation of oligonucleotides, oligonucleotide sequencing, and detection of hybridization using labels. Specific illustrations of suitable techniques can be had by reference to the examples herein, and of course equivalents thereof. Such conventional techniques and descriptions are also set forth in the following standard laboratory manuals: Green et al. (eds.), Genome Analysis: A Laboratory Manual Series (VoIs. 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 Methods from Molecular Cloning: A Laboratory Manual, (2006); and Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition, (2012) (all published by Cold Spring Harbor Laboratory Press); Stryer L, Biochemistry, 4th Edition, W.H. Freeman Company, New York (1995); Gait, Oligonucleotide Synthesis: A Practical Approach, IRL Press, London (1984); Nelson and Cox, Lehninger Principles of Biochemistry, 6th Edition, W.H. Freeman Publishing Company, New York (2012); R.I. Freshney, Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition, Wiley-Blackwell (2010); and Berg et al., Biochemistry, 5th Edition, W.H. Freeman Publishing Company, New York (2002). All of the above references are incorporated herein by reference in their entireties for all purposes. Before the present compositions, research tools, systems and methods are described, it is to be understood that this disclosure is not limited to the particular systems, methods, compositions, targets, and uses described as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0184] The term "chimeric antigen receptor," or simply "CAR," refers to a group of peptides (typically two in the simplest embodiment) that, when present in immune effector cells, enable those cells to be specific to target cells (typically cancer cells) and to generate intracellular signals. In some embodiments, a 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"), wherein the cytoplasmic signaling domain comprises a functional signaling domain of a stimulatory molecule and / or a co-stimulatory molecule. In some cases, the group of peptides is contiguous (e.g., located in the same polypeptide chain, such as in chimeric fusion proteins). In some embodiments, the group of peptides is discontinuous (e.g., located in different polypeptide chains). In some embodiments, the group of peptides includes a dimerization switch that, in the presence of a dimerizing molecule, allows the peptides to couple to each other (e.g., allows the antigen-binding domain to couple to the intracellular signaling domain). In some cases, the stimulatory molecule is a ζ-chain associated with a T-cell receptor complex. In some cases, the cytoplasmic signaling domain also comprises a functional signaling domain of at least one co-stimulatory molecule as defined below. In some cases, the co-stimulatory molecule is selected from those described herein, such as 4-1BB (i.e., CD137), CD27, and / or CD28. In some cases, the CAR comprises a chimeric fusion protein containing an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain.

[0185] "Cytokine release syndrome" and "CRS" refer to acute systemic inflammatory syndromes characterized by fever and multiple organ dysfunction, which are associated with chimeric antigen receptor (CAR)-T cell therapy, therapeutic antibodies and haploidentical allogeneic transplantation, as described by Frey N and Porter D in Biological Blood and Bone Marrow Transplantation, Vol. 25, p. 123, 2019.

[0186] In this disclosure, "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target (such as carbohydrates, polynucleotides, lipids, polypeptides, etc.) through at least one antigen recognition site in the variable region of an immunoglobulin molecule. As used herein, this term includes immunoglobulin molecules capable of specifically binding to antigens and containing an Fc receptor binding site (which may or may not be functional). As used herein, this term includes not only complete polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, biantibodies), Fv fragments, and single-chain (scFv) mutants (which contain antigen recognition or antigen binding sites and have the ability to bind antigens). Antigen-binding antibodies or immunoglobulin fragments are well known in the art, and such fragments may have functional or non-functional Fc receptor binding sites. Furthermore, the term used herein is not limited to complete polyclonal antibodies or monoclonal antibodies, but also includes bispecific and other multispecific antibodies (produced from at least two complete antibodies), humanized antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, grafted antibodies, human antibodies, and any other modified immunoglobulin molecule containing an antigen-binding site, provided that the antibody has the desired biological activity.

[0187] "Specific binding" to an epitope, or CAR, is a well-known term in the art, as are methods for determining such specific binding. A molecule is said to exhibit "specific binding" if it reacts or binds to a particular cell, protein, or substance more frequently, more rapidly, for a longer duration, and / or with a higher affinity than with other cells, proteins, or substances. An antibody is said to "specifically bind" or "selectively bind" to a target if it has a higher affinity, is more readily bound, and / or binds for a longer duration to that target. For example, an antibody that specifically or selectively binds to CD19 has a higher affinity, is more readily bound, and / or binds for a longer duration to the CD19 epitope compared to other epitopes. As another example, an antibody (or other part thereof) that specifically or selectively binds to a first target may or may not bind to a second target. Therefore, "specific binding" or "selective binding" does not necessarily require (but may include) exclusive binding. Usually (but not necessarily), when binding is mentioned, selective binding is referred to.

[0188] When used to describe proteins or peptides, "fragment" refers to a truncated form of a naturally occurring biologically active protein or peptide that may or may not retain at least a portion of its therapeutic and / or biological activity.

[0189] The terms “TeIL,” “anchored interleukin,” “membrane-anchored cytokine,” “membrane-bound cytokine,” or “membrane-bound IL” are used interchangeably to refer to the specific cytokine-anchoring structures described herein.

[0190] The term "sIL" refers to secreted interleukins or secreted cytokines, which are interleukins or cytokines in non-membrane-anchored or non-membrane-bound forms.

[0191] Sequence identity

[0192] The “sequence identity” of a cytokine-anchoring substance or any other amino acid or nucleic acid sequence described herein is defined as the percentage of amino acid residues (or nucleotides) in the query sequence that are identical to those in the reference sequence after aligning the query sequence with a second reference polypeptide sequence (or a portion thereof) or a second reference nucleic acid sequence (or a portion thereof), introducing gaps where necessary to achieve the maximum percentage of sequence identity, without considering conserved substitutions as part of the sequence identity. Those skilled in the art can perform sequence alignment to determine amino acid sequence identity or nucleic acid sequence identity in various ways, such as using publicly available computer software (e.g., BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software). Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms required to achieve maximum alignment of the full length of the sequences to be compared. The percentage of sequence identity can be calculated based on the length of the complete defined polypeptide or nucleic acid sequence, or on a shorter length (e.g., based on a fragment extracted from a longer defined polypeptide or nucleic acid sequence, the fragment being at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues, base pairs, or nucleotides). Such lengths are merely examples, and it should be understood that any fragment length supported by the sequences shown in the tables, figures, or sequence listings herein can be used as a benchmark for calculating the percentage of sequence identity. In some embodiments, the percentage of sequence identity is determined based on the full length of a provided reference sequence (such as the sequence provided herein). For example, the sequence comparison of two amino acid sequences (or shorter fragments thereof) of this disclosure can be performed using the Blastp (protein-protein BLAST) computer program available online at the National Center for Biotechnology Information (NCBI). The percentage of amino acid sequence identity between a given amino acid sequence A and a given amino acid sequence B (also expressed as "a given amino acid sequence A has a specific percentage of amino acid sequence identity with a given amino acid sequence B") is calculated according to the following formula:

[0193] (X / Y)×100%

[0194] Where X is the number of amino acid residues that the sequence alignment program BLAST determined to be identical matches in the alignment results of A and B, and Y is the total number of amino acid residues in A or B (the shorter one is taken).

[0195] Two polynucleotide or polypeptide sequences are said to be "identical" when they align in the following manner to achieve maximum correspondence. When comparing two sequences, local regions of sequence similarity are typically identified and compared using an alignment window.

[0196] signal peptide

[0197] In some aspects, the signal peptide provided in this disclosure is a portion of the polypeptides described in this disclosure (including polypeptides in single-cytokine-anchored substances, first and second polypeptides in dual-cytokine-anchored substances, and first, second, and third polypeptides in tri-cytokine-anchored substances). In some cases, during protein processing, the signal peptide is cleaved from the polypeptide of a single-cytokine-anchored substance to form a protein; cleaved from the first and second polypeptides of a dual-cytokine-anchored substance, respectively, to form a first protein and a second protein; and cleaved from the first, second, and third polypeptides of a tri-cytokine-anchored substance, respectively, to form a first protein, a second protein, and a third protein.

[0198] The signal peptide may be encoded by a signal peptide nucleic acid sequence. This signal peptide nucleic acid sequence may exist as part of the nucleic acid sequences described in this disclosure (including nucleic acid sequences in single-cytokine-anchoring substances, first and second nucleic acid sequences in dual-cytokine-anchoring substances, and first, second, and third nucleic acid sequences in triple-cytokine-anchoring substances).

[0199] Signal peptides can be any suitable peptide capable of guiding the polypeptides described herein to a specific compartment of the cell containing the polypeptide (such as the cell membrane). In some embodiments, signal peptides include peptides capable of guiding the peptide and any polypeptides linked thereto to specific organelles (such as the endoplasmic reticulum) and / or the cell surface. As used herein, the terms "signal peptide" or "signal peptide sequence" refer to a peptide sequence that may be present at the N-terminus of a newly synthesized polypeptide to be secreted or a newly synthesized transmembrane polypeptide. Signal peptides can guide polypeptides across or into the cell membrane, where they can subsequently be cleaved. Specifically, signal peptides can guide polypeptides into the cell's secretory pathway.

[0200] In some cases, the signal peptide can be a peptide of any secretory or transmembrane protein, capable of guiding the delivery of the polypeptide described herein to the cell membrane and cell surface, and enabling the polypeptide to achieve proper localization. In some cases, the signal peptide guides the polypeptide described herein to the cell membrane, causing the extracellular portion of the polypeptide to be displayed on the cell surface. In some cases, the transmembrane portion spans the plasma membrane, and the active domain is located in the cytoplasm or intracellular region. In some embodiments, the signal peptide is cleaved after passing through the endoplasmic reticulum (referred to as a cleavable signal peptide). In some cases, the terminal end of the signal peptide may contain amino acids that can be recognized and cleaved by a signal peptidase. The signal peptidase can cleave the peptide during or after transport, producing a free signal peptide and a mature protein. The free signal peptide can then be degraded by a specific protease.

[0201] 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 a signal peptide of an immunoglobulin heavy chain.

[0202] In some cases, when another component of a cytokine-anchored substance (such as cytokines and / or CARs) is expressed within a cell (such as engineered immune cells), a signal peptide co-expressed in the same polypeptide as that component can guide the nascent protein to the endoplasmic reticulum and then to the cell surface, enabling the expression of that component of the cytokine-anchored substance at the cell surface. In some cases, the core of the signal peptide consists of a long segment of hydrophobic amino acids that tend to form an α-helix. In some cases, the initiating portion of the signal peptide is a short segment of positively charged amino acids, which helps ensure the correct topological structure of the polypeptide during transport. The signal peptide may be located at the N-terminus of the polypeptide. In some embodiments, when cells express membrane-anchored cytokines and / or CARs, the signal peptide sequence can be cleaved from the membrane-anchored cytokines and / or CARs.

[0203] Without being bound by any specific theory, signal peptides can promote the expression of membrane-anchored cytokines and / or CARs, and the presence of signal peptides in expressed membrane-anchored cytokines and / or CARs may contribute to the membrane anchoring of cytokines and / or CARs; however, the presence or absence of signal peptides may be irrelevant to the function of cytokines and / or CARs anchored to the cell membrane.

[0204] In some embodiments, the signal peptide is a transmembrane protein signal peptide. In some embodiments, the signal peptide comprises a combination of CD4 signal peptide, CD8α signal peptide, CD28 signal peptide, CD33 signal peptide, CD137(4-1BB) signal peptide, IL-2 signal peptide, IgE signal peptide, IgG1 signal peptide, GM-CSF signal peptide, HLA-A signal peptide, HLA signal peptide, TCR signal peptide, or β2M signal peptide, or combinations thereof. In some embodiments, the signal peptide comprises a combination of CD4 signal peptide, CD8α signal peptide, CD28 signal peptide, CD33 signal peptide, CD137(4-1BB) signal peptide, IL-2 signal peptide, IgE signal peptide, IgG1 signal peptide, GM-CSF signal peptide, HLA-A signal peptide, HLA signal peptide, TCR signal peptide, or β2M signal peptide, or variants 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 GM-CSF 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 HLA 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.

[0205] In some embodiments, the signal peptide is a naturally occurring signal peptide of a wild-type cytokine. In some embodiments, the signal peptide comprises a subset selected from IL-2, IL-4, IL-7, IL-9, IL-10, IL-12p40, IL-15, IL-18, IL-21, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α, or functional variants thereof. In some embodiments, the signal peptide comprises IL-4 or a variant thereof. In some embodiments, the signal peptide comprises IL-10 or a variant thereof. In some embodiments, the signal peptide comprises IL-7 or a variant thereof. In some embodiments, the signal peptide comprises IL-9 or a variant thereof. In some embodiments, the signal peptide comprises IL-12p40 or a variant thereof. In some embodiments, the signal peptide comprises IL-15 or a variant thereof. In some embodiments, the signal peptide comprises IL-18 or a variant thereof. In some embodiments, the signal peptide comprises IL-21 or a variant thereof. In some embodiments, the signal peptide comprises IL-23 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises IL-27 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises IL-36γ signal peptide or a variant thereof. In some embodiments, the signal peptide comprises IL-23p19 signal peptide or a variant thereof. In some embodiments, the signal peptide comprises IL-1α signal peptide or a variant thereof.

[0206] In some embodiments, the signal peptide and the cytokine peptide are encoded by the same gene. In some embodiments, the signal peptide and the 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.

[0207] In some embodiments, the signal peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:2-7 or 113-114. In some embodiments, the signal peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:2. In some embodiments, the signal peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:3. In some embodiments, the signal peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:4. In some embodiments, the signal peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:5. In some embodiments, the signal peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:6. In some embodiments, the signal peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:7. In some embodiments, the signal peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:113. In some embodiments, the signal peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:114.

[0208] In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:52-59. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:52. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:53. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:54. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 55. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 56. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 57. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 58. In some embodiments, the signal peptide is encoded by a signal peptide nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:59.

[0209] Table 1A. Exemplary signal peptide amino acid sequences

[0210]

[0211]

[0212] Table 1B. Exemplary signal peptide nucleic acid sequences

[0213]

[0214] Cytokines

[0215] In some aspects, the cytokines or cytokine peptides provided in this disclosure are part of the polypeptides described in this disclosure (including polypeptides in single-cytokine-anchoring substances, first and second polypeptides in dual-cytokine-anchoring substances, and first, second, and third polypeptides in triple-cytokine-anchoring substances). The cytokines or cytokine peptides, as part of the polypeptide, are operatively linked to the anchoring structure described herein. This anchoring structure enables the cytokines to attach to the cell membrane.

[0216] Cytokines can be encoded by cytokine nucleic acid sequences. These cytokine nucleic acid sequences can exist as part of the nucleic acid sequences described in this disclosure (including nucleic acid sequences in single-cytokine-anchored substances, first and second nucleic acid sequences in dual-cytokine-anchored substances, and first, second, and third nucleic acid sequences in triple-cytokine-anchored substances).

