Compositions and methods for improving immunological responses in engineered immunological cells

Abstract

Provided herein includes compositions and methods for sustaining the surface expression of a target chimeric antigen receptor (CAR) and / or a target T cell receptor (TCR) in engineered immunological cells (e.g. T cells). In some embodiments, the methods include: co-expressing a polypeptide that comprises a wildtype or a certain mutant of c-Jun, or the functional variant thereof in the engineered immunological cells. Provided also includes a composition comprising a certain c-Jun mutant with disrupted delta region. Immunological cells that co-expressing the c-Jun mutant show improved immunological response, such as a reduced level of basal activation, an increased responsiveness, an increased expansion capability, and / or an increased cytolytic toxicity against target cells.

Description

[0001] COMPOSITIONS AND METHODS FOR IMPROVING IMMUNOLOGICAL RESPONSES IN ENGINEERED IMMUNOLOGICAL CELLS

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application App. No. 63 / 477,071, filed on December 23, 2022, and U.S. Provisional Application App. No. 63 / 480,825, filed on January 20, 2023. The entire contents of the foregoing applications are incorporated herein by reference.

[0004] BACKGROUND

[0005] Engineered T cells expressing an engineered CAR or TCR that can specifically target certain target tumor cells have shown great promises in the antitumor therapies, but dysfunction due to issues such as T cell exhaustion and gradual loss of expression of engineered CAR / TCR is a critical barrier to progress. Thus, there is a need to enhance expansion potential, increase functional capacity, diminish terminal differentiation and improve anti-tumor potency for these engineered cells.

[0006] SUMMARY

[0007] The present disclosure provides compositions and methods for sustaining the surface expression of a target chimeric antigen receptor (CAR) or a target T cell receptor (TCR) in engineered immune cells (e.g., T cells). The methods substantially include: co-expressing, in the immune cells (e.g., T cells) that express the target CARs / TCRs, a polypeptide comprising a wildtype or a certain mutant of c-Jun (eJun or c-JUN), such that the cell surface expression of the target CAR / TCR in the immune cells is better sustained or elevated. Throughout the disclosure, the polypeptide can optionally be termed “sustaining polypeptide” (i.e., a polypeptide whose co-expression sustains the expression of, or supports sustained expression of a target CAR / TCR in immunological cells), and the c-Jun fragment in the sustaining polypeptide can optionally be termed “actuating c-Jun fragment”. In some embodiments, the expression of the sustaining polypeptide (e.g., c-Jun or its variant thereof) has no effect on the expression of one or more exhaustion-associated markers (e.g., LAG3, PD1 and Tim3).

[0008] In one aspect, the disclosure is related to an engineered c-Jun polypeptide comprising a disrupted delta domain, in some embodiments, when the engineered c-Jun polypeptide is expressed in an immune cell, the immune cell has at least one of the following features: (a) a sustained expression of a chimeric antigen receptor (CAR) or a T cell receptor (TCR) on the immune cell, (b) a reduced level of basal activation, (c) an increased responsiveness, (d) an increased expansion capability, or (e) an increased cytolytic toxicity; as compared to an immune cell that does not express the engineered c-Jun polypeptide. In some embodiments, the delta domain is disrupted by a deletion within the delta domain. In some embodiments, the deletion is within a sequence that corresponds to amino acids 31-59 of SEQ ID NO: 37. In some embodiments, a sequence corresponds to amino acids 30-50 of SEQ ID NO: 37 is deleted. In some embodiments, the engineered c-Jun polypeptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 39. In some embodiments, a sequence corresponds to amino acids 34-47 of SEQ ID NO: 37 is deleted. In some embodiments, the engineered c-Jun polypeptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53. In some embodiments, the engineered c-Jun polypeptide comprises one or more of the following: (a) the amino acid that corresponds to S63 of SEQ ID NO: 37 is hydrophobic; and (b) the amino acid that corresponds to S73 of SEQ ID NO: 37 is hydrophobic. In some embodiments, the amino acid that corresponds to S63 of SEQ ID NO: 37 is Ala, Vai, He, Leu, Met, Phe, Try, or Trp. In some embodiments, the amino acid that corresponds to S73 of SEQ ID NO: 37 is Ala, Vai, He, Leu, Met, Phe, Try, or Trp. In some embodiments, the amino acid that corresponds to S63 of SEQ ID NO: 37 is Ala, and the amino acid that corresponds to S73 of SEQ ID NO: 37 is Ala. In some embodiments, the engineered c-Jun polypeptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 54.

[0009] In some embodiments, when the engineered c-Jun polypeptide is expressed in an immune cell, the immune cell has a sustained expression of a CAR or a TCR on the immune cell as compared to an immune cell that does not express the engineered c-Jun polypeptide. In some embodiments, when the engineered c-Jun polypeptide is expressed in an immune cell, the immune cell has a reduced level of basal activation as compared to an immune cell that does not express the engineered c-Jun polypeptide. In some embodiments, when the engineered c-Jun polypeptide is expressed in an immune cell, the immune cell has an increased responsiveness as compared to an immune cell that does not express the engineered c-Jun polypeptide. In some embodiments, when the engineered c-Jun polypeptide is expressed in an immune cell, the immune cell has an increased expansion capability as compared to an immune cell that does not express the engineered c-Jun polypeptide. In some embodiments, when the engineered c-Jun polypeptide is expressed in an immune cell, the immune cell has an increased cytolytic toxicity as compared to an immune cell that does not express the engineered c-Jun polypeptide.

[0010] In one aspect, the disclosure is related to a polynucleotide encoding the engineered c- Jun polypeptide described herein.

[0011] In one aspect, the disclosure is related to a polynucleotide comprising: (a) a first sequence encoding a CAR or a TCR; and (b) a second sequence encoding the engineered c- Jun polypeptide described herein.

[0012] In some embodiments, the first sequence encodes a CAR, in some embodiments, the CAR comprises, from N-terminus to C-terminus: (a) a leader sequence: (b) an antigenbinding fragment; (c) a hinge region comprising a membrane-proximal region from IgG, CD8, or CD28; (d) a transmembrane region comprising a transmembrane region of CD4, CD8, or CD28; (e) a costimulatory region comprising a functional signaling domain from MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-I, LFA-1, CDI la / CDI8, 4-1BB (CD137), B7-H3, CDS, ICAM- 1 , ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD 103, ITGAL, CD 11 a, LFA-1, ITGAM, CD 11b, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, or CD19a; and (!) an intracellular region comprising a functional signaling domain of CD3Z

[0013] In some embodiments, the antigen-binding fragment binds to or recognizes alkaline phosphatase, placental type (ALPP). In some embodiments, the antigen-binding fragment comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3. In some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence; the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence; and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence. The antigen-binding fragment further comprises a light chain variable region (VL) comprising CDRs 1, 2, and 3. In some embodiments, the VL CDRI region comprises an amino acid sequence that is at least 80% identical to a selected VL CDRI amino acid sequence; the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence; and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence. Herein according to different embodiments, the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences can be one of the following: (1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 61, 62, and 63, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 64, 65, and 66, respectively; (2) the selected VH CDRs 1, 2, 3 ammo acid sequences are set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 70, 71, and 72, respectively; (3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 73, 74, and 75, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 76, 77, and 78, respectively; (4) the selected VH CDRs 1 , 2, 3 amino acid sequences are set forth in SEQ ID NOs: 79, 80, and 81, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 82, 83, and 84, respectively; and (5) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 85, 86, and 87, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 88, 89, and 90, respectively.

[0014] In some embodiments, the antigen-binding fragment comprises a VH comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a VL comprising an amino acid sequence that is at least 90% identical to a selected VL sequence. Herein according to different embodiments, the selected VH sequence and the selected VL sequence can be one of the following: (1) the selected VH sequence is SEQ ID NO: 91, and the selected VL sequence is SEQ ID NO: 92; (2) the selected VH sequence is SEQ ID NO: 93, and the selected VL sequence is SEQ ID NO: 94; (3) the selected VH sequence is SEQ ID NO: 95, and the selected VL sequence is SEQ ID NO: 96; (4) the selected VH sequence is SEQ ID NO: 97, and the selected VL sequence is SEQ ID NO: 98; and (5) the selected VH sequence is SEQ ID NO: 99, and the selected VL sequence is SEQ ID NO: 100. In some embodiments, the antigen-binding fragment is a single-chain variable fragment (scFv), e g., an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 17, 18, 19, 20, or 21.