[0217] In various embodiments, the term "cytokine" may include full-length cytokines or interleukins, or fragments (e.g., truncated forms) or variants thereof, wherein the fragments or variants substantially retain the biological activity of the corresponding wild-type cytokine or interleukin (e.g., 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 may be secreted by immune cells (e.g., monocytes, macrophages, T cells, B cells, NK cells, etc.). Cytokines may also be secreted by certain non-immune cells (e.g., endothelial cells, epidermal cells, fibroblasts, etc.). Cytokines may be produced by monocytes, macrophages, B cells, dendritic cells, TH1 cells, TH2 cells, mast cells, NK cells, and bone marrow stromal cells. Cytokines may have the ability to regulate a variety of cellular and organismal functions, including innate and adaptive immunity, hematopoiesis, cell growth, APSC pluripotent cell function, and damaged tissue repair. Cytokines may include interleukins, interferon-α (IFN-α), interferon-β (IFN-β), or tumor necrosis factor (TNF).

[0218] In some embodiments, the cytokines used may be derived from any mammalian species. In some embodiments, the cytokines are derived from the following species: humans, horses, cattle, mice, pigs, rabbits, cats, dogs, rats, goats, sheep, or non-human primates. In some embodiments, the cytokines are derived from humans. In some embodiments, the cytokines may be in their natural or wild-type mutant forms.

[0219] In some cases, the cytokines specifically selected are interleukins (ILs). Interleukins are a class of cytokines produced by and acting on a variety of cells. Interleukins play roles in signal transduction, activation and regulation of immune cells, mediating the activation, proliferation and differentiation of T cells and B cells, and mediating inflammatory responses. Interleukins can be produced by helper CD4+ T lymphocytes. They can also be synthesized by monocytes, macrophages, and endothelial cells. Interleukins can bind to receptors and influence the activation and suppression of the immune system as well as cell division.

[0220] In some embodiments of the present invention, the cytokines comprise ILs of the interleukin-1 (IL-1) family. The IL-1 family may include IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ, IL-1Ra (IL-1 receptor antagonist), IL-36Ra (IL-36 receptor antagonist), IL-38, and an anti-inflammatory cytokine (IL-37). In some cases, the cytokines comprise pro-inflammatory cytokines. The pro-inflammatory cytokines may comprise 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. In some cases, the cytokines comprise anti-inflammatory cytokines. The anti-inflammatory cytokines may comprise IL-4, IL-10, or IL-27.

[0221] In some embodiments of the present invention, the cytokines comprise ILs of the IL-2 family. The IL-2 family, also known as the common γ-chain family, may include IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The IL-2 family can bind to the common γ-chain receptor (also known as CD132). The IL-2 family can function as growth and proliferation factors for progenitor and mature cells.

[0222] In some embodiments of the present invention, the cytokine comprises at least a portion thereof selected from 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. In some embodiments of the present invention, the cytokine comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:100-112. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:150-162.

[0223] In some embodiments of the present invention, the cytokine comprises IL-2 or a variant thereof. IL-2 is primarily produced by CD4+ T cells and CD8+ T cells, and can also be expressed by dendritic cells and NK cells. IL-2 can bind to the IL-2 receptor (IL-2R), which comprises three subunits (CD25, CD122, and a common γ chain), all of which are essential for IL-2 binding. IL-2 can play a role in the development of regulatory T cells (Tregs), act as a B cell growth factor, stimulate antibody synthesis, and promote the proliferation and differentiation of NK cells and helper T cells. In some embodiments of the present invention, the cytokine comprises human IL-2 or a variant thereof. In some embodiments of the present invention, the cytokine comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:100. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:150.

[0224] In some embodiments of the present invention, 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: type I IL-4 receptor (containing CD124 (IL-4Rα) and CD132) and type II IL-4 receptor (containing IL-4Rα and IL-13Rα1). IL-4 can play a variety of roles, including regulating allergic diseases, activating immune responses against extracellular parasites, and stimulating Th2 cell development. In some embodiments of the present invention, the cytokine comprises human IL-4 or a variant thereof. In some embodiments of the present invention, the cytokine comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:101. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:151. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:163.

[0225] In some embodiments of the present invention, the cytokine comprises IL-7 or a variant thereof. IL-7 is a homeostatic cytokine that can be present in T cells, B cell progenitors, and bone marrow macrophages. IL-7 can bind to its receptor IL-7R, which comprises a γ-chain fragment and IL-7Rα (CD127). IL-7 can participate in the survival and proliferation of thymocytes, and also in the development of naive B cells, memory B cells, naive T cells, memory T cells, mature T cells, and NK cells. In some embodiments of the present invention, the cytokine comprises human IL-7 or a variant thereof. In some embodiments of the present invention, the cytokine comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:102. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:152. In some embodiments of the present invention, the human IL-7 or its variants comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of GenBank accession numbers 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, and ANQ68335.1.

[0226] In some embodiments of the present invention, 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 subunit. IL-9 is a potent growth factor for T cells and mast cells, and can inhibit cytokine production, IgE production, and mucus secretion from bronchial epithelial cells. In some embodiments of the present invention, the cytokine comprises human IL-9 or a variant thereof. In some embodiments of the present invention, the cytokine comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:103. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:153.

[0227] In some embodiments of the present invention, the cytokine comprises IL-10 or a variant thereof. In some embodiments of the present invention, the cytokine comprises human IL-10 or a variant thereof. In some embodiments of the present invention, the cytokine comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 104. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 154. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 164.

[0228] In some embodiments of the present invention, the cytokine comprises IL-12 or a variant thereof. IL-12 comprises two subunits, IL-12p35 and IL-12p40, and co-expression of the IL-12p35 and IL-12p40 subunits can produce a disulfide-linked, biologically active IL-12p70. IL-12 can be produced by various hematopoietic cell types and antigen-presenting cells (such as dendritic cells and macrophages). IL-12 can bind to its receptors IL-12Rβ1 / IL-12Rβ2, which can be expressed on activated T cells, NK cells, and dendritic cells. The binding of IL-12 to its receptors can activate tyrosine kinase 2 (TYK2), Janus kinase 2 (JAK2), and the signal transduction and activating transcription (STAT) pathway. In some embodiments of the present invention, the cytokine comprises human IL-12 or a variant thereof. In some embodiments of the present invention, the human IL-12 or a variant thereof comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of GenBank accession numbers AAM34792.1, AAG32620.1, AAG32620.1, CCA63965.1, AJQ18452.1, AAD56386.1, AAL05890.1, AAL05891.1, AAH67502.1, AAH67498.1, AAH67498.1, AAH67500.1, AAH67501.1, AAH74723.1, ABM53138.1, AAA35695.1, and AAA59938.1.

[0229] In some embodiments of the present invention, the cytokine comprises IL-12p40 or a variant thereof. In some embodiments of the present invention, the cytokine comprises human IL-12p40 or a variant thereof. In some embodiments of the present invention, the cytokine comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:105. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:155.

[0230] In some embodiments of the present invention, the cytokine comprises IL-15 or a variant thereof. IL-15 can bind to its receptor IL-15R, which comprises the CD132 subunit, the IL-15Rα subunit, and the IL-2Rβ chain. IL-15 can be produced by keratinocytes, skeletal muscle cells, monocytes, and activated CD4+ T cells in response to signals triggering innate immunity. IL-15 has a similar structure and some of the same functions as IL-2 (such as T cell activation and stimulation of NK cell proliferation), and can also participate in the homeostasis regulation of CD8+ memory cells, NK cells, and NKT cells. In some embodiments of the present invention, the cytokine comprises human IL-15 or a variant thereof. In some embodiments of the present invention, the cytokine comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:106. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:156. In some embodiments of the present invention, the human IL-15 or a variant thereof comprises a sequence with GenBank accession numbers EAX05083.1, EAX05084.1, EAX05085.1, EAX05086.1, EAX05087.1, NP_751915.1, NP_000576.1, CAJ13397.1, CAJ32191.1, CAL40354.1, CAS97649.1, CBI67687.1, AAI00963.1, AAI00964.1, and AAI00962. The sequence shown in any one of 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 has an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity.

[0231] In some embodiments of the present invention, the cytokine comprises IL-18 or a variant thereof. IL-18 can promote responses of Th1 and Th2 cells, and when acting in conjunction with IL-2, can induce T cells and NK cells to produce IL-13. It can also enhance the cytotoxicity of NK cells by promoting the expression of Fas ligand in NK cells. IL-18 can be involved in the development of various autoimmune diseases, myocardial infarction, metabolic syndrome, and other diseases. In some embodiments of the present invention, the cytokine comprises human IL-18 or a variant thereof. In some embodiments of the present invention, the cytokine comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:107. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:157.

[0232] In some embodiments of the present invention, the cytokine comprises IL-21 or a variant thereof. IL-21 can be produced by T cells, NKT cells, and Th17 cells, can bind to its receptors (including CD132 and IL-21R), participate in the regulation of B cell function, and can also promote the proliferation of CD8+ T cells, NK cells, and NKT cells. In some embodiments of the present invention, the cytokine comprises human IL-21 or a variant thereof. In some embodiments of the present invention, the cytokine comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:108. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:158. In some embodiments of the present invention, the human IL-21 or its variants include those associated with GenBank accession numbers AAU88182.1, EAX05226.1, CA194500.1, CAJ47524.1, CAL81203.1, CAN87399.1, CAS03522.1, CAV33288.1, CBE74752.1, CB170418.1, CBI85469.1, CB185472.1, and CBL. The sequence shown in any one of 93962.1, CCA63962.1, AAG29348.1, AAH66258.1, AAH66259.1, AAH66260.1, AAH66261.1, AAH66262.1, AAH69124.1, ABG36529.1, and BBA22643.1 has an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity.

[0233] In some embodiments of the present invention, the cytokine comprises IL-27 or a variant thereof. In some embodiments of the present invention, the cytokine comprises human IL-27 or a variant thereof. IL-27 can bind to its receptor IL-27R, which comprises the widely expressed gp130 protein and WSX-1 / TCCR (T cell cytokine receptor). The biological effects of IL-27 can be mediated by activation of the JAK1, JAK2, TYK2, STAT1, and STAT3 pathways. In some embodiments of the present invention, the cytokine comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 109. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 159.

[0234] In some embodiments of the present invention, the cytokine comprises IL-36γ or a variant thereof. In some embodiments of the present invention, the cytokine comprises human IL-36γ or a variant thereof. In some embodiments of the present invention, the cytokine comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 110. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 160.

[0235] In some embodiments of the present invention, the cytokine comprises IL-23 or a variant thereof. In some embodiments of the present invention, the cytokine comprises IL-23p19 or a variant thereof. IL-23 can bind to its receptor, which is composed of IL-12Rβ1 and a unique IL-23 receptor subunit (IL-23R). The biological effects of IL-23 on its target cells can be mediated by activation of the TYK2, JAK2, STAT3, and STAT4 pathways. In some embodiments of the present invention, the cytokine comprises human IL-23 or a variant thereof. In some embodiments of the present invention, the cytokine comprises human IL-23p19 or a variant thereof. In some embodiments of the present invention, the cytokine comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:111. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:161.

[0236] In some embodiments of the present invention, the cytokine comprises IL-1α or a variant thereof. In some embodiments of the present invention, the cytokine comprises human IL-1α or a variant thereof. In some embodiments of the present invention, the cytokine comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 112. In some embodiments of the present invention, the cytokine is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 162.

[0237] Table 2A. Exemplary Cytokine Amino Acid Sequences

[0238]

[0239]

[0240]

[0241] Table 2B. Exemplary Cytokine Nucleic Acid Sequences

[0242]

[0243]

[0244]

[0245] Anchoring structure

[0246] In some aspects of this invention, the provided anchoring structure exists as part of the polypeptide described in this invention, including a polypeptide in a single-cytokine-anchoring substance, a first polypeptide and a second polypeptide in a dual-cytokine-anchoring substance, and a first polypeptide, a second polypeptide, and a third polypeptide in a triple-cytokine-anchoring substance. The anchoring structure can be any structure capable of anchoring itself and any peptide linked to it to the cell membrane.

[0247] In some embodiments of the present invention, the cell membrane anchoring structure is a polypeptide cell membrane anchoring structure comprising an amino acid sequence. The anchoring structure can attach cytokines to the cell membrane. In various embodiments of the present invention, the cytokines described herein are operatively linked to the anchoring structure. The anchoring structure may comprise a peptide anchoring structure or a non-peptide anchoring structure. The non-peptide anchoring structure can attach to the polypeptide via a non-peptide anchoring attachment signal. The non-peptide anchoring attachment signal may be part of the polypeptide. In some cases, the non-peptide anchoring attachment signal is operatively linked to the cytokines described herein. In some cases, the peptide anchoring structure is operatively linked to the cytokines described herein.

[0248] The peptide anchoring structure provided by this invention can be encoded by a peptide anchoring structure nucleic acid sequence. The peptide anchoring structure nucleic acid sequence can exist as part of the nucleic acid sequence described in this invention, including the nucleic acid sequence in a single-cytokine-anchoring substance, the first and second nucleic acid sequences in a dual-cytokine-anchoring substance, and the first, second, and third nucleic acid sequences in a triple-cytokine-anchoring substance.

[0249] The non-peptide anchoring attachment signal can be encoded by a non-peptide anchoring attachment signal nucleic acid sequence. The non-peptide anchoring attachment signal nucleic acid sequence can exist as part of the nucleic acid sequence described in this invention, including nucleic acid sequences in single-cytokine-anchoring substances, first and second nucleic acid sequences in dual-cytokine-anchoring substances, and first, second, and third nucleic acid sequences in triple-cytokine-anchoring substances.

[0250] Non-peptide anchored structures

[0251] In some embodiments of the present invention, the non-peptide anchoring structure does not contain an amino acid sequence. In some embodiments of the present invention, the non-peptide anchoring structure can attach to the protein during protein processing. In some embodiments of the present invention, the non-peptide anchoring structure can attach to the protein by replacing the non-peptide anchoring attachment signal located at the C-terminus of the protein.

[0252] The non-peptide-anchored structure provided by this invention may include lipids (such as glycolipids). Glycolipids are lipids linked to carbohydrates via glycosidic bonds, which can maintain cell membrane stability, promote cell recognition, and are present on the surface of all eukaryotic cell membranes, extending from the phospholipid bilayer to the extracellular environment. Glycolipids may include glycosylglycerols and glycosphingolipids. Glycosylglycerols may contain acetylated or unacetylated glycerol with at least one fatty acid as a lipid complex, and may further contain galactolipids and thiolipins.

[0253] In some cases, the glycolipid comprises glycosylphosphatidylinositol (GPI). In some cases, the non-peptide-anchored structure comprises a GPI-anchored structure. In some cases, the non-peptide-anchored structure is attached to a non-peptide-anchored attachment signal. In some cases, the non-peptide-anchored attachment signal comprises a glycolipid attachment signal. In some cases, the non-peptide-anchored attachment signal comprises a GPI attachment signal. In some cases, the C-terminal GPI attachment signal comprises 20-30 amino acids, originating from the amino acid that binds to the GPI after the GPI attachment signal is cleaved. In some cases, the GPI-binding amino acid comprises serine (Ser), asparagine (Asn), aspartic acid (Asp), alanine (Ala), glycine (Gly), cysteine ​​(Cys), or threonine (Thr). The GPI attachment signal peptide may comprise about 10 hydrophilic amino acid fragments and may also comprise about 20 hydrophobic amino acid fragments.