[0015] In some embodiments, the first sequence encodes a TCR. In some embodiments, the TCR binds to or recognizes a peptide epitope from NY-ESO-1. In some embodiments, the TCR is an Aspire-TCR. In some embodiments, the Aspire-TCR binds to or recognizes a peptide epitope from IL13Ra2. In some embodiments, the Aspire-TCR comprises an IL13 (E13Y) ligand region.

[0016] In some embodiments, the first sequence and the second sequence are connected by a third sequence encoding a linker, in some embodiments, the linker comprises a self-cleaving peptide (e.g., P2A or T2A) and / or a protease recognition site (e g., furin).

[0017] In one aspect, the disclosure is related to a vector comprising the polynucleotide as described herein. In one aspect, the disclosure is related to an engineered cell comprising the polynucleotide or the vector described herein.

[0018] In one aspect, the disclosure is related to an engineered cell comprising a first vector comprising a polynucleotide encoding a CAR or a TCR, and a second vector comprising a polynucleotide encoding the engineered c-Jun polypeptide described herein.

[0019] In one aspect, the disclosure is related to an engineered cell expressing the engineered c-Jun polypeptide as described herein. In some embodiments, the engineered cell described herein further expresses a CAR or a TCR that binds to or recognizes a peptide epitope from an antigen on a target cell.

[0020] In some embodiments, the engineered cell expresses a CAR that binds to or recognizes a peptide epitope from ALPP, LYPD3, IL13Ra2 (IL13 receptor alpha 2), BCMA, CD19, CD20, CD22, CD138, CD33, CD123, CD171, CD70, CD7, CS-1, PSMA, PSCA, ROR1, GD2, MUC1, MUC16, HER2 (ErbB2), MET, EphA2, EpCAM, CEA, CSPG4, Lewis Y antigen, Mesothelin, NKG2D, Glypican-3 (GPC-3), FAP, FRa (folate receptor alpha), EGFR, EGFR vIII, IL-1 IRa (IL11 receptor alpha), or VEGFR-II. In some embodiments, the engineered cell expresses a TCR that binds to or recognizes a peptide epitope from NY-ESO- 1, EBV LMP2, EBV antigen, HPV16 E6 / E7, KRAS, H3K27M, WT-1, or PRAME. In some embodiments, the engineered cell expresses an Aspire-TCR that binds to or recognizes a peptide epitope from IL13Ra2.

[0021] In some embodiments, the engineered cell is a T cell (e.g., a CD3+T cell, a CD4+T cell, a CD8+T cell, a natural killer (NK) T cell, an alpha beta T cell, a gamma delta T cell, or a memory T cell (e.g. a central memory T cell or an effector memory T cell)), a tumor infiltrating lymphocyte (TIL), a microphage, or a natural killer (NK) cell.

[0022] In one aspect, the disclosure is related to a method for improving the surface expression of a CAR or a TCR in an immune cell, comprising modifying the immune cell to express: (a) a CAR or a TCR that binds or recognizes a peptide epitope from an antigen; and (b) a sustaining polypeptide. In some embodiments, the immune cell has an improved sustaining of the expression of the CAR or TCR as compared to an immune cell that does not express the sustaining polypeptide. In some embodiments, the sustaining polypeptide is an AP-1 transcription factor. In some embodiments, the sustaining polypeptide is a wildtype c- Jun or a variant thereof. In some embodiments, sustaining polypeptide is Jun (e.g., c-Jun, JunB, or JunD), Fos (e.g., c-Fos, FosB, Fral, and Fra2), activating transcription factor (ATF), Jun dimerization protein (JDP), or a variant thereof. In some embodiments, the sustaining polypeptide is a wildtype c-Jun. In some embodiments, the sustaining polypeptide is a c-Jun variant. In some embodiments, the c-Jun variant has a disrupted delta domain. In some embodiments, the c-Jun variant does not comprise one or more amino acids that correspond to all or a portion of the delta domain in a wildtype c-Jun. In some embodiments, one or more amino acids in the JNK sites of the c-Jun variant are hydrophobic. In some embodiments, the c-Jun or the variant thereof comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to any one of SEQ ID NOs: 37-54. In some embodiments, the c-Jun or the variant thereof comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to any one of SEQ ID NOs: 37, 38, 39, 46, 50, 51, 52, 53, and 54. In some embodiments, the c-Jun or the variant thereof comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 53 or SEQ ID NO: 54.

[0023] In some embodiments, the method further comprises: stimulating the immune cell by the antigen for at least 1, 2, 3, 4, 5, or 6 times. In some embodiments, the immune cell has an improved immunological response (e.g., a reduced level of basal activation; an increased responsiveness; an increased expansion capability, and / or an increased cytolytic toxicity) as compared to an immune cell that does not express the sustaining polypeptide. In some embodiments, the expression of the sustaining polypeptide has no effect on the expression of one or more exhaustion-associated markers (e.g., LAG3, PD1 and / or Tim3) in the immune cell.

[0024] In some embodiments, the immune cell expresses a CAR that binds to or recognizes a peptide epitope from ALPP, LYPD3, IL13Ra2 (IL13 receptor alpha 2), BCMA, CD19, CD20, CD22, CD138, CD33, CD123, CD171, CD70, CD7, CS-1, PSMA, PSCA, R0R1, GD2, MUC1, MUC16, HER2 (ErbB2), MET, EphA2, EpCAM, CEA, CSPG4, Lewis Y antigen, Mesothelin, NKG2D, Glypican-3 (GPC-3), FAP, FRa (folate receptor alpha), EGFR, EGFR vIII, IL-1 IRa (IL11 receptor alpha), or VEGFR-II. In some embodiments, the CAR binds to or recognizes a peptide epitope from ALPP, CD19, or IL13Ra2. In some embodiments, the immune cell expresses a TCR that binds to or recognizes a peptide epitope from NY-ESO-1, EBV LMP2, EBV antigen, HPV16 E6 / E7, KRAS, H3K27M, WT-1, or PRAME. In some embodiments, the TCR binds to or recognizes a peptide epitope from NY- ESO-1. In some embodiments, the immune cell expresses an Aspire-TCR (e.g., an Aspire- TCR that binds to IL13Ra2).

[0025] In some embodiments, the method further comprises: modifying the immune cell to express an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the checkpoint inhibitor can inhibit or block PD-1, PD-L1, PD-L2, 2B4 (CD244), 4-1BB, A2aR, B7.1, B7.2, B7-H2, B7- H3, B7-H4, B7-H6, BTLA, butyrophilins, CD160, CD48, CTLA4, GITR, gp49B, HHLA2, HVEM, ICOS, ILT-2, ILT-4, KIR family receptors, LAG-3, OX-40, PIR-B, SIRPalpha (CD47), TFM-4, TIGIT, TIM-1, TIM-3, TIM-4, VISTA, or combinations thereof. In some embodiments, the additional therapeutic agent is a cytokine or chemokine (e.g., IL 12 or IL- 7 / CCL19), or a bifunctional trap fusion protein. In some embodiments, the immune cell is a T cell (e.g., a CD3+T cell, a CD4+T cell, a CD8+T cell, a natural killer (NK) T cell, an alpha beta T cell, a gamma delta T cell, or a memory T cell (e.g. a central memory T cell or an effector memory T cell). In some embodiments, the immune cell is a tumor infiltrating lymphocyte (TIL), a microphage, or a natural killer (NK) cell. In some embodiments, the CAR or TCR is heterologous to the immune cell. In some embodiments, the immune cell is a cell line. In some embodiments, the immune cell is a primary cell obtained from a subject (e.g., a human subject).

[0026] In one aspect, the disclosure is related to a method for producing the engineered cell, comprising introducing the vector described herein into a cell in vitro or ex vivo. In some embodiments, the vector is a viral vector and the introducing is carried out by transduction.

[0027] In one aspect, the disclosure is related to a method of treating a disease or disorder, comprising administering the engineered cell described herein to a subject having the disease or disorder.

[0028] In one aspect, the disclosure is related to a method of treating a disease or disorder in a subject, the method comprising administering to the subject in need thereof, (a) an engineered T cell, comprising: a nucleic acid encoding a CAR or a TCR; and (b) the engineered c-Jun polypeptide described herein. In some embodiments, the disease or disorder is a cancer. In some embodiments, the disease or disorder is a non-cancerous disease.

[0029] In one aspect, the disclosure is related to an antibody or antigen-binding fragment thereof that specifically binds to ALPP. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3. In some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence; the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 ammo acid sequence; and the VH CDR3 region comprises an ammo acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence. The antibody or antigen-binding fragment thereof further comprises a light chain variable region (VL) comprising CDRs 1, 2, and 3. In some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence; the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence; and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence. Herein according to different embodiments, the selected VH CDRs 1, 2, and 3 ammo acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following: (1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 61, 62, and 63, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 64, 65, and 66, respectively; (2) the selected VH CDRs 1, 2, 3 ammo acid sequences are set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 70, 71, and 72, respectively; (3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 73, 74, and 75, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 76, 77, and 78, respectively; (4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 79, 80, and 81, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 82, 83, and 84, respectively; and (5) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 85, 86, and 87, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 88, 89, and 90, respectively. In some embodiments, the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 61, 62, and 63, respectively, and the VL comprises CDRs I, 2, 3 with the ammo acid sequence set forth in SEQ ID NOs: 64, 65, and 66, respectively. In some embodiments, the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequence set forth in SEQ ID NOs: 70, 71, and 72, respectively. In some embodiments, the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 73, 74, and 75, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequence set forth in SEQ ID NOs: 76, 77, and 78, respectively. In some embodiments, the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively, and the VL comprises CDRs 1, 2, 3 with the ammo acid sequence set forth in SEQ ID NOs: 82, 83, and 84, respectively. In some embodiments, the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 85, 86, and 87, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequence set forth in SEQ ID NOs: 88, 89, and 90, respectively.

[0030] In some embodiments, the VH consists of or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 91, 93, 95, 97, or 99; and the VL consists of or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 92, 94, 96, 98, or 100. In some embodiments, the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 91 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 92. In some embodiments, the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 93 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 94. In some embodiments, the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 95 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 96. In some embodiments, the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 97 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 98. In some embodiments, the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 99 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 100.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human ALPP. In some embodiments, the antibody or antigen-binding fragment thereof is an scFv. In some embodiments, the VH and VL are connected with a linker peptide (e g., SEQ ID NO: 25). In some embodiments, the scFv comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 17, 18, 19, 20, or 21.

[0032] In one aspect, the disclosure is related to a chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof described herein. In one aspect, the disclosure is related to a polynucleotide encoding the antibody or antigen-binding fragment thereof or the CAR described herein. In one aspect, the disclosure is related to a vector comprising the polynucleotide described herein. In one aspect, the disclosure is related to an engineered cell comprising the CAR, the polynucleotide, or the vector described herein.

[0033] In some embodiments, the CAR described herein further comprises: a leader sequence (e.g., any of the leader sequences described herein), a hinge region derived from CD8, a transmembrane region derived from CD4, a costimulatory region derived from 4-1BB, and / or an intracellular signaling region derived from CD3C. In some embodiments, the leader sequence comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 60. In some embodiments, the hinge region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 14. In some embodiments, the transmembrane region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 15. In some embodiments, the costimulatory region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 8. In some embodiments, the intracellular signaling region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 9.

[0034] In some embodiments, the CAR described herein comprises an scFv that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 17, 18, or 21. In some embodiments, the CAR can be stably expressed on T cell surface.

[0035] In some embodiments, the CAR described herein comprises an scFv that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 17 or 18. In some embodiments, the CAR, when expressed on the surface of T cells, can activate the T cells (e.g., by inducing IFN-y expression) that are co-cultured with ALPP-expressing tumor cells (e.g., SiHa cells).

[0036] In some embodiments, the disclosure is related to an engineered c-Jun polypeptide, comprising a first sequence that is at least 80% identical to amino acids 1-30 of SEQ ID NO: 37, and a second sequence that is at least 80% identical to amino acids 60-331 of SEQ ID NO: 37, wherein the engineered c-Jun polypeptide does not comprise one or more amino acids from a sequence corresponding to amino acids 31-59 of SEQ ID NO: 37. In some embodiments, the disclosure is related to an engineered c-Jun polypeptide, comprising a first sequence that is at least 80% identical to amino acids 1-29 of SEQ ID NO: 37, and a second sequence that is at least 80% identical to amino acids 51-331 of SEQ ID NO: 37, wherein the engineered c-Jun polypeptide does not comprise one or more amino acids from a sequence corresponding to amino acids 30-50 of SEQ ID NO: 37. In some embodiments, the disclosure is related to an engineered c-Jun polypeptide, comprising a first sequence that is at least 80% identical to amino acids 1-33 of SEQ ID NO: 37, and a second sequence that is at least 80% identical to amino acids 48-331 of SEQ ID NO: 37, wherein the engineered c-Jun polypeptide does not comprise one or more amino acids from a sequence corresponding to amino acids 34.47 of SEQ ID NO: 37.

[0037] As used herein, the term “basal activation” refers to activation of immune cells in the absence of any antigens. In some embodiments, the basal activation is indicated by the secretion level of one or more proinflammatory cytokines (e.g., IFN-y), and / or the expression level of one or more activation markers (e.g., 4- IBB), when the immune cells are cultured in the absence of antigen stimulation (e.g., specific target cells). In some embodiments, “reduced basal activation” indicates no greater than 80% of the IFN-y secretion level by reference T cells that are armored with a wildtype c-Jun protein.

[0038] As used herein, the term “expansion capability ” refers to the capability of immune cells (e.g., immune cells expressing a CAR / TCR and a sustaining polypeptide described herein) to proliferate (e.g., after about 12, 15, 18, 21, or 26 days post transfection). In some embodiments, “increased expansion capability” indicates no less than 1.2-fold increase of the proliferation rate of reference immune cells that are armored with a wildtype c-Jun protein.

[0039] As used herein, the term “cytolytic toxicity” refers to the capability to of immune cells to kill specific target cells when the immune cells are co-cultured with the specific target cells. As used herein, the term “specific target cells” refer to cells that express antigens that can be specifically recognized by the CAR or TCR (e.g., Aspire-TCR) on the immune cells. In some embodiments, “increased cytolytic toxicity” indicates no less than 1.2-fold increase of killing efficiency of specific target cells.

[0040] As used herein, the term “responsiveness” or “responsiveness to antigen stimulation” refers to an increase of the secretion level of one or more proinflammatory cytokines (e.g., IFN-y) and / or the expression level of one or more activation markers (e.g., 4-1BB), when the immune cells are cultured in the presence of antigen stimulation (e.g., specific target cells) as compared to those in the absence of antigen stimulation (e.g., specific target cells). In some embodiments, “increased responsiveness to antigen stimulation" indicates no less than 1.2- fold increase of IFN-y secretion level as compared to the IFN-y secretion level when T cells are armored with a wildtype c-Jun protein.

[0041] As used herein, the phrase “obtain a sustained expression”, “obtain a better sustained surface expression”, “obtain an improved sustaining of the expression”, or alike, means that after transduction of the target CAR or the target TCR into the immunological cells, any one of the following two scenarios is met:

[0042] Scenario (1): expression of the target CAR or the target TCR on the transduced cell surface drops both when the sustaining polypeptide is not co-expressed and when the sustaining polypeptide is co-expressed, wherein the drop of the expression at a later timepoint relative to an earlier reference timepoint when the sustaining polypeptide is co-expressed is less than or equal to a first predetermined threshold of the drop when the sustaining polypeptide is not co-expressed, wherein the first predetermined threshold can be a percentage that is <=80%, such as 80%, preferably 70%, more preferably 60%, and more preferably 50%, etc.;

[0043] Scenario (2): expression of the target CAR or the target TCR on the transduced cell surface drops when the sustaining polypeptide is not co-expressed, but remains substantially unchanged or increases when the sustaining polypeptide is co-expressed. According to certain embodiments, the phrase “obtain a better sustained surface expression” or alike may further mean that after transduction of the target CAR or the target TCR into the immunological cells, the following scenario (3) is met:

[0044] Scenario (3): expression of the target CAR or the target TCR on the transduced cell surface remains substantially unchanged or increases when the sustaining polypeptide is not co-expressed, but increases when the sustaining polypeptide is co-expressed, wherein the increase of the expression at a later timepoint relative to an earlier reference timepoint when the sustaining polypeptide is co-expressed is greater than or equal to a second predetermined threshold of the increase when the sustaining polypeptide is not co-expressed, wherein the second predetermined threshold is a percentage that is no less than 120%, such as 120%, preferably 130%, more preferably 140%, and more preferably 150%, etc.

[0045] As used herein, the phrase “substantially unchanged” means that a level of a variable under examination (such as the expression level of the target CAR / TCR) changes by less than 5% if comparing a later timepoint with an earlier reference timepoint. If a change is greater than or equal to 5%, such a change can be deemed as an “increase” or a “drop” as mentioned.