[0254] In some embodiments of the present invention, the non-peptide anchoring structure can attach to the C-terminus of the polypeptide during post-translational modification and anchor cytokines to the cell membrane. In some embodiments of the present invention, the non-peptide anchoring structure is a glycosylated phosphatidylinositol anchoring structure (GPI anchoring structure). The GPI anchoring structure may include a phosphate ethanolamine linker, a core glycan, and a phospholipid tail. The structure of the core glycan may be EtNP-6Manα2-Manα6-(EtNP)2Manα4-GlcNα6-myoIno-P-lipid (where EtNP is phosphate ethanolamine, Man is mannose, GlcN is glucosamine, and Ino is inositol). In some cases, the GPI anchoring structure can be linked to the C-terminus of the polypeptide via an amide bond formed by the C-terminal carboxyl group of the polypeptide and the amino group of the terminal EtNP. In some cases, the core glycan may have side chain modifications, such as modifications selected from phosphate ethanolamine groups, mannose, galactose, sialic acid, or other sugars. Examples of GPI-anchored structures, their synthesis, structure, and function can be found in "Biosynthesis and biology of mammalian GPI-anchored proteins" by Kinoshita Taroh, published in Open Biol., Vol. 10, pp. 190-290 in 2020, and in the paper by Paulick MG et al., published in Biochemistry, Vol. 47, No. 27, pp. 6991-7000 in 2008. The full text of the above references is incorporated herein by reference for all purposes.

[0255] GPI-anchored structures are a type of post-translational modification of proteins that enables them to anchor to the extracellular surface of the cell membrane by adding glycosylated phosphatidylinositol. In some cases, wild-type proteins with GPI-anchored structures do not contain transmembrane or cytoplasmic domains. These GPI-anchored structures can be from different protein families, including membrane-associated enzymes, adhesion molecules, and proteins covering the outer surface of protoparasites such as Trypanosoma brevicornu.

[0256] GPI-anchored structures connect to the peptide via a GPI attachment signal located at the C-terminus. After translocation via the endoplasmic reticulum, the GPI attachment signal can be cleaved and removed by a specific transaminases, replaced by the GPI-anchored structure. Modifying proteins by adding GPI-anchored structures can endow them with specific properties because the added lipid moiety allows the protein to insert into the cell membrane, thereby achieving protein anchoring. In some embodiments, the GPI anchoring structure is derived from rat brain Thy-1 protein, human erythrocyte acetylcholinesterase (AChE), hamster brain-sheep scrapie prion protein, human urine CD59 protein, mouse skeletal muscle neuronal cell adhesion molecule (NCAM), bovine liver 5'-nucleotidase, human placental alkaline phosphatase (APase), human CD52 protein, porcine renal membrane dipeptidase, human renal membrane dipeptidase, Trypanosoma brucei variant surface glycoprotein (VSG), Trypanosoma cruzi IG7 protein, Trypanosoma cruzi mucin, Trypanosoma cruzi NETNES protein, Leishmania major gp63 protein, Saccharomyces cerevisiae gp125 protein, Aspergillus fumigatus PhoAp protein, Pyrus communis arabinogalactan protein, and Dictyostelium The PsA protein of *discoideum*, the VSG protein of *Trypanosoma congolense*, or the AChE protein of *Torpedo*.

[0257] In some embodiments of the present invention, the GPI attachment signal comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:200. In some embodiments of the present invention, the GPI attachment signal is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:250-252. In some embodiments of the present invention, the GPI attachment signal is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:250. In some embodiments of the present invention, the GPI attachment signal is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:251. In some embodiments of the present invention, the GPI attachment signal is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:252.

[0258] Table 3A. Exemplary non-peptide anchoring attachment signal amino acid sequences

[0259]

[0260]

[0261] Table 3B. Exemplary non-peptide anchoring attachment signal nucleic acid sequences

[0262]

[0263] Peptide anchoring structure

[0264] In some embodiments of the present invention, the peptide anchoring structure comprises an amino acid sequence comprising at least a portion of a transmembrane domain derived from any suitable transmembrane protein. In some embodiments of the present invention, the peptide anchoring structure comprises a transmembrane peptide sequence.

[0265] In some embodiments of the present invention, the peptide anchoring structure comprises a portion of a transmembrane peptide sequence. In some embodiments of the present invention, the transmembrane peptide sequence comprises a sequence selected from the B7-1 transmembrane amino acid sequence, the B7-2 transmembrane amino acid sequence, the B7-H1 transmembrane amino acid sequence, the B7-H3 transmembrane amino acid sequence, the tumor necrosis factor receptor 2 (TNFR2) transmembrane amino acid sequence, the CD8α transmembrane amino acid sequence, the CD28 transmembrane amino acid sequence, the CD3ζ transmembrane amino acid sequence, the CTLA-4 (CD152) transmembrane amino acid sequence, or the PD-L1 transmembrane amino acid sequence, or any variant thereof. In some embodiments of the present invention, the peptide anchoring structure comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:203. In some embodiments of the present invention, the peptide anchoring structure is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:253. In some embodiments of the present invention, the peptide anchoring structure comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:204. In some embodiments of the present invention, the peptide anchoring structure is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:254. In some embodiments of the present invention, the peptide anchoring structure is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:255.

[0266] In some embodiments of the present invention, the peptide anchoring structure comprises at least a portion of a transmembrane domain selected from B7-1, B7-2, CD8, and CD28.

[0267] In some embodiments of the present invention, the peptide anchoring structure comprises at least a portion of the B7-1 transmembrane-intracellular amino acid sequence. In some embodiments of the present invention, the peptide anchoring structure comprises at least a portion of the B7-2 transmembrane-intracellular amino acid sequence. In some embodiments of the present invention, the peptide anchoring structure comprises at least a portion of the CD8α transmembrane-intracellular amino acid sequence. In some embodiments of the present invention, the peptide anchoring structure comprises at least a portion of the B7-1 transmembrane amino acid sequence. In some embodiments of the present invention, the peptide anchoring structure comprises at least a portion of the B7-2 transmembrane amino acid sequence. In some embodiments of the present invention, the peptide anchoring structure comprises at least a portion of the CD8α transmembrane amino acid sequence. In some embodiments of the present invention, the peptide anchoring structure comprises the B7-H1 transmembrane amino acid sequence or any fragment or variant thereof. In some embodiments of the present invention, the peptide anchoring structure comprises the B7-H3 transmembrane amino acid sequence or any fragment or variant thereof. In some embodiments of the present invention, the peptide anchoring structure comprises the tumor necrosis factor receptor 2 (TNFR2) transmembrane amino acid sequence or any fragment or variant thereof. In some embodiments of the present invention, the peptide anchoring structure comprises a CD28 transmembrane amino acid sequence or any fragment or variant thereof. In some embodiments of the present invention, the peptide anchoring structure comprises a CD3ζ transmembrane amino acid sequence or any fragment or variant thereof. In some embodiments of the present invention, the peptide anchoring structure comprises a CTLA-4 (CD152) transmembrane amino acid sequence or any fragment or variant thereof. In some embodiments of the present invention, the peptide anchoring structure comprises a PD-L1 transmembrane amino acid sequence or any fragment or variant thereof.

[0268] In some embodiments of the present invention, the peptide anchoring structure is located at the C-terminus of the polypeptide. In some embodiments of the present invention, the signal peptide and the cytokine peptide described herein are operatively linked to the peptide anchoring structure in a direction from the N-terminus of the polypeptide to the C-terminus of the polypeptide.

[0269] Table 4A. Exemplary peptide anchoring structure amino acid sequences

[0270]

[0271] Table 4B. Exemplary peptide-anchored nucleic acid sequences

[0272]

[0273] Targeted portion

[0274] In some aspects of this invention, the provided targeting portion exists as part of the polypeptide described in this invention, including a polypeptide in a single-cytokine-anchoring substance, a first polypeptide and a second polypeptide in a dual-cytokine-anchoring substance, and a first polypeptide, a second polypeptide, and a third polypeptide in a triple-cytokine-anchoring substance. The targeting portion may comprise a chimeric antigen receptor (CAR), a T-cell receptor (TCR), a B-cell receptor (BCR), or a fragment thereof.

[0275] In some embodiments of the present invention, the targeting portion comprises a TCR or a fragment thereof. The TCR may be a recombinant TCR, generated by recombinant expression of one or more exogenous TCR α, β, γ, and / or δ chain encoding genes. The recombinant TCR may be a chimeric or heterozygous TCR, containing TCR amino acid sequences derived from two or more mammalian species. The TCR may be a humanized TCR. The TCR may contain an α chain, a β chain, or a γ or δ chain of the TCR. The polypeptide chain of the TCR is known in the art.

[0276] The targeting portion can be linked to the polypeptide described herein via a cleavable adapter. The targeting portion is encoded by a targeting sequence, which can be linked to a nucleic acid sequence via a cleavable adapter. In some cases, the targeting portion recognizes an antigen; in other cases, the targeting portion binds to an antigen.

[0277] In some cases, the targeting component recognizes tumor-specific antigens. These tumor-specific antigens may be molecules (including proteins, polypeptides, peptides, lipids, carbohydrates, etc.) primarily expressed or overexpressed by tumor cells, and thus can be considered specifically associated with a tumor or cancer. The tumor-specific antigens may also be expressed in normal non-tumor or non-cancerous cells, but at lower levels or with lower intensity than in tumor cells. Tumor cells may overexpress the tumor-specific antigens, or their expression levels may be significantly higher than in normal non-cancerous cells. The tumor-specific antigens may be expressed by cells at different developmental or maturation stages, such as cells at the embryonic or fetal stage (such cells are typically not present in adult subjects), or by stem cells or progenitor cells (such cells are typically not present in adult subjects). In some cases, the tumor-specific antigen may be a mutant antigen, primarily expressed or overexpressed by tumor or cancer cells, but not expressed or expressed at significantly lower levels in normal non-cancerous cells.

[0278] Examples of tumor-specific antigens include mesothelin, gp100, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, CD138, CD276, CD171, CD5, CD7, mucin 1 (MUC1), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), interleukin-13 receptor α2 (IL13Rα2), disialotyl-ganglioside (GD2), natural killer cell family 2 member D (NKG2D), epidermal growth factor receptor variant III (EGFRvIII), CS1, and chemokines (CC motif). Ligand 1 (CCL1), B cell maturation antigen (BCMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), FMS-like tyrosine kinase 3 (FLT3), fibroblast activation protein (FAP), tumor-associated glycoprotein 72 (TAG72), CD44 variant 6 (CD44v6), epithelial cell adhesion molecule (EPCAM), B7 homolog 3 (B7H3), stem cell factor receptor (KIT), protease serine 21 (PRSS21), vascular endothelial growth factor receptor 2 (VEGFR2), Lewis Y antigen (LewisY), CD24, platelet-derived growth factor receptor β (PDGFR-β), stage-specific embryonic antigen 4 (SSEA-4), neural cell adhesion molecule (NCAM), tight junction protein 18.2 (Claudin18).2) Phosphatidylinositol proteoglycan 3 (GPC3), ganglioside GM3 (GM3), T cell recognition glycoprotein 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), tumor endothelial marker 7 receptor (TEM7R), tight junction protein 6 (CLDN6), G protein-coupled receptor C5 family D member (GPRC5D), X chromosome open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), placenta-specific 1 (PLAC1), globulin H (GloboH), New York breast cancer 1 (NY-BR-1), urokinase-type plasminogen activator receptor 2 (UPK2), liver glycoprotein A receptor 2 (HAVCR1), β3-adrenergic receptor (ADRB3), pan-connecting protein 3 (PANX3), G protein-coupled receptor 20 (GP... R20), Lymphocyte antigen 6 complex locus K (LY6K), olfactory receptor 51E2 (OR51E2), tumor-associated phosphatase (TRAP), Wilms' tumor 1 (WT1), New York esophageal squamous cell carcinoma 1 (NY-ESO-1), melanoma antigen gene 1 (LAGE-1a), melanoma antigen A1 (MAGE-A1), MAGE-A2, breast cancer susceptibility gene (BRCA), MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, EGFRVIII, VEGFR-2, tyrosinase-associated protein 1 (TRP-1), TRP-2, tyrosinase, human papillomavirus (HPV) 16E6, HPV 16E7, HPV 18E6, HPV 18E7, KK-LC-1, NY-BR-1, CAG-3 (i.e., NY-ESO-1), synovial sarcoma X breakpoint protein 2 (SSX-2), SSX-3, SSX-4, SSX-5, SSX-9, SSX-1. In some embodiments of the present invention, the tumor-specific antigen is CD19.

[0279] In some embodiments of the present invention, the targeting portion comprises a chimeric antigen receptor (CAR). The CAR may include a ligand-binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signal transduction domain.

[0280] In some embodiments of the present invention, the ligand-binding domain recognizes the tumor-specific antigens described herein. In some embodiments of the present invention, the ligand-binding domain binds to the tumor-specific antigens described herein. In some embodiments of the present invention, the ligand-binding domain is derived from an antibody or antibody fragment (e.g., a murine antibody, a human antibody, or a humanized antibody) that recognizes the tumor-specific antigens described herein.

[0281] In some embodiments of the present invention, the CAR targets CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, CD5, CD7, MUC1, AFP, CEA, PSCA, PSMA, HER2, EGFR, IL13Rα2, GD2, NKG2D, EGFRvIII, CS1, CCL1, BCMA, mesothelin, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Rα2, and PRSS21. , VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, AFP, NCAM, Claudin18.2, GM3, TGS5, HMWMAA, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179 a, 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.

[0282] In some embodiments of the present invention, the ligand-binding domain of the CAR binds to CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, CD5, CD7, MUC1, AFP, CEA, PSCA, PSMA, HER2, EGFR, IL13Rα2, GD2, NKG2D, EGFRvIII, CS1, CCL1, BCMA, mesothelin, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Rα2, PR SS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, AFP, NCAM, Claudin18.2, GM3, TGS5, HMWMAA, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD1 79a, 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.

[0283] In some embodiments of the present invention, the ligand-binding domain is a single-stranded variable region fragment (scFv). In some embodiments of the present invention, the ligand-binding domain targets CD19. In some embodiments of the present invention, the ligand-binding domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:401. In some embodiments of the present invention, the ligand-binding domain comprises an amino acid sequence having at least 75% identity with the sequence shown in SEQ ID NO:401. In some embodiments of the present invention, the ligand-binding domain comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO:401. In some embodiments of the present invention, the ligand-binding domain comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:401. In some embodiments of the present invention, the ligand-binding domain comprises an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:401. In some embodiments of the present invention, the ligand-binding domain comprises an amino acid sequence having at least 95% identity with the sequence shown in SEQ ID NO:401. In some embodiments of the present invention, the ligand-binding domain comprises an amino acid sequence having at least 98% identity with the sequence shown in SEQ ID NO:401. In some embodiments of the present invention, the ligand-binding domain comprises an amino acid sequence having at least 99% identity with the sequence shown in SEQ ID NO:401. In some embodiments of the present invention, the ligand-binding domain comprises an amino acid sequence having 100% identity with the sequence shown in SEQ ID NO:401. In some embodiments of the present invention, the ligand-binding domain is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:451. In some embodiments of the present invention, the ligand-binding domain is encoded by a nucleic acid sequence having at least 75% identity with the sequence shown in SEQ ID NO:451. In some embodiments of the present invention, the ligand-binding domain is encoded by a nucleic acid sequence having at least 80% identity with the sequence shown in SEQ ID NO:451. In some embodiments of the present invention, the ligand-binding domain is encoded by a nucleic acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:451. In some embodiments of the present invention, the ligand-binding domain is encoded by a nucleic acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:451. In some embodiments of the present invention, the ligand-binding domain is encoded by a nucleic acid sequence having at least 95% identity with the sequence shown in SEQ ID NO:451.In some embodiments of the present invention, the ligand-binding domain is encoded by a nucleic acid sequence having at least 98% identity with the sequence shown in SEQ ID NO:451. In some embodiments of the present invention, the ligand-binding domain is encoded by a nucleic acid sequence having at least 99% identity with the sequence shown in SEQ ID NO:451. In some embodiments of the present invention, the ligand-binding domain is encoded by a nucleic acid sequence having 100% identity with the sequence shown in SEQ ID NO:451.

[0284] In some cases, the CAR comprises a signal peptide. The signal peptide may be located at the N-terminus of the CAR and may be linked to a ligand-binding domain. In some embodiments of the present invention, the signal peptide comprises a CD8α signal peptide or a variant thereof. In some embodiments of the present invention, the signal peptide comprises a β2M signal peptide or a variant thereof. In some embodiments of the present invention, the signal peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:400. In some embodiments of the present invention, the signal peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:4. In some embodiments of the present invention, the signal peptide is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:450. In some embodiments of the present invention, the signal peptide is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:54.

[0285] In some cases, the CAR includes a hinge region. This hinge region connects a ligand-binding domain to a transmembrane domain. In some cases, the hinge region is derived from a human protein. In some embodiments of the present invention, the hinge region includes a human immunoglobulin (Ig) hinge region (such as the hinge regions of IgG1, IgG4, or IgD), a FcγRIIIα hinge region, a KIR2DS2 hinge region, or a CD8α hinge region. In some embodiments of the present invention, the hinge region includes a polypeptide linker (such as a GS linker) as described herein. In some embodiments of the present invention, the hinge region includes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:402. In some embodiments of the present invention, the hinge region is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:452.

[0286] In some cases, the CAR includes a transmembrane domain. This transmembrane domain may be derived from the transmembrane domains of the TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD123, CD134, CD137, CD154, or any combination thereof. In some embodiments of the present invention, the transmembrane domain comprises any segment or variant thereof selected from the TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD123, CD134, CD137, CD154.

[0287] In some embodiments of the present invention, the transmembrane domain comprises a transmembrane domain of CD8α or a fragment thereof. In some embodiments of the present invention, the transmembrane domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:403. In some embodiments of the present invention, the transmembrane domain is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:453.

[0288] In some cases, the CAR includes a co-stimulatory domain. In some cases, the co-stimulatory domain includes at least a portion of, or any combination thereof, selected from any one of Toll-like receptor 1 (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, and CD3ε. In other cases, the co-stimulatory domain comprises at least a portion of another co-stimulatory domain having an activation motif based on an immune receptor tyrosine (ITAM). In some embodiments of the invention, the co-stimulatory domain is a CD28 co-stimulatory domain. In some embodiments of the invention, the co-stimulatory domain is a CD137(4-1BB) co-stimulatory domain. In some embodiments of the invention, the co-stimulatory domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:404. In some embodiments of the invention, the co-stimulatory domain is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:454.

[0289] In some cases, the CAR includes an intracellular signal transduction domain. The intracellular signal transduction domain may include at least a portion of an intracellular signal transduction domain selected from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, or CD66d. In some embodiments of the present invention, the intracellular signal transduction domain includes the intracellular signal transduction domain of CD3ζ. In some embodiments of the present invention, the intracellular signal transduction domain includes an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:405. In some embodiments of the present invention, the intracellular signal transduction domain is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:455.

[0290] In some embodiments of the present invention, the CAR comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:4, 400-407. In some embodiments of the present invention, the CAR comprises an amino acid sequence having at least 75% identity with the sequence shown in any one of SEQ ID NO:4, 400-407. In some embodiments of the present invention, the CAR comprises an amino acid sequence having at least 80% identity with the sequence shown in any one of SEQ ID NO:4, 400-407. In some embodiments of the present invention, the CAR comprises an amino acid sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO:4, 400-407. In some embodiments of the present invention, the CAR comprises an amino acid sequence having at least 90% identity with the sequence shown in any one of SEQ ID NO:4, 400-407. In some embodiments of the present invention, the CAR comprises an amino acid sequence having at least 95% identity with the sequence shown in any one of SEQ ID NO:4, 400-407. In some embodiments of the present invention, the CAR comprises an amino acid sequence having at least 98% identity with the sequence shown in any one of SEQ ID NO:4, 400-407. In some embodiments of the present invention, the CAR comprises an amino acid sequence having at least 99% identity with the sequence shown in any one of SEQ ID NO:4, 400-407. In some embodiments of the present invention, the CAR comprises an amino acid sequence having 100% identity with the sequence shown in any one of SEQ ID NO:4, 400-407. In some embodiments of the present invention, the CAR comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:406. In some embodiments of the present invention, the CAR comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO:406. In some embodiments of the present invention, the CAR comprises an amino acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:406. In some embodiments of the present invention, the CAR comprises an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:406. In some embodiments of the present invention, the CAR comprises an amino acid sequence having at least 95% identity with the sequence shown in SEQ ID NO:406. In some embodiments of the present invention, the CAR comprises an amino acid sequence having 100% identity with the sequence shown in SEQ ID NO:406.In some embodiments of the present invention, the CAR comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:407.

[0291] In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 54, 450-457. In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having at least 75% identity with the sequence shown in any one of SEQ ID NO: 54, 450-457. In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having at least 80% identity with the sequence shown in any one of SEQ ID NO: 54, 450-457. In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having at least 85% identity with the sequence shown in any one of SEQ ID NO: 54, 450-457. In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having at least 90% identity with the sequence shown in any one of SEQ ID NO: 54, 450-457. In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having at least 95% identity with the sequence shown in any one of SEQ ID NO:54, 450-457. In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having at least 98% identity with the sequence shown in any one of SEQ ID NO:54, 450-457. In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having at least 99% identity with the sequence shown in any one of SEQ ID NO:54, 450-457. In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having 100% identity with the sequence shown in any one of SEQ ID NO:54, 450-457. In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having at least 80% identity with the sequence shown in SEQ ID NO:456. In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having at least 85% identity with the sequence shown in SEQ ID NO:456. In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:456. In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having at least 95% identity with the sequence shown in SEQ ID NO:456. In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having 100% identity with the sequence shown in SEQ ID NO:456. In some embodiments of the present invention, the CAR is encoded by a nucleic acid sequence having 100% identity with the sequence shown in SEQ ID NO:457.

[0292] Table 5A. Amino acid sequences of exemplary CAR sequences

[0293]

[0294]

[0295]

[0296] Table 5B. Nucleic acid sequences of exemplary CAR sequences

[0297]

[0298]

[0299]

[0300] Cuttable connector sequence

[0301] In some aspects of the invention, this document provides a cuttable connector that can connect one or more components of the invention, including a targeting portion and peptide in a single-cytokine-anchoring substance, a targeting portion, a first peptide, and a second peptide in a dual-cytokine-anchoring substance, and a targeting portion, a first peptide, a second peptide, and a third peptide in a triple-cytokine-anchoring substance.

[0302] In some embodiments, the cleavable connector links the target portion to a signal peptide of the polypeptide. In some embodiments, the cleavable connector links the target portion to a peptide anchoring structure of the polypeptide. In some embodiments, the cleavable connector links the target portion to a non-peptide anchoring attachment signal. In some embodiments, the cleavable connector links a first polypeptide and a second polypeptide. In some embodiments, the cleavable connector links a first polypeptide and a third polypeptide. In some embodiments, the cleavable connector links a third polypeptide and a second polypeptide.

[0303] The cleavable adapter may be encoded by a cleavable adapter nucleic acid sequence. The cleavable adapter nucleic acid sequence may link two or more nucleic acid sequences as described in this invention, including the target sequence and nucleic acid sequence in a single cytokine-anchoring substance, the target sequence, the first nucleic acid sequence and the second nucleic acid sequence in a dual cytokine-anchoring substance, and the target sequence, the first nucleic acid sequence, the second nucleic acid sequence and the third nucleic acid sequence in a triple cytokine-anchoring substance.

[0304] In some embodiments, the second nucleic acid sequence is linked to the first nucleic acid sequence via a cleavable adapter nucleic acid sequence. In some embodiments, the third nucleic acid sequence is linked to the first nucleic acid sequence via a cleavable adapter nucleic acid sequence. In some embodiments, the second nucleic acid sequence is linked to the third nucleic acid sequence via a cleavable adapter nucleic acid sequence. In some embodiments, the target sequence is linked to the first nucleic acid sequence via a cleavable adapter nucleic acid sequence. In some embodiments, the target sequence is linked to the second nucleic acid sequence via a cleavable adapter nucleic acid sequence. In some embodiments, the target sequence is linked to the third nucleic acid sequence via a cleavable adapter nucleic acid sequence.

[0305] In some embodiments, a 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 to generate two polypeptides: a first polypeptide comprising a first cytokine and a first anchoring structure; and a second polypeptide comprising a second cytokine and a second anchoring structure. In some embodiments, a 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 to generate two polypeptides: a second polypeptide comprising a second cytokine and a second anchoring structure; and a third polypeptide comprising a third cytokine and a third anchoring structure. In some embodiments, a 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 to generate two polypeptides: a first polypeptide comprising a first cytokine and a first anchoring structure; and a third polypeptide comprising a third cytokine and a third anchoring structure.

[0306] In some embodiments, a cleavable linker sequence between a first nucleic acid sequence encoding a first polypeptide and a target sequence encoding a target portion can be cleaved to produce two polypeptides: a first polypeptide comprising a first cytokine and a first anchoring structure; and a target portion comprising a chimeric antigen receptor (CAR) (e.g., CAR19 having the sequence shown in SEQ ID NO:406). In some embodiments, a cleavable linker sequence between a second nucleic acid sequence encoding a second polypeptide and a target sequence encoding a target portion can be cleaved to produce two polypeptides: a second polypeptide comprising a second cytokine and a second anchoring structure; and a target portion comprising a CAR (e.g., CAR19). In some embodiments, a cleavable linker sequence between a third nucleic acid sequence encoding a third polypeptide and a target sequence encoding a target portion can be cleaved to produce two polypeptides: a third polypeptide comprising a third cytokine and a third anchoring structure; and a target portion comprising a CAR (e.g., CAR19).

[0307] The cuttable connector can be any cuttable connector that connects two peptides.

[0308] In some embodiments, the length of the cuttable connector is not limited. The cuttable connector may contain about 20 to about 30 amino acid residues, such as about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues.

[0309] In some cases, the cleavable adapter includes an internal ribosome entry site (IRES) element, such as an IRES element derived from encephalomyocarditis virus (EMCV). In some cases, the cleavable adapter includes a self-cleaving 2A peptide. The 2A peptide may be a viral oligopeptide that mediates peptide cleavage during eukaryotic translation. Here, the term "2A" refers to a specific region of the viral genome.

[0310] Without being bound by any specific theory, the 2A-mediated self-cleavage mechanism may be that the ribosome skips the formation of the glycyl-prolyl peptide bond at the C-terminus of the 2A peptide, rather than actual proteolytic cleavage.

[0311] In some embodiments, the cleavable adapter comprises a 2A self-cleaving peptide or a 2A-like peptide derived from foot-and-mouth disease virus or heart virus. In some embodiments, the cleavable adapter comprises a 2A peptide sequence, including the amino acid sequence of porcine swine choriovirus type 1 2A (P2A), equine rhinitis virus A 2A (E2A), β-tetrasomic virus 2A (T2A), or foot-and-mouth disease virus 2A (F2A). In some embodiments, the cleavable adapter comprises a P2A peptide sequence. In some embodiments, the cleavable adapter comprises a T2A peptide sequence. In some embodiments, the cleavable adapter comprises an E2A peptide sequence. In some embodiments, the cleavable adapter comprises an F2A peptide sequence. In some embodiments, the cleavable adapter comprises an IRES peptide. In some embodiments, the cleavable adapter comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:300-303. In some embodiments, the cleavable linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:300. In some embodiments, the cleavable linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:301. In some embodiments, the cleavable linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:302. In some embodiments, the cleavable linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:303.

[0312] In some embodiments, the cleavable adapter nucleic acid sequence encoding the cleavable adapter comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:350-355. In some embodiments, the cleavable adapter nucleic acid sequence encoding the cleavable adapter comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:350. In some embodiments, the cleavable adapter nucleic acid sequence encoding the cleavable adapter comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:351. In some embodiments, the cleavable adapter nucleic acid sequence encoding the cleavable adapter comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:352. In some embodiments, the cleavable adapter nucleic acid sequence encoding the cleavable adapter comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:353. In some embodiments, the cleavable adapter nucleic acid sequence encoding the cleavable adapter comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:354. In some embodiments, the cleavable adapter nucleic acid sequence encoding the cleavable adapter comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:355.

[0313] In other cases, the cleavable adapter comprises a furin protease cleavage site or a tobacco etch virus cleavage site. The furin protease cleavage site and its sequence are detailed in the literature by Klimstra et al., published in the *Journal of Virology*, Volume 73, Issue 8, pp. 6299-6306, August 1999, and in U.S. Patent No. 8,871,906; the full text of the above literature is incorporated herein by reference for all purposes.

[0314] Table 6A. Exemplary cleavable linker amino acid sequences

[0315]

[0316]

[0317] Table 6B. Exemplary cleavable adapter nucleic acid sequences

[0318]

[0319] peptide linkers

[0320] In some aspects of the present invention, peptide linkers are provided herein that can link one or more components described in the present invention, including cytokines and anchoring structures of peptides in single-cytokine-anchoring substances, cytokines and anchoring structures of first and second peptides in dual-cytokine-anchoring substances, and cytokines and anchoring structures of first, second, and third peptides in triple-cytokine-anchoring substances.

[0321] In some embodiments, the peptide linker links a cytokine to an anchoring structure. In some embodiments, the peptide linker links a cytokine to a peptide anchoring structure. In some embodiments, the peptide linker links a cytokine to a non-peptide anchoring attachment signal. In some embodiments, the peptide linker links a peptide anchoring structure to a cleavable connector. In some embodiments, the peptide linker links a non-peptide anchoring attachment signal to a cleavable connector.