[0046] As used herein, the term “Aspire-TCR” can be any Aspire-TCRs constructs described herein.

[0047] As used herein, the term “increased” or “reduced” refers to the level change of no less than 10% compared to a reference level.

[0048] In some embodiments, one or more domains of an enzyme can be “disrupted.” In some embodiments, the disrupted domain includes one or more deletions, mutations, and / or substitutions of its amino acid sequence. In some embodiments, the disrupted domain is functionally defective as compared to a wildtype domain.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0050] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. DESCRIPTION OF DRAWINGS

[0051] FIG. 1 shows schematic structures of A02. A02-cJun, A02-8H, and A02-8H eJun constructs.

[0052] FIG. 2A shows the percentage expression of different ALPP CARs (“A02” and “A02- cJun”) in human T cells at different timepoints after transduction (Day 4, Day 12, and Day 19).

[0053] FIG. 2B shows the percentage expression of different ALPP CARs (i.e. “8H” and “8H eJun”) in human T cells at different timepoints after transduction (Day 5, Day 7 and Day 20).

[0054] FIGS. 3A-3B show the memory phenotyping of different ALPP CARs in human T cells.

[0055] FIGS. 4A-4B show the A02 and A02 eJun CAR-T cell activation upon antigenspecific activation. Both live CD8+(FIG. 4A) and CD4+(FIG. 4B) T cell populations were analyzed.

[0056] FIGS. 4C-4D show the A02-8H and A02-8H eJun CAR-T cell activation upon antigen-specific activation. Both live CD8+(FIG. 4C) and CD4+(FIG. 4D) T cell populations were analyzed.

[0057] FIGS. 5A-5B show the activation of A02 and A02 eJun CAR-T cells upon repeated antigen-specific stimulation. Both live CD8+(FIG. 5A) and CD4+(FIG. 5B) T cell populations were analyzed.

[0058] FIGS. 5C-5D show the activation of A02-8H and A02-8H eJun CAR-T cells upon repeated antigen-specific stimulation. Both live CD8+(FIG. 5C) and CD4+(FIG. 5D) T cell populations were analyzed.

[0059] FIGS. 6A-6B show the proliferation of A02, A02 eJun CAR-T cells (FIG. 6 A) and A02-8H and A02-8H eJun CAR-T cells (FIG. 6B) upon repeated antigen-specific stimulation.

[0060] FIG. 7 shows the in vivo efficacy of ALPP CAR-T cells in SKOV3-ALPP subcutaneous NSG mouse model.

[0061] FIG. 8 shows the in vivo efficacy of ALPP CAR-T cells in SiHa intraperitoneal NSG mouse model.

[0062] FIG. 9A shows effects of c-Jun co-expression on the expression of NY-ESO-1 TCR in the engineered TCR-T cells either under regular culturing condition.

[0063] FIGS. 9B-9E show effects of c-Jun co-expression on the expression of exhaustion- associated markers LAG3, PD1 and Tim3. Flow cytometry was performed 21 days post transduction for unstimulation groups (FIG. 9B and FIG. 9D), or 3 days post transduction for stimulation groups (FIG. 9C and FIG. 9E).

[0064] FIG. 10 shows functional domains of human c-Jun, where "5" stands for the 8 (delta) domain, "DB" for the DNA-binding domain, and "LZ" for the leucine zipper dimerization domain. S63, S73, T91, and T93 are amino acid residues in the JNK phosphorylation sites.

[0065] FIGS. 11 A-l ID show effects of various c-Jun variants on the expression and / or activation of TCR and CAR in the engineered T cells. FIS. 11 A shows the effects on antiNY -ESO- 1 TCR expression. FIG. 11B shows the effects on ALPP CAR expression in a regular culturing condition. FIG. 11 C shows the effects on ALPP CAR expression in an antigen stimulation and re-stimulation condition. FIG. 1 ID shows the activation of the CAR- T cells. "NT", non-treated cells; "Before sti" indicates before stimulation; "1st sti" indicating after 1st stimulation and before 2nd re-stimulation; "2nd sti" indicating after 2nd restimulation and before 3rd re-stimulation; and "3rd sti" indicating after 3rd re-stimulation.

[0066] FIGS. 12A-12B show effects of the various c-Jun variants on the expression of ALPP CAR in the engineered T cells either under regular culturing condition (FIG. 12 A) or under re-stimulation condition (FIG. 12B).

[0067] FIGS. 12C-12F show effects of the various c-Jun variants on the activation of the engineered T cells.

[0068] FIGS. 12G-12H show effects of the various c-Jun variants on the cytolytic toxicity against target tumor cells.

[0069] FIGS. 13A-13B show effects of various c-Jun variants on the Aspire-TCR expression.

[0070] FIGS. 13C-13D show effects of various c-Jun variants on the expansion (FIG. 13C) and basal activation (FIG. 13D) of, the engineered Aspire-T cells.

[0071] FIG. 14A shows the binding curves of anti-hALPP scFv-Fc fusion proteins C9, F7, F8, H5, and A3 to 293T cells expressing human ALPP (hALPP-293T). The EC50 value of each binding curve is also provided.

[0072] FIG. 14B shows the binding curves of anti-hALPP scFv-Fc fusion proteins C9, F7, F8, H5, and A3 to SiHa cells. The EC50 value of each binding curve is also provided.

[0073] FIG. 14C shows the binding curves of anti-hALPP scFv-Fc fusion proteins C9, F7, F8, H5, and A3 to 293T cells expressing human ALPL (hALPL-293T).

[0074] FIG. 14D shows the binding curves of anti-hALPP scFv-Fc fusion proteins C9, F7, F8, H5, and A3 to 293T cells expressing human ALPI (hALPI-293T). FIG. 14E shows the binding curves of anti-hALPP scFv-Fc fusion proteins C9, F7, F8, H5, and A3 to parental 293T cells (control).

[0075] FIGS. 15A-15B show the percentage expression of different ALPP CARs (A3 CAR, C9 CAR, F7 CAR, F8 CAR, and F9 CAR) in primary human T cells on Day 4 post transduction. “UT” stands for untransduced T cells.

[0076] FIGS. 15C-15D show the percentage expression of different ALPP CARs (A3 CAR, C9 CAR, F7 CAR, F8 CAR, and F9 CAR) in primary human T cells on Day 11 post transduction. “UT” stands for untransduced T cells.

[0077] FIGS. 16A-16C show the untransduced (“UT”), F8, and H5 CAR-T cell activation upon antigen-specific activation. Live CD8+T cell populations were analyzed.

[0078] FIGS. 17A-17B show SDS-PAGE results of anti-hALPP scFv-Fc fusion proteins C9, F7, F8, H5, and A3 in non-reduced and reduced conditions.

[0079] FIG. 18 shows VH and VL CDR sequences in F8, H5, C9, F7, and A3 antibodies.

[0080] FIG. 19 shows VH and VL sequences in F8, H5, C9, F7, and A3 antibodies.

[0081] FIG. 20 lists sequences discussed in the disclosure.

[0082] DETAILED DESCRIPTION

[0083] The present disclosure provides an approach for improving the immunological responses in immunological cells (e.g., T cells), especially those that have been engineered to express a target chimeric antigen receptor (CAR) and / or a target T cell receptor (TCR). Substantially, the approach comprises the armoring or co-expression of an immunological cell with a c-Jun fragment, which can result in improved phenotypes or performance of the immunological cell. Herein, the improvements may include one or more of the following aspects.

[0084] (1) The improvement may be on the aspect of the expression of the engineered CAR / TCR on the immunological cells, which can be better sustained in the immunological cell in the presence of such an armor compared with otherwise (i.e., in the absence of the armor). For example, in certain cases, the expression of certain engineered CAR / TCR, such as the NY-ESO-1 TCR in Example 9 or the IL 13 Aspire-TCR in Example 12, may undergo a continuous decrease or gradual loss upon transduction to the T cells in the absence of a c-Jun armor, while the armoring of such engineered T cells with a c-Jun fragment, such as wildtype eJun, or eJun AA (i.e., a eJun variant with S63A and S73A substitutions), a eJun deletion mutant at its delta domain (e.g., dJun 30-50 (i.e., c-Jun deletion at positions 30-50), or dJun 34-47 (i.e., c-Jun deletion at position 34-47), or a combination (e.g. dJun 34-47 AA), can beter sustain the expression of the CAR / TCR on the engineered T cells. In some other cases, under the more in wvo-mimicking antigen stimulation and re-stimulation conditions, the expression of certain engineered CAR / TCR, such as the ALPP CAR in Example 11, may gradually decrease or lose in the absence of a c-Jun armor, but the armoring of such engineered T cells with a c-Jun fragment, such as wildtype c-Jun, dJun 34-47, or dJun 34-47 AA, can beter sustain the expression of the CAR / TCR on the engineered T cells. It is expected that the beter sustaining of the CAR / TCR expression can in turn translate into an improved therapy of these engineered CAR-T or TCR-T cells against target tumor cells. It is important to note that the improved expression of CAR / TCR resulting from the armoring of any of the above c-Jun fragment is independent from the reduced exhaustion as previously reported in Lynn RC et al. Nature. 2019 Dec;576(7786):293-300.