[0322] The peptide linkers provided herein are not specifically limited. In some cases, the peptide linkers in the peptides described herein are flexible peptide linkers. In some cases, the peptide linkers in the peptides described herein are rigid peptide linkers. In some cases, the peptide linkers in the peptides described herein are non-self-cleaving linkers. The peptide linker can be any suitable linker sequence connecting an anchoring structure to any other component. The peptide linker can be any suitable linker sequence connecting an anchoring structure to a cytokine. The peptide linker can be any suitable linker sequence connecting an anchoring structure to a cleavable linker (which further connects to another peptide or targeting moiety).

[0323] In some embodiments, the length of the polypeptide linker is not particularly limited. In some embodiments, the polypeptide linker comprises about 2 to about 10 amino acid residues. In some embodiments, the polypeptide linker comprises 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 polypeptide linker comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 amino acid residues.

[0324] In some embodiments, the polypeptide linker comprises a selection of GS linker, Lr1 linker, or Lr8 linker.

[0325] In some embodiments, the peptide linker comprises glycine and serine residues. In some embodiments, the peptide linker comprises one or more G4S or G3S repeating units, for example, about 3 to about 15 or about 5 to about 12 G4S and G3S repeating units. In some embodiments, the peptide linker comprises about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G4S repeating units. In some embodiments, the peptide linker comprises about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G3S repeating units.

[0326] In some embodiments, the peptide linker comprises a glycine polymer (G)n. In some embodiments, the peptide linker comprises a glycine-serine polymer (G1-5S1-5)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, where 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 LRQRDGERP. In some embodiments, the peptide linker comprises LRQKDGGGSERP. In some embodiments, the peptide linker comprises LRQKD(GGGS)2ERP.

[0327] In some embodiments, the polypeptide linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 500, 501, 504, 506, and 507, or with any one of LE, AS, GSG, and EF. In some embodiments, the polypeptide linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 500. In some embodiments, the polypeptide linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 501. In some embodiments, the polypeptide linker comprises the LE sequence. In some embodiments, the polypeptide linker comprises the AS sequence. In some embodiments, the polypeptide linker comprises the amino acid sequence shown in SEQ ID NO: 504. In some embodiments, the polypeptide linker comprises a GSG sequence. In some embodiments, the polypeptide linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:506. In some embodiments, the polypeptide linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:507. In some embodiments, the polypeptide linker comprises an EF sequence. In some embodiments, the nucleic acid sequence encoding the polypeptide linker comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the gagtc sequence. In some embodiments, the nucleic acid sequence encoding the polypeptide linker comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:509. In some embodiments, the nucleic acid sequence encoding the polypeptide linker comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:520. In some embodiments, the nucleic acid sequence encoding the polypeptide linker comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the ggctccggc sequence. In some embodiments, the nucleic acid sequence encoding the polypeptide linker comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the ggaagcgga sequence.

[0328] Other examples of polypeptide linkers can be found in Liu et al., 1997, Proceedings of the National Academy of Sciences (PNAS), pp. 5525-5530; Pomerantz et al., October 1995, PNAS, Vol. 92, pp. 9752-9756; Kim et al., 1996, PNAS, Vol. 93, pp. 1156-1160; Chaudhary et al., 1990, PNAS, Vol. 87, pp. 1066-1070; and Bird et al., 1988, Science, Vol. 242, pp. 423-426. All of the above references are incorporated herein by reference in their entirety for all purposes.

[0329] Table 7A. Exemplary peptide linker amino acid sequences

[0330]

[0331] Table 7B. Exemplary peptide linker nucleic acid sequences

[0332]

[0333]

[0334] Nucleic acid molecules, vectors and systems

[0335] According to aspects of the present invention, this document provides nucleic acid molecules encoding the polypeptides described herein.

[0336] Based on the primary amino acid sequence of the polypeptide (or any component thereof) encoding the cytokine-anchored protein construct provided herein, those skilled in the art can determine the appropriate nucleotide sequence encoding the polypeptide; if necessary, codon-optimized nucleotide sequences can also be determined (see, for example, the literature by Mauro and Chappell published in Trends in Molecular Medicine, Vol. 20, No. 11, pp. 604-613, 2014).

[0337] According to some embodiments of the present invention, the nucleic acid molecule encoding a polypeptide in a cytokine-anchoring substance may be part of a vector (such as a plasmid vector, granular vector, or viral vector, or artificial chromosome), which may contain other functional regions (elements), such as one or more promoters, one or more origins of replication, one or more selection markers, and one or more other elements typically present in expression vectors. Cloning and expression techniques for nucleic acids encoding proteins (including CARs and cytokines) are well-established and well-known to those skilled in the art.

[0338] In some embodiments, the nucleic acid molecule purity of the cytokine-anchoring substance is greater than 80%, for example greater than 90%, greater than 95%, greater than 97%, or greater than 99%.

[0339] In some aspects of this invention, vectors comprising one or more nucleic acid sequences described herein are provided. The vector may be a transfer vector, referring to a composition of material containing isolated nucleic acids that can be used to deliver said isolated nucleic acids into cells. Various vectors are known in the art, including linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, and viruses. "Transfer vector" includes autonomously replicating plasmids or viruses. The term should also be understood to include non-plasmid and non-viral compounds that facilitate the delivery of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral transfer vectors include adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, etc. The vector may also comprise an expression vector, referring to a vector containing a recombinant polynucleotide that includes an expression control sequence operatively linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements to achieve expression; other elements required for expression may be provided by a host cell or an in vitro expression system. Expression vectors include all types known in the art, including viscera, plasmids (such as naked plasmids or plasmids contained in liposomes), and viruses (such as lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), all of which contain the recombinant polynucleotide.

[0340] In some embodiments, the nucleic acid molecule described herein is a vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector.

[0341] In some embodiments, the vector is a retroviral vector. Retroviral vectors typically refer to a class of RNA viruses that can reverse transcribe a complementary DNA strand within infected cells, and then synthesize a second DNA strand using this single-stranded DNA as a template, subsequently integrating it into the cell's genome. Retroviral vectors can utilize host cell enzymes to transcribe and replicate RNA to synthesize proteins, package the virus, and release it from the cell, forming an infectious virus. Retroviruses have high transduction efficiency, and retroviral vectors can effectively improve gene transfection rates.

[0342] In some embodiments, the vector is a lentiviral vector. A 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 simultaneously, and can effectively infect almost all mammalian cells, including nerve cells and hepatocytes, with high infection efficiency. Lentivirals can efficiently integrate exogenous genes into the host chromosome, achieving sustained gene expression.

[0343] In some embodiments, the vector is a transposon plasmid. A transposon plasmid is generally a basic unit located on chromosomal DNA that is capable of autonomous replication and translocation. Transposon plasmids can "jump" from one location in the genome to another through a series of processes, including cutting and recombination.

[0344] In some embodiments, the vector is an expression vector. In some embodiments, the expression vector contains a nucleic acid sequence encoding a targeting moiety (such as a CAR). In some embodiments, the expression vector contains a nucleic acid sequence encoding a cytokine and an anchoring structure. In some embodiments, the expression vector contains a nucleic acid sequence encoding a CAR, a cytokine, and an anchoring structure. In some embodiments, the expression vector contains a second nucleic acid sequence encoding a second cytokine and a second anchoring structure. In some embodiments, the expression vector contains a second nucleic acid sequence encoding a second CAR, a second cytokine, and a second anchoring structure. In some embodiments, the expression vector contains a third nucleic acid sequence encoding a third cytokine and a third anchoring structure. In some embodiments, the nucleic acid sequences within the expression vector may be arranged upstream or downstream. Specifically, the nucleic acids encoding the CAR molecule may be arranged upstream or downstream of the CAR molecule. The nucleic acid encoding the CAR and the nucleic acid encoding the cytokine may be linked by a cleavable adapter nucleic acid sequence encoding a 2A peptide or IRES.

[0345] In some embodiments, the nucleic acid molecules and / or vectors of the present invention are introduced into host cells. For eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-glucan transfection, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses (such as vaccinia virus); for insect cells, baculoviruses may be used for transduction. In some cases, the introduction of nucleic acids into host cells (especially eukaryotic cells) may employ viral or plasmid systems. In some cases, the plasmid system is maintained in episome form. In other cases, the plasmid system is integrated into the host cell or into an artificial chromosome. In one specific embodiment, the integration is by randomly integrating one or more copies into one or more loci. In some embodiments, the integration is by targeted integration into one or more loci. For bacterial cells, suitable techniques include calcium chloride conversion, electroporation, and transfection using bacteriophages.

[0346] In some embodiments, nucleic acid sequences are located on the same vector. In some embodiments, two or more nucleic acid sequences are encoded by the same nucleic acid molecule, located in the same reading frame, and expressed as a single polypeptide chain. In some cases, the targeting moiety (e.g., CAR) and the cytokine-anchored structure can be separated by one or more peptide cleavage sites (e.g., self-cleavage sites or intracellular protease substrates). In some cases, the targeting sequence and the nucleic acid sequence or exogenous nucleic acid sequence are regulated by different promoters. In some cases, the targeting sequence and the nucleic acid sequence or exogenous nucleic acid sequence are regulated by the same promoter. In some cases, the targeting sequence and the first nucleic acid sequence are regulated by the same promoter. In some cases, the targeting sequence and the first nucleic acid sequence are regulated by two different promoters. In some cases, the targeting sequence and the second nucleic acid sequence are regulated by the same promoter. In some cases, the targeting sequence and the second nucleic acid sequence are regulated by two different promoters. In some cases, the targeting sequence and the third nucleic acid sequence are regulated by the same promoter. In some cases, the targeting sequence and the third nucleic acid sequence are regulated by two different promoters. In some cases, the second nucleic acid sequence and the first nucleic acid sequence are regulated by the same promoter. In some cases, the second nucleic acid sequence and the first nucleic acid sequence are regulated by two different promoters. In some cases, the second nucleic acid sequence and the third nucleic acid sequence are regulated by the same promoter. In some cases, the second nucleic acid sequence and the third nucleic acid sequence are regulated by two different promoters. In some cases, the third nucleic acid sequence and the first nucleic acid sequence are regulated by the same promoter. In some cases, the third nucleic acid sequence and the first nucleic acid sequence are regulated by two different promoters.

[0347] In some cases, the first, second, and third nucleic acid sequences are operably ligated in a 5' to 3' orientation.

[0348] In some cases, the target 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' orientation.

[0349] In some cases, the first, second, third, and target nucleic acid sequences are operably linked in a 5' to 3' orientation.

[0350] In other cases, the nucleic acid sequence is located on different vectors.

[0351] In some embodiments, the nucleic acids of the present invention are integrated into the genome (e.g., chromosome) of a host cell. In one specific embodiment, integration may be achieved by including sequences that promote recombination with the genome, according to standard techniques. In some embodiments, the target sequence and the nucleic acid sequence are present in the genome of an immune cell. In some embodiments, the target sequence, the first nucleic acid sequence, and the second nucleic acid sequence are present in the genome of the cell. In some embodiments, the target 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.

[0352] According to aspects of the present invention, systems relating to the cytokine-anchoring substances described herein are provided. The systems may comprise the nucleic acid molecules or vectors described herein. In some embodiments, the system comprises a nucleic acid molecule or a plasmid encoding a targeting portion and one or more cytokines, each linked to an anchoring structure. In some embodiments, a system comprising two nucleic acid molecules (e.g., a dual-plasmid system) is provided, wherein the first plasmid contains a targeting sequence encoding a CAR, and the second plasmid contains a nucleic acid sequence encoding a cytokine and an anchoring structure. For example, the first and second nucleic acid molecules may be co-delivered to host cells (e.g., engineered immune cells). In some embodiments, a system comprising two or more nucleic acid molecules is provided, wherein the two or more plasmids each contain at least one targeting sequence encoding a CAR, or at least two or more nucleic acid sequences encoding a cytokine and an anchoring structure. For example, three or more nucleic acid molecules may be co-delivered to host cells (e.g., engineered immune cells). In some cases, the first plasmid contains a target sequence, the second plasmid contains a first nucleic acid sequence encoding a first cytokine and a first anchoring structure (which may or may not contain the target sequence), and the third plasmid contains a second nucleic acid sequence encoding a second cytokine and a second anchoring structure (which may or may not contain the target sequence). In some cases, the first plasmid contains a first nucleic acid sequence encoding a first cytokine and a first anchoring structure (which may or may not contain the target sequence), the second plasmid contains a second nucleic acid sequence encoding a second cytokine and a second anchoring structure (which may or may not contain the target sequence), and the third plasmid contains a third nucleic acid sequence encoding a third cytokine and a third anchoring structure (which may or may not contain the target sequence). In some cases, the first plasmid contains a first nucleic acid sequence encoding a first cytokine and a first anchoring structure, and a second nucleic acid sequence encoding a second cytokine and a second anchoring structure (which may or may not contain the target sequence), the second plasmid contains the target sequence, and the third plasmid contains a third nucleic acid sequence encoding a third cytokine and a third anchoring structure (which may or may not contain the target sequence).

[0353] In some embodiments, the target sequence and the first nucleic acid sequence are present in the same plasmid within the system. In some embodiments, the target sequence and the first nucleic acid sequence are present in two different plasmids within the system. In some embodiments, the target sequence and the second nucleic acid sequence are present in the same plasmid within the system. In some embodiments, the target sequence and the second nucleic acid sequence are present in two different plasmids within the system. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present in the same plasmid within the system. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present in two different plasmids within the system. In some embodiments, the target sequence and the first nucleic acid sequence are present in the same plasmid within the system. In some embodiments, the target sequence and the second nucleic acid sequence are present in the same plasmid within the system. In some embodiments, the target sequence and the second nucleic acid sequence are present in two different plasmids within the system. In some embodiments, the target sequence and the second nucleic acid sequence are present in two different plasmids within the system. In some embodiments, the target sequence and the third nucleic acid sequence are present in two different plasmids within the system. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present in the same plasmid within the system. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present in two different plasmids within the system. In some embodiments, the first and third nucleic acid sequences are present in the same plasmid within the system. In some embodiments, the first and third nucleic acid sequences are present in two different plasmids within the system. In some embodiments, the second and third nucleic acid sequences are present in the same plasmid within the system. In some embodiments, the second and third nucleic acid sequences are present in two different plasmids within the system.

[0354] The system of the present invention may comprise a protein or polypeptide encoded by a nucleic acid sequence or nucleic acid molecule within the system described herein.

[0355] host cells

[0356] Another aspect of the present invention provides a host cell comprising the nucleic acid molecules, vectors, or systems disclosed herein. In some embodiments, the host cell is an in vitro cell. In some embodiments, the host cell is in a culture state.

[0357] In some cases, 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 human cells or rodent cells), such as engineered T cells or engineered NK cells. In some cases, the host cell can be any T cell, such as cultured T cells, primary T cells, T cells derived from cultured T cell lines (such as Jurkat cell lines, SupT1 cell lines, etc.), or T cells derived from mammals. In some cases, the T cells are derived from mammals. In some cases, the T cells can be obtained from multiple sources, including blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. The T cells can be enriched or purified. In some cases, the T cells are human T cells. In some cases, the T cells are T cells isolated from the human body. The T cells can be any type of T cell and can be T cells at any developmental stage, including CD4+. + / CD8 + Double-positive T cells, CD4 + Helper T cells (such as Th1 cells, Th2 cells), CD4 + T cells, CD8 + T cells (such as cytotoxic T cells), tumor-infiltrating lymphocytes (TILs), memory T cells (such as central memory T cells and effector memory T cells), naive T cells, etc.