[0085] (2) The improvement may be on the aspect of the basal activation of the engineered immunological cells. As shown by Examples 10-12 below, the armoring of the engineered T cells with certain c-Jun fragment, such as wildty pe c-Jun and dJun 30-50, unfavorably causes a relatively high level of basal activation (i. e. , antigen-independent secretion of interferon gamma) of the engineered T cells, suggesting that such armored engineered T cells may have unfavorable off-target toxicity if administered to a subject in need thereof. In contrast, the engineered T cells armored with certain other c-Jun fragment, such as the dJun 34-47 and dJun 34-47 AA, unexpectedly exhibit reduced basal activation (i.e., about 1 / 4- 1 / 2 as estimated by the level of antigen-independent secretion of IFN-y or by the level of T cell activation marker 41BB) of the engineered T cells compared with those armored with the wildtype c-Jun. It is expected that the engineered T cells armored with dJun 34-47 and dJun 34-47 AA can favorably exhibit lower off-target toxicity compared with those armored with wildtype c-Jun.

[0086] (3) The improvement may be on the aspect of the antigen-responsiveness of the engineered immunological cells. As at least shown by Example 11, the engineered T cells armored with dJun 34-47 and dJun 34-47 AA exhibited unexpectedly higher antigenresponsiveness (i.e., about 1.7-2.4 fold higher) compared with engineered T cells armored with wildtype c-Jun. It is expected that the engineered T cells armored with dJun 34-47 and dJun 34-47 AA can favorably exhibit higher specificity against the target tumor cells compared with those armored with wildtype c-Jun. (4) The improvement may be on the aspect of the expansion capability of the engineered immunological cells. As shown by Example 12, the engineered T cells armored with wildtype c-Jun exhibited reduced expansion capability compared with those cells without any armor, yet the engineered T cells armored with dJun 34-47 and dJun 34-47 AA exhibited favorably higher expansion capability. It is expected that the engineered T cells armored with dJun 34-47 and dJun 34-47 AA can favorably exhibit better expansion capability, and thus better druggability , compared with those armored with wildtype c-Jun.

[0087] (5) The improvement may be on the aspect of the cytolytic toxicity of the engineered immunological cells against target tumor cells. As show n by Example 11, the engineered T cells armored with dJun 34-47 and dJun 34-47 AA exhibited higher cytolytic toxicity against target tumor cells compared with engineered T cells armored with wildtype c-Jun.

[0088] In some embodiments, the engineered cells expressing the sustaining polypeptide (e.g., a c-Jun variant) described herein expresses a CAR or a TCR at a level that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than reference cells that do not express the sustaining polypeptide (e.g., a c-Jun variant), or expresses a wildtype c-Jun protein.

[0089] In some embodiments, the engineered cells expressing the sustaining polypeptide (e.g., a c-Jun variant) described herein exhibit a basal activation level (e.g., antigenindependent activation level) that is less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less 25%, less than 20%, less than 15%, less than 10%, or less than 5% as compared to that of reference cells expressing a wildtype c-Jun protein. In some embodiments, the basal activation is determined by measuring the secretion of one or more proinflammatory cytokines (e.g., IFN-y) and / or one or more immune cell (e.g., T cell) activation markers (e.g., 4-1BB).

[0090] In some embodiments, the engineered cells expressing the sustaining polypeptide (e.g., a c-Jun variant) described herein exhibit a responsiveness level to antigen stimulation that is great than 110%, greater than 120%, greater than 130%, greater than 140%, greater than 150%, greater than 160%, greater than 170%, greater than 180%, greater than 190%, greater than 2-fold, greater than 3-fold, greater than 4-fold, greater than 5-fold, greater than 6- fold, greater than 7-fold, greater than 8-fold, greater than 9-fold, or greater than 10-fold, as compared to that of reference cells expressing a wildtype c-Jun protein. In some embodiments, the responsiveness is determined by measuring the secretion of one or more proinflammatory cytokines (e.g., IFN-y).

[0091] In some embodiments, the engineered cells expressing the sustaining polypeptide (e.g., a c-Jun variant) described herein exhibit an expansion capability that is great than 110%, greater than 120%, greater than 130%, greater than 140%, greater than 150%, greater than 160%, greater than 170%, greater than 180%, greater than 190%, greater than 2-fold, greater than 3 -fold, greater than 4-fold, greater than 5 -fold, greater than 6-fold, greater than 7- fold, greater than 8-fold, greater than 9-fold, or greater than 10-fold, as compared to that of reference cells expressing a wildtype c-Jun protein.

[0092] In some embodiments, the engineered cells expressing the sustaining polypeptide (e.g., a c-Jun variant) described herein exhibit a cytolytic activity that is great than 110%, greater than 120%, greater than 130%, greater than 140%, greater than 150%, greater than 160%, greater than 170%, greater than 180%, greater than 190%, greater than 2-fold, greater than 3 -fold, greater than 4-fold, greater than 5 -fold, greater than 6-fold, greater than 7-fold, greater than 8-fold, greater than 9-fold, or greater than 10-fold, as compared to that of reference cells expressing a wildtype c-Jun protein. In some embodiments, the cytolytic activity is determined by measuring target tumor cell killing efficiency, either under unstimulated conditions or repeated stimulation conditions.

[0093] An anti-ALPP CAR was used as an illustrating example where T cells armored with co-expression of c-Jun (i.e. the sustaining polypeptide) showed a much improved sustaining of the expression for the anti-ALPP CAR compared with otherwise (i.e. not armored with c- Jun expression). Thus such c-Jun armored CAR-T cells are expected to realize an improved efficacies in treating ALPP-positive cancers, e.g., ovarian cancer, cervical cancer, or testicular cancer. In some embodiments, the subject has testicular seminoma, primary intracranial germinoma, epithelial ovarian carcinoma, ovarian adenocarcinoma, serous cystadenocarcinoma, undifferentiated carcinoma, dysgerminoma, ovarian cancer, uterus cancer, endometrial cancer, cervical cancer, urothelial cancer, stomach cancer, lung cancer, pancreatic cancer, testis cancer, osteosarcoma, and / or gastric cancer. In some embodiments, the anti-ALPP CAR described herein is a murine ALPP CAR, a human ALPP CAR, or a chimeric ALPP CAR.

[0094] Sustaining polypeptide In one aspect, provided herein is a sustaining polypeptide, when co-expressed with a CAR or TCR in an immune cell (e.g., T cell), can improve the sustaining of surface expression of the CAR or the TCR over time. For example, at a certain timepoint post transfection of the immune cells to co-express the CAR / TCR and the sustaining polypeptide, the surface expression of the CAR / TCR is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20- fold, 50-fold, or 100-fold higher than the surface expression of the CAR / TCR in a reference immune cell that does not express the sustaining polypeptide. In some embodiments, the sustaining can occur after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days post transfection. In some embodiments, the immune cell is unstimulated. In some embodiments, the immune cell is stimulated by an antigen for at least 1, 2, 3, 4, 5, or 6 times. In some embodiments, the immune cell is restimulated by the same or a different antigen for at least once, at least twice, or at least three times.

[0095] In some embodiments, the sustaining polypeptide is an AP-1 transcription factor. Activator protein 1 (AP-1) is a transcription factor that regulates gene expression in response to a variety of stimuli, including cytokines, growth factors, stress, and bacterial and viral infections. AP-1 controls a number of cellular processes including differentiation, proliferation, and apoptosis. The structure of AP-1 is a heterodimer composed of proteins belonging to the c-Fos, c-Jun, ATF and JDP families.