[0358] In some cases, the host cells are treated to induce or allow nucleic acid molecules to express proteins with cytokine-anchored structures (e.g., by culturing host cells under conditions suitable for the expression of nucleic acid sequences). In some embodiments, the expression product is purified using methods known to those skilled in the art. For example, the expression vector can be introduced into the host cells by physical, chemical, or biological methods.

[0359] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, and electroporation. Methods for preparing cells containing vectors and / or exogenous nucleic acid molecules are well-known in the art (see, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, Volumes 1–4, Cold Spring Harbor Laboratory Press, New York, 2012). In some cases, calcium phosphate transfection is used to introduce polynucleotides into host cells.

[0360] Biological methods for introducing the polynucleotides of the present invention into host cells include the use of DNA vectors and RNA vectors. Viral vectors (especially retroviral vectors) have become the most widely used method for introducing genes into mammalian cells (such as human cells). Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, and adeno-associated viruses, etc. (see, for example, U.S. Patents 5,350,674 and 5,585,362).

[0361] Chemical methods for introducing polynucleotides into host cells include colloidal dispersions (such as macromolecular complexes, nanocapsules, microspheres, and microbeads) and lipid-based systems (including oil-in-water emulsions, micelles, hybrid micelles, and liposomes). An exemplary colloidal system used as a delivery carrier in vitro and in vivo is liposomes (such as artificial membrane vesicles). Other targeted nucleic acid delivery methods exist in the prior art, such as delivering polynucleotides using targeted nanoparticles or other suitable submicron-scale delivery systems.

[0362] In the use of non-viral delivery systems, liposomes are an exemplary delivery vector. The present invention contemplates the use of lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, the nucleic acid may bind to lipids. Lipid-bound nucleic acids may be encapsulated within the aqueous interior of liposomes, dispersed in a lipid bilayer of liposomes, attached to liposomes by linkers that simultaneously bind to liposomes and oligonucleotides, embedded in liposomes, complexed with liposomes, dispersed in a lipid-containing solution, mixed with lipids, bound to lipids, present in lipids as a suspension, contained or complexed in micelles, or otherwise bound to lipids. Lipids, lipid / DNA, or lipid / expression vector-related compositions are not limited to a specific structural form in solution; for example, they may be bilayers, micelles, or “folded” structures; they may also be dispersed only in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances and may be naturally occurring or synthetic lipids, such as naturally occurring lipid droplets in the cytoplasm, and compounds comprising long-chain aliphatic hydrocarbons and their derivatives (such as fatty acids, alcohols, amines, amino alcohols, and aldehydes).

[0363] The lipids suitable for use in this invention are commercially available. For example, dimyristicophosphatidylcholine (DMPC) is available from Sigma-Aldrich (St. Louis, Missouri, USA); diceryl phosphate (DCP) is available from K&K Laboratories (Plainview, New York, USA); cholesterol (Choi) is available from Calbiochem-Behring; dimyristicophosphatidylglycerol (DMPG) and other lipids are available from Avanti Polar Lipids (Birmingham, Alabama, USA). The lipids can be stored in chloroform or chloroform / methanol stock solutions at approximately -20°C. Chloroform is used only as a solvent because it evaporates more readily than methanol. "Liposome" is a general term encompassing various monolayer and multilayer lipid carriers formed from enclosed lipid bilayers or aggregates. Liposomes are characterized by a vesicle structure having a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium and can spontaneously form when phospholipids are suspended in excess aqueous solution. Lipid components undergo self-reorganization before forming a closed structure, encapsulating water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991, *Glycobiology*, Vol. 5, pp. 505-510). However, this invention also covers compositions having structures in solution that differ from conventional vesicle structures; for example, lipids may exist in a micellar structure or as heterogeneous aggregates of lipid molecules. This invention also covers lipid-transfected amine-nucleic acid complexes.

[0364] Regardless of the method used to introduce the nucleic acid molecules, vectors, or systems described herein into host cells, various tests can be performed to confirm the presence of recombinant DNA sequences in the host cells. Such tests include, for example, "molecular biology" tests (such as Southern blotting, Northern blotting, RT-PCR, PCR) known to those skilled in the art, and "biochemical" tests (such as confirming the presence of specific peptides by immunological methods (ELISA, Western blotting) or by the detection methods described herein to identify reagents falling within the scope of this invention).

[0365] This invention provides a vector comprising a targeting sequence encoding a CAR and nucleic acid sequences encoding cytokines and anchoring structures. In some cases, the vector can be directly transduced into cells (such as T cells or NK cells). In some cases, the vector is capable of expressing a CAR construct in mammalian T cells or NK cells. In one aspect, the mammalian T cells are human T cells.

[0366] In some embodiments, the nucleic acid molecules of the present invention (including vector nucleic acids comprising nucleic acid sequences encoding the CAR or cytokine-anchored polypeptides described herein) are present in isolated host cells. In some cases, the host cells are part of a host cell clonal population. Referring to a host cell herein also encompasses a clonal population of that cell; a clonal population refers to a population obtained from a single parental host cell. In some cases, the host cells are derived from any suitable organism. In some embodiments, the host cells are, for example, bacterial cells, yeast cells, fungal cells, or mammalian cells. In some embodiments, the host cells are immune cells or tumor cells. In some embodiments, the host cells are engineered immune cells. In some embodiments, the host cells are T cells. In some embodiments, the host cells are tumor-infiltrating lymphocytes (TILs). In some embodiments, the engineered immune cells are natural killer (NK) cells.

[0367] In some embodiments, the target sequence and the first nucleic acid sequence are present in the same plasmid within the cell. In some embodiments, the target sequence and the first nucleic acid sequence are present in two different plasmids within the cell. In some embodiments, the target sequence and the second nucleic acid sequence are present in the same plasmid within the cell. In some embodiments, the target sequence and the second nucleic acid sequence are present in two different plasmids within the cell. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present in the same plasmid within the cell. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present in two different plasmids within the cell. In some embodiments, the target sequence and the first nucleic acid sequence are present in the same plasmid within the cell. In some embodiments, the target sequence and the second nucleic acid sequence are present in the same plasmid within the cell. In some embodiments, the target sequence and the second nucleic acid sequence are present in two different plasmids within the cell. In some embodiments, the target sequence and the second nucleic acid sequence are present in two different plasmids within the cell. In some embodiments, the target sequence and the third nucleic acid sequence are present in two different plasmids within the cell. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present in the same plasmid within the cell. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present in two different plasmids within the cell. In some embodiments, the first and third nucleic acid sequences are present in the same plasmid within the cell. In some embodiments, the first and third nucleic acid sequences are present in two different plasmids within the cell. In some embodiments, the second and third nucleic acid sequences are present in the same plasmid within the cell. In some embodiments, the second and third nucleic acid sequences are present in two different plasmids within the cell.

[0368] Cell source

[0369] Prior to amplification, gene modification, or other modifications, cell sources (such as T cells or natural killer (NK) cells) can be obtained directly or indirectly from the subject. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and their transgenic species. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some aspects of this invention, immune effector cells (such as T cells) can be obtained using any method known to those skilled in the art (such as Ficoll). TM Apheresis is used to obtain cells from units of blood donated by a subject. In some cases, cells from an individual's circulating blood are obtained via apheresis. The apheresis product may contain lymphocytes (including T cells, monocytes, granulocytes, and B cells), other nucleated leukocytes, erythrocytes, and platelets. In some cases, the cells collected via apheresis may be washed to remove plasma components, and optionally placed in a suitable buffer or culture medium for subsequent processing steps. In some cases, phosphate-buffered saline (PBS) is used to wash the cells. In other cases, the wash solution is calcium-free and may be magnesium-free, or may be free of most (if not all) divalent cations; performing the initial activation step under calcium-free conditions may enhance activation. Those skilled in the art will recognize that the washing step can be performed using methods known in the art, such as using a semi-automatic "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate cell processor, or a Haemonetics Cell Saver 5 cell processor), operated according to the manufacturer's instructions. After washing, cells can be resuspended in a variety of biocompatible buffers (e.g., calcium- and magnesium-free PBS, PlasmaLyteA solution, or other saline solutions with or without buffers); or unwanted components in a single sample can be removed and the cells can be resuspended directly in the culture medium.

[0370] In some cases, this is achieved by lysing red blood cells and removing monocytes (e.g., via PERCOLL). TM T cells are isolated from peripheral blood lymphocytes using gradient centrifugation or countercurrent centrifugation washing method.

[0371] The method of the present invention may include, for example, using negative selection techniques to select specific subsets of immune effector cells (such as T cells) (e.g., depleted regulatory T cell populations, CD25...). + (Exhausted cells). Preferably, the exhausted regulatory T cell population contains CD25. + The percentage of cells is below 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.

[0372] In some embodiments, regulatory T cells (such as CD25 antibodies) are removed from the population using an anti-CD25 antibody (or a fragment thereof) or a CD25-binding ligand (IL-2). + (T cells). In some embodiments, the anti-CD25 antibody (or a fragment thereof) or CD25-binding ligand is conjugated to a matrix (such as microbeads) or otherwise coated onto the surface of a matrix (such as microbeads). In some embodiments, the anti-CD25 antibody (or a fragment thereof) is conjugated to the matrix described herein.

[0373] In some embodiments, Miltenyi is used TM The company's CD25 depletion reagent removes regulatory T cells (such as CD25) from the population. + cell).

[0374] In some embodiments, the CliniMAC system equipped with exhaustion tubing kits (such as the 162-01 tubing) is used to remove regulatory T cells (such as CD25 cells) from the population. + (Cells). In some embodiments, the CliniMAC system is set to run in exhaustion mode (such as DEPLETION 2.1 mode).

[0375] The methods described herein may include more than one selection step (e.g., more than one exhaustion step). T cell populations can be enriched by negative selection, for example, using a mixture of antibodies targeting surface markers specific to negatively selected cells. One approach is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry, using a mixture of monoclonal antibodies targeting surface markers of negatively selected cells. For example, for enriching CD4 by negative selection... + Cellular monoclonal antibody mixtures may contain antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0376] Without being limited by any specific theory, administering the immune cells containing CAR and cytokine-anchored structures described herein to subjects can reduce the risk of relapse. This also reduces or eliminates the need to lower the levels of negative regulatory factors in immune cells (e.g., reducing the number of unwanted immune cells, such as Treg cells) before apheresis or during the preparation of CAR-expressing cell products, further reducing the risk of relapse. In some cases, administering the immune cells containing CAR and cytokine-anchored structures described herein to subjects can reduce the risk of relapse without removing specific immune cells from the subject (i.e., without undergoing lymphocyte removal procedures). Conventional methods for removing Treg cells are known in the art, including cyclophosphamide treatment, anti-GITR antibody treatment (the anti-GITR antibody described herein), CD25 depletion treatment, and combinations thereof.

[0377] In some embodiments, the preparation method does not include a step of reducing (e.g., depleting) the number of Treg cells before preparing CAR-expressing cells. In some embodiments, the preparation method includes a step of reducing (e.g., partially depleting) the number of Treg cells before preparing CAR-expressing cells. For example, the preparation method includes contacting a sample (e.g., a single sample) with an anti-GITR antibody and / or an anti-CD25 antibody (or a fragment thereof, or a CD25 binding ligand), for example, partially depleting Treg cells before preparing CAR-expressing cell (e.g., T cell, NK cell) products.

[0378] In some embodiments, subjects did not receive one or more treatments that reduced Treg cells before collecting cells used to prepare CAR-expressing cell products, but the risk of relapse after receiving CAR-expressing cell therapy was still reduced due to the co-expression of CAR and cytokine-anchoring structures. In some embodiments, subjects received one or more treatments that partially reduced Treg cells before collecting cells used to prepare CAR-expressing cell products, thereby further reducing the risk of relapse after receiving CAR-expressing cell therapy.

[0379] In some embodiments, subjects did not receive cyclophosphamide pretreatment before collecting cells for preparing CAR-expressing cell products, but the risk of relapse after receiving CAR-expressing cell therapy was still reduced due to the co-expression of CAR and cytokine-anchoring structures. In some embodiments, subjects received cyclophosphamide pretreatment before collecting cells for preparing CAR-expressing cell products, which, combined with the co-expression of CAR and cytokine-anchoring structures, further reduced the risk of relapse after receiving CAR-expressing cell therapy. In some embodiments, subjects did not receive anti-GITR antibody pretreatment before collecting cells for preparing CAR-expressing cell products, but the risk of relapse after receiving CAR-expressing cell therapy was still reduced due to the co-expression of CAR and cytokine-anchoring structures. In some embodiments, subjects received anti-GITR antibody pretreatment before collecting cells for preparing CAR-expressing cell products, which, combined with the co-expression of CAR and cytokine-anchoring structures, further reduced the risk of relapse after receiving CAR-expressing cell therapy.

[0380] In some embodiments, other cell populations (such as cells that negatively affect the expansion and / or function of CAR-T cells) may be optionally removed, for example, cells expressing CD14, CD11b, CD33, CD15 or other potential immunosuppressive cell expression markers.

[0381] The method of the present invention may 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 to obtain a depleted population of regulatory T cells (such as CD25) suitable for expressing CARs (such as CAR19) and cytokine-anchored structures. + Both tumor antigen-depleted populations and tumor antigen-depleted populations can be removed simultaneously. In some embodiments, cells expressing tumor antigens and regulatory T cells (such as CD25 cells) can be removed at the same time. + (Cells). For example, an anti-CD25 antibody (or a fragment thereof) and an anti-tumor antigen antibody (or a fragment thereof) can be linked to the same matrix (such as microbeads) for cell removal; or, an anti-CD25 antibody (or a fragment thereof) and an anti-tumor antigen antibody (or a fragment thereof) can be linked to different microbeads, and the microbead mixture can be used for cell removal. In some embodiments, regulatory T cells (such as CD25 cells) are removed. + The steps of removing cells expressing tumor antigens and removing cells can be performed sequentially, and the order is arbitrary.

[0382] This invention also provides methods including removing expression of checkpoint inhibitors (such as PD1) from a population. + Cells, LAG3 + Cells, TIM3 + A method involving one or more cells in a cell to obtain a depleted population of regulatory T cells (such as CD25). + Exhausted populations and checkpoint inhibitor exhaustion populations (such as PD1) + LAG3 + and / or TIM3 + Exhausted population). Exemplary checkpoint inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, carcinoembryonic antigen-associated cell adhesion molecules (CEACAMs, such as CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLA, and LAIR1. In some embodiments, cells expressing checkpoint inhibitors and regulatory T cells (such as CD25) may be simultaneously depleted. + (Cells). For example, an anti-CD25 antibody (or a fragment thereof) and an anti-checkpoint inhibitor antibody (or a fragment thereof) can be linked to the same microbead for cell removal; or, an anti-CD25 antibody (or a fragment thereof) and an anti-checkpoint inhibitor antibody (or a fragment thereof) can be linked to separate microbeads, and the microbead mixture can be used for cell removal. In some embodiments, regulatory T cells (such as CD25 cells) are removed. + The steps of removing cells expressing checkpoint inhibitors can be performed sequentially, and the order is arbitrary.

[0383] The methods described herein may include a positive selection step. For example, T cells may be conjugated with anti-CD3 / anti-CD28 (e.g., 3×28) microbeads (e.g., ... M-450 CD3 / CD28 T beads are co-incubated for a sufficient time to positively select target T cells. In one aspect, the incubation time is approximately 30 minutes. In another aspect, the incubation time ranges from 30 minutes to 36 hours or longer, covering all integer time values ​​within this range. In yet another aspect, the incubation time is at least 1, 2, 3, 4, 5, or 6 hours. In another preferred aspect, the incubation time is 10 to 24 hours. In one aspect, the incubation time is 24 hours. For cases where the proportion of T cells, such as tumor-infiltrating lymphocytes (TILs), is low from tumor tissue or immunocompromised individuals, a longer incubation time can be used to isolate T cells; furthermore, a longer incubation time can increase CD8+... + T cell capture efficiency. Therefore, at the start of culture or at other time points during the process, T cell subsets can be preferentially selected or excluded by simply shortening or lengthening the binding time of T cells to CD3 / CD28 beads, and / or increasing or decreasing the ratio of beads to T cells (further explained below); in addition, T cell subsets can also be preferentially selected or excluded at the start of culture or at other desired time points by increasing or decreasing the proportion of anti-CD3 and / or anti-CD28 antibodies on beads or other surfaces.

[0384] In some embodiments, a population of T cells expressing one or more of the following molecules may be selected: interferon-γ (IFN-γ), tumor necrosis factor-α (TNFα), interleukin-17A (IL-17A), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-10 (IL-10), interleukin-13 (IL-13), granzyme B, perforin, or other suitable molecules (such as other cytokines). The detection method for cell expression can be determined by, for example, the method described in U.S. Patent Publication No. US20220056116A1, the entire contents of which are incorporated herein by reference.

[0385] To isolate a target cell population through positive or negative selection, the concentration of cells and surface particles (such as microbeads) can be adjusted. In some embodiments, the mixing volume of microbeads and cells can be significantly reduced (i.e., the cell concentration is increased) to ensure maximum contact between cells and microbeads. For example, approximately 10 × 10⁻⁶ microbeads can be used. 9 Cells / mL, 9×10 9 1 cell / mL, 8×10 9 1 cell / mL, 7×10 9 Cells / mL, 6×10 91 cell / mL or 5 × 10⁻⁶ 9 A concentration of cells / mL. In one aspect, using 1×10⁻⁶ cells / mL. 9 A concentration of cells per milliliter. In one aspect, use 75, 80, 85, 90, 95, or 100 × 10⁻⁶ cells / mL. 6 A concentration of cells / mL. Alternatively, 125 or 150 × 10⁻⁶ cells / mL can be used. 6 Concentration per cell per milliliter.

[0386] Using high concentrations can improve cell yield, cell activation efficiency, and cell expansion efficiency. Furthermore, high cell concentrations allow for more efficient capture of cells weakly expressing target antigens (such as CD28-negative T cells) or from samples rich in tumor cells (such as leukemia blood or tumor tissue). Such cell populations may have therapeutic value and are therefore necessary to obtain. For example, high cell concentrations allow for more efficient selection of CD8 cells, which typically have weak CD28 expression. + T cells.

[0387] In some embodiments, lower cell concentrations can be used. By significantly diluting the mixture of T cells and surface particles (such as microbeads), particle-cell interactions can be reduced, thereby selecting cells that highly express the target antigen to bind to the particles. For example, CD4 + T cells showed higher CD28 expression levels than CD8 at dilution concentrations. + T cells are more easily captured. In some embodiments, a cell concentration of 5 × 10⁻⁶ is used. 6 Cells / mL. In other embodiments, cell concentrations in the range of 1 × 10⁻⁶ cells / mL were used. 5 Cells / mL to 1×10 6 Cells / mL, and covering all integer concentration values ​​in between.

[0388] In some embodiments, the cells are incubated on a rotator for different incubation times and rotation speeds, and the incubation temperature is 2-10°C or room temperature.

[0389] T cells used for activation can also be cryopreserved after the washing step. Without theoretical limitations, freezing and subsequent thawing steps can yield a more homogeneous product by removing granulocytes and some monocytes. After the washing step to remove plasma and platelets, the cells can be resuspended in a cryogenic solution. Although various cryogenic solutions and freezing parameters are known in the art for this purpose, one approach is to use PBS containing 20% ​​dimethyl sulfoxide (DMSO) and 8% human serum albumin, or a medium containing 10% dextran 40, 5% glucose, 20% human serum albumin, and 7.5% DMSO, or a medium containing 31.25% Plasmalyte-A solution, 31.25% 5% glucose solution, 0.45% sodium chloride, 10% dextran 40, 5% glucose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing media (such as a medium containing hydroxyethyl starch (Hespan) and Plasmalyte-A solution); the cells are then frozen to -80°C at a rate of 10°C per minute and stored in the gas phase of a liquid nitrogen tank. Other controlled freezing methods may also be used, or uncontrolled freezing may be performed directly at -20°C or in liquid nitrogen.

[0390] In some embodiments, the cryopreserved cells are thawed and washed as described herein, left to stand at room temperature for 1 hour, and then activated using the method of the present invention.

[0391] This invention also considers obtaining blood samples or apheresis products from subjects at any point in time prior to the need for the amplified cells described herein. Therefore, the source of cells to be amplified can be collected at any necessary point in time, target cells (such as T cells) can be isolated and cryopreserved, and subsequently used for immune effector cell therapy (applicable to a variety of diseases or conditions from which one can benefit, as described herein). In one aspect, blood samples or apheresis products are obtained from healthy subjects. In some cases, blood samples or apheresis products are obtained from healthy subjects at risk of disease but who have not yet developed the disease, target cells are isolated and cryopreserved for later use. In some cases, T cells can be amplified, cryopreserved, and subsequently used when needed. In some cases, samples are collected from patients after a diagnosis of a specific disease (such as that described herein) and before any treatment is initiated. In another embodiment, prior to cell isolation from the subject, the subject had not received any of the following treatments, including natamizumab, efazolin, antiviral drugs, chemotherapy, radiotherapy, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, FK506), antibodies, or other immune scavengers (such as CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate mofetil, steroids, FR901228, and radiotherapy).

[0392] In another embodiment of the invention, T cells can be obtained directly from the patient after treatment that preserves functional T cells in the subject. In some cases, after certain cancer treatments (especially those using drugs that damage the immune system), patients are often in a recovery phase where the quality of the T cells obtained may be optimal or their in vitro expansion capacity may be enhanced; similarly, after ex vivo manipulation using the methods described herein, these cells may be in a state more conducive to enhanced transplantation and in vivo expansion. Therefore, the present invention considers collecting blood cells (including T cells, dendritic cells, or other hematopoietic lineage cells) during this recovery phase. Furthermore, in some embodiments, mobilization protocols (such as those using GM-CSF) and pretreatment protocols can be employed to create conditions in the subject that favor the reproliferation, recycling, regeneration, and / or expansion of specific cell types (especially within a specific time window after treatment), exemplary cell types including T cells, B cells, dendritic cells, and other immune cells.

[0393] In some embodiments, immune effector cells expressing a CAR molecule (such as CAR19 co-expressed with a cytokine-anchored structure) may be obtained from subjects who have received low-dose immunomodulatory mTOR inhibitor therapy. In some embodiments, an in vitro treatment population of immune effector cells (such as T cells or NK cells) may be exposed to a certain amount of mTOR inhibitor, which may increase the number of PD1-negative immune effector cells (such as T cells) or increase the ratio of PD1-negative immune effector cells (such as T cells or NK cells) to PD1-positive immune effector cells (such as T cells or NK cells); the immune effector cell population has been engineered to express a CAR (such as CAR19 co-expressed with a cytokine-anchored structure) or is to be engineered to express the CAR.

[0394] In some embodiments, the T cell population is a diglyceride kinase (DGK) deficient population. DGK deficient cells include cells that do not express DGK RNA or protein, or whose DGK activity is reduced or inhibited. DGK deficient cells can be prepared by genetic methods (such as applying RNA interference agents (such as siRNA, shRNA, miRNA) to reduce or prevent DGK expression); or, DGK deficient cells can be prepared by treatment with the DGK inhibitors described herein.

[0395] In some embodiments, the T cell population is an Ikaros-deficient population. Ikaros-deficient cells include cells that do not express Ikaros RNA or protein, or whose Ikaros activity is reduced or suppressed. Ikaros-deficient cells can be prepared by genetic methods (such as applying RNA interference agents (such as siRNA, shRNA, miRNA) to reduce or prevent Ikaros expression); or, Ikaros-deficient cells can be prepared by treating with Ikaros inhibitors (such as lenalidomide).

[0396] In this embodiment, the T cell population is a DGK-deficient and Ikaros-deficient population, for example, a population that does not express DGK and Ikaros, or whose DGK and Ikaros activities are reduced or suppressed. Such DGK and Ikaros-deficient cells can be prepared by any of the methods described herein.

[0397] In some embodiments, the NK cells are obtained from the subject. In another embodiment, the NK cells are NK cell lines (such as the NK-92 cell line, purchased from Conkwest).

[0398] Pharmaceutical Composition

[0399] This document provides pharmaceutical compositions comprising nucleic acid molecules, cells, immune cells, or systems associated with cytokine-anchoring structures. In this regard, the present invention provides a pharmaceutical composition comprising any of the cytokine-anchoring structure substances described herein, and pharmaceutically acceptable excipients or carriers. In some embodiments, the pharmaceutical composition comprises cells described herein (e.g., engineered immune cells expressing CARs and cytokine-anchoring structures), and pharmaceutically acceptable excipients or carriers.

[0400] In some embodiments, the carrier is a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can be any carrier conventionally used for cell delivery and is well known to those skilled in the art; the pharmaceutically acceptable carrier has no harmful side effects or toxicity under the conditions of use.

[0401] The choice of carrier may depend in part on the specific method used to administer the particular cytokine-anchoring substance. The pharmaceutical compositions of the present invention are available in a variety of suitable formulations for parenteral, subcutaneous, intravenous, intramuscular, arterial, intrathecal, intratumoral, or intraperitoneal administration. The IL-21 / 15 substance of the present invention can be administered via more than one route, and in some cases, a particular route of administration may produce a faster and more effective response than other routes.

[0402] In some embodiments, one or more CAR-expressing cells described herein may be administered or delivered to a subject via a biopolymer scaffold (such as a biopolymer implant). The biopolymer scaffold may support or enhance the delivery, expansion, and / or dispersion of CAR-expressing cells described herein. The biopolymer scaffold comprises a biocompatible (e.g., substantially non-inducing of inflammation or immune responses) and / or biodegradable polymer, which may be a naturally occurring or synthetic polymer.

[0403] Suitable examples of 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-hydroxyhexanoate) (PHBHHx), polylactide, polycaprolactone (PCL), poly(lactide-co-glycolic acid) (PLG), polyethylene oxide (PEO), poly(lactic acid-co-glycolic acid) (PLGA), polypropylene oxide (PPO), polyvinyl alcohol (PVA), silk fibroin, soy protein, and soy protein isolate, which can be used alone or in combination with any other polymer in any concentration and proportion. Biopolymers can be enhanced or modified by adding adhesion-promoting or migration-promoting molecules (such as collagen mimic peptides that bind to lymphocyte collagen receptors) and / or stimulating molecules (to enhance the delivery, expansion, or function of the cells to be delivered (such as anticancer activity)). Biopolymer scaffolds can be in injectable formulations (such as gels or semi-solid formulations) or solid formulations.

[0404] In some embodiments, cells expressing CARs and cytokine-anchored structures as described herein may be seeded onto a biopolymer scaffold prior ...

Claims

1. A nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein: (a) The first nucleic acid sequence encodes a first polypeptide, the first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchoring attachment signal or (ii) a first peptide anchoring structure; (b) The second nucleic acid sequence encodes a second polypeptide, the second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchoring attachment signal or (ii) a second peptide anchoring structure; and The first cytokine peptide and the second cytokine peptide each independently comprise at least a portion or a variant thereof selected from any one 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.

2. A nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein: (a) The first nucleic acid sequence encodes a first polypeptide, the first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchoring attachment signal or (ii) a first peptide anchoring structure; (b) The second nucleic acid sequence encodes a second polypeptide, the second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchoring attachment signal or (ii) a second peptide anchoring structure; and The first cytokine peptide and the second cytokine peptide each independently comprise at least a portion or a variant thereof selected from any one 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.

3. A system comprising a first nucleic acid sequence and distinct second nucleic acid sequences, wherein: (a) The first nucleic acid sequence encodes a first polypeptide, the first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchoring attachment signal or (ii) a first peptide anchoring structure; (b) The second nucleic acid sequence encodes a second polypeptide, the second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchoring attachment signal or (ii) a second peptide anchoring structure; and The first cytokine peptide and the second cytokine peptide each independently comprise at least a portion or a variant thereof selected from any one 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.

4. A system comprising a first nucleic acid sequence and distinct second nucleic acid sequences, wherein: (a) The first nucleic acid sequence encodes a first polypeptide, the first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchoring attachment signal or (ii) a first peptide anchoring structure; (b) The second nucleic acid sequence encodes a second polypeptide, the second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchoring attachment signal or (ii) a second peptide anchoring structure; and The first cytokine peptide and the second cytokine peptide each independently comprise at least a portion or a variant thereof selected from any one 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.

5. A nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein: (a) The first nucleic acid sequence encodes a first polypeptide, the first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchoring attachment signal or (ii) a first peptide anchoring structure; (b) The second nucleic acid sequence encodes a second polypeptide, the second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchoring attachment signal or (ii) a second peptide anchoring structure; and 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 distinct second nucleic acid sequences, wherein: (a) The first nucleic acid sequence encodes a first polypeptide, the first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchoring attachment signal or (ii) a first peptide anchoring structure; (b) The second nucleic acid sequence encodes a second polypeptide, the second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchoring attachment signal or (ii) a second peptide anchoring structure; and 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 anchored structure or (ii) a first peptide anchored structure. The second protein comprises a second cytokine peptide and (i) a second non-peptide-anchored structure or (ii) a second peptide-anchored structure; and The first cytokine peptide and the second cytokine peptide each independently comprise at least a portion or a variant thereof selected from any one 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.

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 anchored structure or (ii) a first peptide anchored structure. The second protein comprises a second cytokine peptide and (i) a second non-peptide-anchored structure or (ii) a second peptide-anchored structure; and The first cytokine peptide and the second cytokine peptide each independently comprise at least a portion or a variant thereof selected from any one 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.

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 anchored structure or (ii) a first peptide anchored structure. The second protein comprises a second cytokine peptide and (i) a second non-peptide-anchored structure or (ii) a second peptide-anchored structure; and The first cytokine peptide is a pro-inflammatory cytokine peptide, and the second cytokine peptide is an anti-inflammatory cytokine peptide.