[0096] The AP-1 proteins function as dimers. Both homodimers and heterodimers are found; although not all proteins can homodimerize and not all heterodimers are possible. Dimerization is mediated by the leucine zipper. This domain adopts an alpha helical structure where leucine side chains interact with the alpha helix of the leucine zipper of the other family member to mediate dimerization. The basic domain is important for interacting with DNA. AP-1 proteins bind 12-O-tetradecanoylphorbol- 13 -acetate (TP A) responsive elements (TRE), cAMP responsive elements (CRE), and related sequences. Individual dimers differ in their DNA binding and transcriptional activities. For example, c-Jun:c-Fos dimers prefer TRE sites, whereas c-Jun:ATF dimers prefer CRE sites. Moreover, c-Jun:c-Fos heterodimers have higher affinity for TRE sites than c-Jun: c-Jun homodimers, and dimers containing JunB are less transcriptionally active than those containing c-Jun. While these proteins are primarily thought to function as transcriptional activators, there are situations where they appear to function as repressors. Thus, the AP-1 family is a diverse collection of proteins that generate an even greater collection of dimers with varied DNA binding and transcriptional activities. Not surprisingly, AP-1 family proteins regulate a wide range of cellular and biological activities. These include the cell cycle and proliferation, programmed cell death including apoptosis and autophagy, and lipid synthesis. As well, AP-1 proteins regulate migration and invasion through modulation of the cytoskeleton, and are implicated in inflammatory diseases, bone development, the nervous system, immune cell development and activation, and cancer. Details of AP-1 transcription factor and its functions can be found, e.g., in Wu, Z. et al. "AP-1 family transcription factors: A diverse family of proteins that regulate varied cellular activities in classical hodgkin lymphoma and ALK+ ALCL." Experimental Hematology & Oncology 10.1 (2021): 1-12; and Garces de los Fay os Alonso, Ines, et al. "The role of activator protein-1 (AP-1) family members in CD30-positive lymphomas." Cancers 10.4 (2018): 93; each of which is incorporated herein by reference in its entirety.

[0097] In some embodiments, the sustaining polypeptide described herein is Jun (e.g., c-Jun, JunB, or JunD), Fos (e.g., c-Fos, FosB, Fral, and Fra2), activating transcription factor (ATF), Jun dimerization protein (JDP), or a variant thereof. In some embodiments, the sustaining polypeptide described herein is a wildtype c-Jun protein or a variant thereof (e.g., any one of the c-Jun variant described herein).

[0098] According to certain embodiments, the sustaining polypeptide comprises a c-Jun fragment having a sequence that is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, or 100%) identical to one of the following versions of c-Jun listed below, including wildtype and certain mutant forms of c-Jun, where certain substitution mutations are underlined, and certain deletion mutations are indicated in their names by the “A” followed by position ranges relative to the wildtype c-Jun (e.g. “Jun-A30-50”: missing a peptide fragment that corresponds to a region from position 30 to position 50 of the wildtype c-Jun). According to certain other embodiments, the c-Jun fragment may substantially be a c-Jun fragment corresponding to a combination of the various mutations in the various c-Jun variants / mutants disclosed herein.

[0099] As used herein, unless otherwise indicated, the sustaining polypeptide (e.g., eJun) in the disclosure includes the wildtype enzymes and the variants thereof. A variant can have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the wildtype enzyme, but the variant retains similar functions or activities or even can have improved functions or activities. In some embodiments, a variant is a truncated form of the wildtype enzyme, optionally with at least 1, 2, 3, 4, 5, or 6 mutations. For example, one or more functional domains of the wildtype enzyme can be deleted, and the remaining portions of the enzyme are connected. In some embodiments, one or more amino acid residues in the wildtype enzyme can be substituted, deleted, or mutated. In some embodiments, a variant includes one or more (e.g., 1, 2, 3, 4, 5, or 6) portions of the wildtype enzyme, but one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300) amino acid sequences are deleted. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or 6) function of the wildtype enzyme is disrupted in the variant. In some embodiments, a variant is a fragment of the wildtype enzyme. For example, the term “eJun” includes the wildtype eJun and the variants thereof, wherein the variants have substantially similar or even better functions or activities, e.g., sustaining the surface expression of the target CAR or TCR. In some embodiments, the eJun described herein includes any one of the eJun variants / mutants discussed in the disclosure. eJun: (SEQ ID NO: 37)

[0100] MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPH LRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFA EGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEP PVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQ ALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAASKCRKRKLERIARLE EKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTF Jun-AA (SEQ ID NO: 38) MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPH LRAKNSDLLTAPDVGLLKLAAPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFA EGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEP PVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQ ALICEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAASKCRKRKLERIARLE EKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTF

[0101] Jun-A30-50 (SEQ ID NO: 39)

[0102] MTAKMETTFYDDALNASFLPSESGPYGYSPHLRAKNSDLLTSPDVGLLKLAS PELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFAEGFVRALAELHSQNTLPSVTS AAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEPPVYANLSNFNPGALSSGGGAP SYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQALKEEPQTVPEMPGETPPLSPI DMESQERIKAERKRMRNRIAASKCRKRKLERIARLEEKVKTLKAQNSELASTANML REQVAQLKQKVMNHVNSGCQLMLTQQLQTF

[0103] Jun-ATAD (2-102) (SEQ ID NO: 40)

[0104] MVTDEQEGFAEGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAG GSGSGGFSASLHSEPPVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPH HLPQQMPVQHPRLQALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAA SKCRKRKLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQ LMLTQQLQTF

[0105] Jun-Abasic (254-280) (SEQ ID NO: 41)

[0106] MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPH LRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFA EGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEP PVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQ ALKEEPQTVPEMPGETPPLSPIDMESQERIEEKVKTLKAQNSELASTANMLREQVAQ LKQKVMNHVNSGCQLMLTQQLQTF

[0107] Jun-ALeu (280-314) (SEQ ID NO: 42)

[0108] MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPH LRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFA EGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEP PVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQ ALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAASKCRKRKLERIARHV NSGCQLMLTQQLQTF

[0109] Jun-AZIP(254-314) (SEQ ID NO: 43)

[0110] MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPH LRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFA EGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEP PVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQ ALKEEPQTVPEMPGETPPLSPIDMESQERIHVNSGCQLMLTQQLQTF

[0111] Jun A3- 102 (SEQ ID NO: 44)

[0112] MTVTDEQEGFAEGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVA GGSGSGGFSASLHSEPPVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPP HHLPQQMPVQHPRLQALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIA ASKCRKRKLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGC QLMLTQQLQTF

[0113] Jun A3- 122 (SEQ ID NO: 45)

[0114] MTSQNTLPSVTSAAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEPPVYA NLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQALKE EPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAASKCRKRKLERIARLEEKVK TLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTF

[0115] Jun A->D265A270-272 (SEQ ID NO: 46)

[0116] MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPH LRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFA EGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEP PVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQ ALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIDASKCKLERIARLEEKV KTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTF

[0117] Jun A->D265A256-258 (SEQ ID NO: 47)

[0118] MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPH LRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFA EGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEP PVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQ ALKEEPQTVPEMPGETPPLSPIDMESQERIKARMRNRIDASKCRKRKLERIARLEEKV KTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTF

[0119] Jun A->D265in(SEQ ID NO: 48)

[0120] MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPH LRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFA EGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEP PVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQ ALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIDDDASKCRKRKLERIAR LEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTF

[0121] Jun A287-331 (SEQ ID NO: 49)

[0122] MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPH LRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFA EGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEP PVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQ ALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAASKCRKRKLERIARLE EKVKT

[0123] Jun-bZIP (SEQ ID NO: 50)

[0124] MTAKKAERKRMRNRIAASKSRKRKLERIARLEEKVKTLKAQNSELASTANM LREQVAQLKQKVMNH

[0125] Jun- 103-209 (SEQ ID NO: 51)

[0126] MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPH LRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTPTPTQFLCPKNHLPQQMPVQ HPRLQALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAASKSRKRKLER IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTF

[0127] Jun- 103-145 (SEQ ID NO: 52)

[0128] MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPH LRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTPTPTQFLCPKNAVASVAGGS GSGGFSASLHSEPPVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHL PQQMPVQHPRLQALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAASKS RKRKLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLML TQQLQTF

[0129] According to certain embodiments, the sustaining polypeptide may further comprise, in addition to the actuating c-Jun fragment as mentioned above, one or more other functional fragments. Examples can include protein tags (e.g., HIS or FLAG).

[0130] According to certain embodiments, the sustaining polypeptide may be configured to realize a controlled expression in the immunological cells expressing the target CAR / TCR. For example, in the vector that encodes and expresses the sustaining polypeptide, the polynucleotide encoding the sustaining polypeptide can be configured to contain an inducible gene expression element (e.g., promoter or enhancer) so as to realize a controllable expression of the sustaining polypeptide. For example, under a first condition, the expression of the sustaining polypeptide is turned on so that it can exert its effect in sustaining the expression of the target CAR / TCR in the immunological cells; whereas under a second condition, the expression of the sustaining polypeptide is turned off, such that the potential adverse effects caused by the long-term expression of the c-Jun containing sustaining polypeptide (e.g., c-Jun induced tumorigenesis) can be effectively avoided.

[0131] It is noted that the sustaining polypeptide is not limited to the above mentioned c-Jun fragments, it may comprise other peptide fragment that has the capability of sustaining the cell surface expression of the target CAR / TCR in the immunological cells. Non-limiting examples of such peptide fragment may include various versions (wildtype, mutants / variants implicating amino acid residue substitutions, deletions, insertions, etc.) of a submit of an AP- 1 dimeric transcription factor, such as Jun (c-Jun, JunB, and JunD), Fos (c-Fos, FosB, Fral, and Fra2), activating transcription factor (ATF), or Jun dimerization protein (JDP), etc.

[0132] In some embodiments, the sustaining polypeptide described herein is wild-type “eJun” or a variant thereof. In some embodiments, the w ild-type eJun or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 37.

[0133] As show n in FIG. 10, the wildtype human transcription factor Jun, also know n as c- Jun, JUN, AP-1, cJUN, or p39 (NCBI Reference Sequence: NP 002219.1; UniProt ID: P05412; SEQ ID NO: 37), is a 331 amino acid protein. The c-Jun protein includes a delta (6) domain that corresponds to amino acids 31-59 of SEQ ID NO: 37, a DNA-binding (DB) domain that corresponds to amino acids 257-276 of SEQ ID NO: 37, and a leucine zipper (LZ) domain that corresponds to amino acids 280-308 of SEQ ID NO: 37. The N-terminal phosphorylation (JNK) sites include S63, S73, T91, and T93 in SEQ ID NO: 37.

[0134] In some embodiments, the sustaining polypeptide described herein is “Jun-AA” or a variant thereof. In some embodiments, the Jun-AA or the variant thereof comprises or consists of the following mutations: (a) the amino acid that corresponds to S63 of SEQ ID NO: 37 is alanine; and (b) the amino acid that corresponds to S73 of SEQ ID NO: 37 is alanine. In some embodiments, the Jun-AA or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 38.

[0135] In some embodiments, the sustaining polypeptide described herein is “Jun- 30-50” (or “dJun 30-50”) or a variant thereof. In some embodiments, a contiguous amino acid sequence that corresponds to N30-K50 of SEQ ID NO: 37 is deleted in the Jun-A30-50 or the variant thereof. In some embodiments, the Jun-A30-50 or the variant thereof comprises or consists of a first amino acid sequence that corresponds to M1-S29 of SEQ ID NO: 37, and a second amino acid sequence that corresponds to P51-F331 of SEQ ID NO: 37. In some embodiments, the Jun-A30-50 or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 39. In some embodiments, the sustaining polypeptide described herein is “Jun-ATAD (2- 102)” or a variant thereof. In some embodiments, the Jun-ATAD (2-102) or the variant thereof does not include all or a portion of the transactivation domain (TAD) domain of wildtype eJun protein. In some embodiments, the Jun-ATAD (2-102) or the variant thereof does not include an amino acid sequence that corresponds to amino acids 2-102 of SEQ ID NO: 37. In some embodiments, the Jun-ATAD (2-102) or the variant thereof comprises or consists of an amino acid sequence that corresponds to VI03-F331 of SEQ ID NO: 37. In some embodiments, the Jun-ATAD (2-102) or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40.

[0136] In some embodiments, the sustaining polypeptide described herein is “Jun-Abasic (254-280)” or a variant thereof. In some embodiments, the Jun-Abasic (254-280) or the variant thereof does not include all or a portion of the basic motif of wildtype eJun protein. In some embodiments, the Jun-Abasic (254-280) or the variant thereof does not include an amino acid sequence that corresponds to amino acids 254-280 of SEQ ID NO: 37. In some embodiments, the Jun-Abasic (254-280) or the vanant thereof compnses or consists of a first amino acid sequence that corresponds to MI-1253 of SEQ ID NO: 37, and a second amino acid sequence that corresponds to E281-F331 of SEQ ID NO: 37. In some embodiments, the Jun-Abasic (254-280) or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41.

[0137] In some embodiments, the sustaining polypeptide described herein is “Jun-ALeu (280- 314)” or a variant thereof. In some embodiments, the Jun-ALeu (280-314) or the variant thereof does not include all or a portion of the Leucine-rich region (“Leu”) of wildtype eJun protein. In some embodiments, the Jun-ALeu (280-314) or the variant thereof does not include an amino acid sequence that corresponds to amino acids 280-314 of SEQ ID NO: 37. In some embodiments, the Jun-ALeu (280-314) or the variant thereof comprises or consists of a first amino acid sequence that corresponds to M1-R279 of SEQ ID NO: 37, and a second ammo acid sequence that corresponds to H315-F331 of SEQ ID NO: 37. In some embodiments, the Jun-ALeu (280-314) or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42. In some embodiments, the sustaining polypeptide described herein is “Jun-AZIP(254- 314)” or a variant thereof. In some embodiments, the Jun-AZIP(254-314) or the variant thereof does not include all or a portion of the Leucine Zipper domain (ZIP) domain of wildtype eJun protein. In some embodiments, the Jun-AZIP(254-314) or the variant thereof does not include an amino acid sequence that corresponds to amino acids 254-314 of SEQ ID NO: 37. In some embodiments, the Jun-AZIP(254-314) or the variant thereof comprises or consists of a first amino acid sequence that corresponds to MI-1253 of SEQ ID NO: 37, and a second amino acid sequence that corresponds to H315-F331 of SEQ ID NOL 37. In some embodiments, the Jun-AZIP(254-314) or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 43.

[0138] In some embodiments, the sustaining polypeptide described herein is “Jun A3-102” or a variant thereof. In some embodiments, the Jun A3 -102 or the variant thereof does not include an amino acid sequence that corresponds to amino acids 3-102 of SEQ ID NO: 37. In some embodiments, the Jun A3-102 or the variant thereof comprises or consists of a first ammo acid sequence that corresponds to M1-T2 of SEQ ID NO: 37, and a second amino acid sequence that corresponds to V103-F331 of SEQ ID NO: 37. In some embodiments, the Jun A3-102 or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44.

[0139] In some embodiments, the sustaining polypeptide described herein is “Jun A3 -122” or a variant thereof. In some embodiments, the Jun A3-122 or the variant thereof does not include an amino acid sequence that corresponds to amino acids 3-122 of SEQ ID NO: 37. In some embodiments, the Jun A3-102 or the variant thereof comprises or consists of a first amino acid sequence that corresponds to M1-T2 of SEQ ID NO: 37, and a second amino acid sequence that corresponds to S123-F331 of SEQ ID NO: 37. In some embodiments, the Jun A3-122 or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 45.

[0140] In some embodiments, the sustaining polypeptide described herein is “Jun A->D265A270-272” (or “dJun 270-272”) or a variant thereof. In some embodiments, the Jun A->D265A270-272 or the variant thereof (1) does not include an amino acid sequence that corresponds to amino acids 270-272 of SEQ ID NO: 37, and (2) includes the following mutation: the amino acid that corresponds to A265 of SEQ ID NO: 37 is aspartic acid (D). In some embodiments, the Jun A->D265A270-272 or the variant thereof includes a first amino acid sequence that corresponds to MI-1265 of SEQ ID NO: 37, a second amino acid sequence that corresponds to A266-C269 of SEQ ID NO: 37, and a third amino acid sequence that corresponds to K273-F331 of SEQ ID NO: 37. In some embodiments, the first amino acid sequence and the second amino acid sequence are connected with a aspartic acid (D). In some embodiments, the Jun A->D265A270-272 or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46.