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

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

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

13. The nucleic acid molecule according to any one of claims 1, 2 or 5, the system according to any one of claims 3, 4 or 6, or the cell according to claim 11, wherein the second nucleic acid sequence is linked to the first nucleic acid sequence by a nucleic acid sequence encoding a cleavable adapter, optionally, the cleavable adapter comprising a peptide selected from P2A, T2A, E2A, F2A or IRES.

14. The nucleic acid molecule according to any one of claims 1, 2, or 5; the system according to any one of claims 3, 4, or 6; or the cell according to any one of claims 7-9, wherein: (a) The first protein or the first polypeptide comprises a first signal peptide; optionally, the first signal peptide, the first cytokine peptide, and (i) the first non-peptide anchoring attachment signal or (ii) the first peptide anchoring structure are operatively 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 the second polypeptide includes a second signal peptide, and optionally, the second signal peptide, the second cytokine peptide, and (i) the second non-peptide anchoring attachment signal or (ii) the second peptide anchoring structure are operatively linked in a direction from the N-terminus of the polypeptide to the C-terminus of the polypeptide.

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

16. The nucleic acid molecule, system, or cell of claim 14, wherein the first signal peptide and the second signal peptide each independently comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:2-7.

17. The nucleic acid molecule, system, or cell of claim 14, wherein the first signal peptide and the second signal peptide are each independently encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:52-57.

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

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

20. The nucleic acid molecule, system, or cell of claim 19, wherein the GPI attachment signal comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:

200.

21. The nucleic acid molecule, system, or cell of claim 19, wherein the GPI attachment signal is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 250-252.

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

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

24. The nucleic acid molecule, system, or cell of claim 22, wherein the transmembrane peptide sequence comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 203-204.

25. The nucleic acid molecule, system, or cell of claim 22, wherein the transmembrane peptide sequence is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 253-255.

26. 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 or a variant thereof selected from any one of IL-4, IL-10, or IL-27, and the pro-inflammatory cytokine peptide comprises at least a portion or a variant thereof selected from any one 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.

27. 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. 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 having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 101, 104, or 109, and the pro-inflammatory cytokine peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 100, 102, 103, 105-108, or 110-112.

29. 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 having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 151, 163, 154, 164, or 159, and the pro-inflammatory cytokine peptide is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 150, 152, 153, 155-158, or 160-162.

30. 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 having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 101, and the pro-inflammatory cytokine peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 100, 102, 103, 105-108, or 110-112.

31. 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 having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 151 or 163, and the pro-inflammatory cytokine peptide is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 150, 152, 153, 155-158, or 160-162.

32. The nucleic acid molecule according to claim 1 or 5, the system according to claim 3 or 6, or the cell according to 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-4 or a variant thereof selected from any one 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.

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

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

35. A nucleic acid molecule according to any one of claims 2 or 5, a system according to any one of claims 4 or 6, or a cell according to any one of claims 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. The nucleic acid molecule according to any one of claims 2 or 5, the system according to any one of claims 4 or 6, or the cell according to any one of claims 8 or 9, wherein the first cytokine peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO: 101, and the second cytokine peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 100, 102-105, or 107-112.

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

38. The nucleic acid molecule according to any one of claims 1 or 5, the system according to any one of claims 3 or 6, or the cell according to any one of claims 7 or 9, wherein the first cytokine peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 100-107 or 109-112.

39. The nucleic acid molecule according to any one of claims 1, 2 or 5, the system according to any one of claims 3, 4 or 6, or the cell according to any one of claims 7-9, wherein the first cytokine peptide is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the sequence shown in any one of SEQ ID NO: 150-157 or 159-164.

40. A nucleic acid molecule according to any one of claims 1 or 5, a system according to any one of claims 3 or 6, or a cell according to any one of claims 7 or 9, wherein the first cytokine peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 100-105 or 107-112.

41. The nucleic acid molecule according to any one of claims 2 or 5, the system according to any one of claims 4 or 6, or the cell according to any one of claims 8 or 9, wherein the first cytokine peptide is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 150-155 or 157-164.

42. The nucleic acid molecule according to any one of claims 1 or 5, the system according to any one of claims 3 or 6, or the cell according to any one of claims 7 or 9, wherein the second cytokine peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 100-107 or 109-112.

43. The nucleic acid molecule according to any one of claims 1 or 5, the system according to any one of claims 3 or 6, or the cell according to any one of claims 7 or 9, wherein the second cytokine peptide is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 150-157 or 159-164.

44. The nucleic acid molecule according to any one of claims 1 or 5, the system according to any one of claims 3 or 6, or the cell according to any one of claims 7 or 9, wherein the second cytokine peptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 100-105 or 107-112.

45. The nucleic acid molecule according to any one of claims 1 or 5, the system according to any one of claims 3 or 6, or the cell according to any one of claims 7 or 9, wherein the second cytokine peptide is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 150-155 or 157-164.

46. ​​The nucleic acid molecule according to any one of claims 1, 2 or 5, the system according to any one of claims 3, 4 or 6, or the cell according to any one of claims 7-9, wherein the nucleic acid molecule, system or cell further comprises a targeting sequence encoding a targeting portion, and the targeting portion comprises a chimeric antigen receptor (CAR), a T cell receptor, a B cell receptor or any combination thereof.

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

48. The nucleic acid molecule, system, or cell of claim 47, wherein the chimeric antigen receptor (CAR) comprises a ligand-binding domain, and the ligand-binding domain targets CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, CD138, CD171, CD5, CD7, MUC1, AFP, CEA, PSCA, PSMA, HER2, EGFR, IL13Rα2, GD2, NKG2D, EGFRvIII, CS1, CCL1, BCMA, mesothelin, ROR1, FLT3, FAP, TAG72, CD44v6, and EPC. AM, B7H3, KIT, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, NCAM, Claudin18.2, GPC3, GM3, TGS5, HMWMAA, TEM7R, CLDN6, GPRC5D, CXORF 61. 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. The nucleic acid molecule, system, or cell of claim 48, wherein the ligand-binding domain targets CD19.

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

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

52. The nucleic acid molecule, system, or cell according to any one of claims 47-50, wherein the CAR is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO:54 or 450-457.

53. 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 adapter, optionally, the cleavable adapter comprising a peptide selected from P2A, T2A, E2A, F2A, or IRES.

54. The nucleic acid molecule, system, or cell of claim 53, wherein the cleavable adapter is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 350-355.

55. The nucleic acid molecule, system, or cell of claim 53, wherein the cleavable adapter (a) connects the CAR to the first signal peptide and / or the second signal peptide; (b) connects the CAR to the first peptide anchoring structure and / or the second peptide anchoring structure; and / or (c) connects the CAR to the first non-peptide anchoring attachment signal and / or the second non-peptide anchoring attachment signal.

56. The nucleic acid molecule, system, or cell of claim 53, wherein the cleavable adapter comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 300-303.

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

58. The nucleic acid molecule, system, or cell according to claim 57, wherein: (a) The peptide connector connects the first cytokine peptide to the first peptide anchoring structure and / or connects the second cytokine peptide to the second peptide anchoring structure; (b) The peptide connector connects the first cytokine peptide to the first non-peptide anchoring attachment signal and / or connects the second cytokine peptide to the second non-peptide anchoring attachment signal; (c) The peptide connector connects the first peptide anchoring structure and / or the second peptide anchoring structure to the cuttable connector; and / or (d) The peptide connector connects the first non-peptide anchoring attachment signal and / or the second non-peptide anchoring attachment signal to the cuttable connector.

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

60. The nucleic acid molecule, system, or cell of claim 57, wherein the peptide linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 500, 501, 504, 506, or 507, or with the sequence shown in any one of LE, AS, GSG, or EF.

61. The nucleic acid molecule, system, or cell of claim 57, wherein the peptide linker is encoded by a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with any one of the sequences shown in ggctccggc, ggaagcgga, gagtc, SEQ ID NO:509, or SEQ ID NO:

520.

62. A nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide, said polypeptide comprising a signal peptide, a cytokine peptide, and a non-peptide anchoring attachment signal, wherein said cytokine peptide comprises at least a portion thereof selected from any one of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19, or IL-1α.

63. An immune cell comprising an exogenous nucleic acid sequence encoding a polypeptide, said polypeptide comprising cytokine peptides and non-peptide anchoring attachment signals, wherein said cytokine peptides comprise at least a portion or variants of any one of IL-2, IL-4, IL-7, IL-9, IL-10, IL-18, IL-23, IL-27, IL-36γ, IL-23p19 or IL-1α.

64. An immune cell comprising a foreign nucleic acid sequence encoding a polypeptide, the polypeptide comprising a cytokine peptide and a non-peptide anchoring attachment signal, wherein the cytokine peptide is at least a portion of IL-12p40 or at least a portion of IL-12p35, and the foreign nucleic acid sequence does not simultaneously encode IL-12p40 and IL-12p35, and the immune cell does not contain a stimulation response element (SRE) derived from PDE5 (phosphodiesterase 5).

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

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

68. A nucleic acid molecule comprising a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence, wherein: (a) The first nucleic acid sequence encodes a first polypeptide, the first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchoring attachment signal or (ii) a first peptide anchoring structure; (b) The second nucleic acid sequence encodes a second polypeptide, the second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchoring attachment signal or (ii) a second peptide anchoring structure; (c) The third nucleic acid sequence encodes a third polypeptide, the third polypeptide comprising a third cytokine peptide and (i) a third non-peptide anchoring attachment signal or (ii) a third peptide anchoring structure; The first cytokine peptide, the second cytokine peptide, and the third cytokine peptide each independently comprise at least a portion or a variant thereof selected from any one 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.

69. A system comprising a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence, wherein: (a) The first nucleic acid sequence encodes a first polypeptide, the first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchoring attachment signal or (ii) a first peptide anchoring structure; (b) The second nucleic acid sequence encodes a second polypeptide, the second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchoring attachment signal or (ii) a second peptide anchoring structure; (c) The third nucleic acid sequence encodes a third polypeptide, the third polypeptide comprising a third cytokine peptide and (i) a third non-peptide anchoring attachment signal or (ii) a third peptide anchoring structure; The first cytokine peptide, the second cytokine peptide, and the third cytokine peptide each independently comprise at least a portion or a variant thereof selected from any one 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, and the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are all different from each other.

70. A cell comprising a first protein, a second protein, and a third protein, wherein: (a) The first protein comprises a first cytokine peptide and (i) a first non-peptide-anchored structure or (ii) a first peptide-anchored structure; (b) The second protein comprises a second cytokine peptide and (i) a second non-peptide-anchored structure or (ii) a second peptide-anchored structure; (c) The third protein comprises a third cytokine peptide and (i) a third non-peptide-anchored structure or (ii) a third peptide-anchored structure; The first cytokine peptide, the second cytokine peptide, and the third cytokine peptide each independently comprise at least a portion or a variant thereof selected from any one 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.

71. A nucleic acid molecule comprising a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 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 having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 515-517, 523-526, 530-532, 534, 603-605, 607, 618-624, 639-651, 655-657, 667-675, 687-697, or 699.

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

74. The nucleic acid molecule according to 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. The nucleic acid molecule according to claim 74, wherein the vector is a viral vector, optionally, the vector is a retroviral vector, a lentiviral vector, an adenovirus vector, or an adeno-associated virus 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. The cell according to 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 according to any one of claims 7-9, 67, 70 or 76-83, wherein the cell is an immune cell or a tumor cell.

86. The cell according to 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.

87. The cell according to claim 86, wherein the engineered immune cell is a natural killer cell.

88. The immune cell according to any one of claims 63-65 or the immune cell according to claim 85, wherein the cytotoxicity of the immune cell is improved relative to the reference immune cell, wherein the reference immune cell: (a) not simultaneously comprising: (1) a first nucleic acid sequence encoding a first polypeptide comprising a first cytokine peptide and (i) a first non-peptide anchoring attachment signal or (ii) a first peptide anchoring structure; and (2) a second nucleic acid sequence encoding a second polypeptide comprising a second cytokine peptide and (i) a second non-peptide anchoring attachment signal or (ii) a second peptide anchoring structure; or (b) Not simultaneously comprising: (1) a first protein comprising a first cytokine peptide and (i) a first non-peptide-anchored structure or (ii) a first peptide-anchored structure; and (2) a second protein comprising a second cytokine peptide and (i) a second non-peptide-anchored structure or (ii) a second peptide-anchored structure.

89. The cell according to claim 88, wherein the degree of increased cytotoxicity of the immune cells is determined by the in vitro cytotoxicity assay method described in Example AD.

90. The cell of claim 88, wherein the degree of cytotoxic enhancement of the immune cells is determined in vitro or in vivo.

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

92. The immune cells according to any one of claims 63-65 or the immune cells according to claim 85, wherein the proliferation duration of the immune cell population is prolonged relative to a reference immune cell population, wherein the reference immune cell population: (a) A first nucleic acid sequence that does not contain a first polypeptide encoding a first cytokine peptide and (i) a first non-peptide anchoring attachment signal or (ii) a first peptide anchoring structure, and / or a second nucleic acid sequence that does not contain a second polypeptide encoding a second cytokine peptide and (i) a second non-peptide anchoring attachment signal or (ii) a second peptide anchoring structure; or (b) does not contain a first protein containing a first cytokine peptide and (i) a first non-peptide-anchored structure or (ii) a first peptide-anchored structure, and / or does not contain a second protein containing a second cytokine peptide and (i) a second non-peptide-anchored structure or (ii) a second peptide-anchored structure.

93. The cell according to claim 92, wherein the proliferation of the immune cell population is determined by the in vitro immune cell proliferation assay method described in Example AE.

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

95. The cell of claim 92, wherein the proliferation duration of the immune cell population is extended by at least about 5%, 10%, 20%, 30%, 40%, 50%, 100%, 2 times, 3 times, 5 times, 10 times or longer than that of the reference immune cell population.

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

97. A reagent kit comprising: (a) the pharmaceutical composition of claim 96; and (b) A data document containing dosage instructions for administering the immune cells, the cells, or the dosage form of the pharmaceutical composition.

98. Use of the immune cells of any one of claims 63-65 or the cells of any one of claims 7-9, 67, 70 or 76-83 in the preparation of a medicament for treating a subject.

99. The use according to claim 98, wherein the immune cells or the cells are used to treat a subject's cancer, optionally, the cancer is selected from solid tumors or melanoma, adrenal cancer, bladder cancer, bone cancer, brain tumors, breast cancer, cervical cancer, colorectal 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 preparing engineered immune cells, comprising introducing a nucleic acid molecule of any one of claims 1, 2, 5, 62, 66, 68, 71 or 73 or a system of any one of claims 3, 4, 6 or 69 into immune cells.

101. A method for preparing immune cells according to any one of claims 63-65 or cells according to any one of claims 7-9, 67, 70 or 76-83.

102. A method for preparing a pharmaceutical composition comprising combining immune cells of any one of claims 63-65 or cells of any one of claims 7-9, 67, 70 or 76-83 with a pharmaceutically acceptable excipient or carrier.

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