[0141] In some embodiments, the sustaining polypeptide described herein is “Jun A->D265A256-258” or a variant thereof. In some embodiments, the Jun A->D265A256-258 or the variant thereof (1) does not include an amino acid sequence that corresponds to amino acids 256-258 of SEQ ID NO: 37, and (2) includes the following mutation: the amino acid that corresponds to A265 of SEQ ID NO: 37 is aspartic acid (D). In some embodiments, the Jun A->D265A256-258 or the variant thereof comprises or consists of a first amino acid sequence that corresponds to M1-A255 of SEQ ID NO: 37, and a second amino acid sequence that corresponds to R259-F331 of SEQ ID NO: 37. In some embodiments, the Jun A->D265A270- 272 or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 47.

[0142] In some embodiments, the sustaining polypeptide described herein is “Jun A->D265in” or a variant thereof. In some embodiments, the Jun A->D265inor the variant thereof (1) includes the following mutation: the amino acid that corresponds to A265 of SEQ ID NO: 37 is aspartic acid (D), and (2) includes an insertion of at least 1 , 2, 3, 4, 5, or 6 (e g., 2) aspartic acid residues between two neighboring amino acids that correspond to A265 and A266 of SEQ ID NO: 37. In some embodiments, the Jun A->D265inor the variant thereof comprises or consists of a first amino acid sequence that corresponds to MI-1264 of SEQ ID NO: 37, and a second amino acid sequence that corresponds to A266-F331 of SEQ ID NO: 37. In some embodiments, the first amino acid sequence and the second amino acid sequence that connected with at least 1, 2, 3, 4, 5, or 6 (e.g., 3) aspartic acid residues. In some embodiments, the Jun A->D265A270-272 or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48.

[0143] In some embodiments, the sustaining polypeptide described herein is “Jun A287-331” or a variant thereof. In some embodiments, the Jun A287-331 or the variant thereof does not include an amino acid sequence that corresponds to amino acids 287-331 of SEQ ID NO: 37. In some embodiments, the Jun A287-331 or the variant thereof comprises or consists of an amino acid sequence that corresponds to M1-T286 of SEQ ID NO: 37. In some embodiments, the Jun A287-331 or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 49.

[0144] In some embodiments, the sustaining polypeptide described herein is “Jun-bZIP” (or “eJun bZIP vl”) or a variant thereof. In some embodiments, the Jun-bZIP or the variant thereof comprises or consists of a first ammo acid that corresponds to M1-R4 of SEQ ID NO: 37; and a second amino acid sequence that corresponds to K254-H315 of SEQ ID NO: 37, in which the amino acid that corresponds to C269 of SEQ ID NO: 37 is mutated to serine (S). In some embodiments, the Jun-bZIP or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 50.

[0145] In some embodiments, the sustaining polypeptide described herein is “Jun-Al 03-209” (or “dJun 103-209”) or a variant thereof. In some embodiments, the Jun-A103-209 or the variant thereof does not include an amino acid sequence that corresponds to amino acids 103- 209 of SEQ ID NO: 37. In some embodiments, the Jun-A103-209 or the variant thereof comprises or consists of a first amino acid sequence that corresponds to M1-N102 of SEQ ID NO: 37, and a second amino acid sequence that corresponds to H210-F331 of SEQ ID NO: 37. In some embodiments, the Jun A287-331 or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 51 .

[0146] In some embodiments, the sustaining polypeptide described herein is “Jun-Al 03 -145” (or “dJun 103-145”) or a variant thereof. In some embodiments, the Jun-A103-145 or the variant thereof does not include an amino acid sequence that corresponds to amino acids 103- 145 of SEQ ID NO: 37. In some embodiments, the Jun-A103-145 or the variant thereof comprises or consists of a first amino acid sequence that corresponds to M1-N102 of SEQ ID NO: 37; and a second amino acid sequence that corresponds to A146-F331 of SEQ ID NO: 37, in which the amino acid that corresponds to C269 of SEQ ID NO: 37 is mutated to serine (S). In some embodiments, the Jun-A103-145 or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52.

[0147] In some embodiments, the sustaining polypeptide described herein is “dJun 34-47” or a variant thereof. In some embodiments, the dJun 34-47 or the variant thereof does not include an amino acid sequence that corresponds to amino acids 34-47 of SEQ ID NO: 37. In some embodiments, the dJun 34-47 or the variant thereof comprises or consists of a first amino acid sequence that corresponds to MI-133 of SEQ ID NO: 37, and a second amino acid sequence that corresponds to S48-F331 of SEQ ID NO: 37. In some embodiments, the dJun 34-47 or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53.

[0148] In some embodiments, the sustaining polypeptide described herein is “dJun 34-47 AA” or a variant thereof. In some embodiments, the dJun 34-47 AA or the variant thereof does not include an amino acid sequence that corresponds to amino acids 34-47 of SEQ ID NO: 37; and the dJun 34-47 AA or the variant thereof comprises or consists of the following mutations: (a) the amino acid that corresponds to S63 of SEQ ID NO: 37 is alanine; and (b) the amino acid that corresponds to S73 of SEQ ID NO: 37 is alanine. In some embodiments, the dJun 34-47 AA or the variant thereof comprises or consists of a first amino acid sequence that corresponds to MI-133 of SEQ ID NO: 37, and a second amino acid sequence that corresponds to S48-F331 of SEQ ID NO: 37, in which the amino acids that correspond to S63 and S67 of SEQ ID NOL 37 are two alanine residues. In some embodiments, the dJun 34-47 AA or the variant thereof includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54.

[0149] Target CARs / TCRs

[0150] As used herein, the CAR includes any transmembrane protein that comprises an extracellular domain, a transmembrane domain, and an intracellular domain; in the transmembrane protein, the intracellular domain may at least include a functional intracellular T cell signaling domain (e.g., a CD3-zeta cytoplasmic domain or a functional variant or fragment thereof) and may further include one or more co-stimulatory domains (e.g., CD28 costimulatory domain, 4-1BB costimulatory domain etc ), the extracellular domain may include one or more target-recognizing domains, and the transmembrane domain can, upon binding of the one or more target-recognizing domains of the extracellular domain to the target molecule on the target cell, transmit the activation signal from the extracellular domain to the intracellular domain to thereby allow the activation or stimulation of the corresponding immunological responses in the immunological cell that expresses the transmembrane protein.

[0151] Further as used herein, the TCR includes any transmembrane protein that comprise one or more extracellular target-recognizing domains and can form a functional TCR complex with other TCR subunit proteins on the cellular surface of the immunological cell. Specifically, the TCR protein may be an engineered transmembrane protein that comprises, in addition to the one or more extracellular target-recognizing domains, a polypeptide domain that corresponds to a functional TCR alpha subunit, TCR beta subunit, CD3 delta subunit, CD3 gamma subunit, or CD3 epsilon subunit, or a functional variant or fragment thereof. Once the one or more extracellular target-recognizing domains of the TCR protein recognize and bind to the target molecule on the target cell, the functional TCR complex that the transmembrane protein forms with other subunits of the TCR complex (including any or a combination of TCR alpha, TCR beta, CD3 delta, CD3 gamma, CD3 epsilon and CD3 zeta) on the immunological cell can activate or stimulate the corresponding immunological responses in the immunological cell that expresses the transmembrane protein.

[0152] Herein, it is noted that in any of the CAR or TCR mentioned above, each such extracellular target-recognizing domain can comprise an antigen-binding domain, a ligand domain, or any domain that can specifically recognize and bind to a target molecule expressed or presented on the surface of a target cell. Non-limiting examples for an antigen- binding domain may include an antibody domain, an Fab region, a single-chain variable fragment (scFv), a nanobody, etc. Non-limiting examples for a ligand domain may include a ligand or a functional variant or fragment thereof that specifically recognizes and binds to a corresponding ligand-binding receptor of a target cell (e.g., the ligand can be an IL-13 variant with E I 3Y mutation, that has selective affinity to the glioma-specific IL-13Ra2; see the IL13 (El 3Y) Aspire-TCR in Example 12). It is noted that other scheme may be possible as long as the extracellular target-recognizing domain can bind to the target molecule expressed or presented on the surface of the target cell.

[0153] According to certain embodiments, the target CAR whose expression in the target immunological cells can be better sustained by co-expressing the aforementioned polypeptide in the target immunological cells can be a CAR that specifically targets one of the following target molecules: ALPP (disclosed in WO2020263796A1), LYPD3 (disclosed in W02020018973 Al), IL13Ra2 (IL13 receptor alpha 2), BCMA, CD19, CD20, CD22, CD138, CD33, CD123, CD171, CD70, CD7, CS-I, PSMA, PSCA, R0R1, GD2, MUCI, MUC16, HER2 (ErbB2), MET, EphA2, EpCAM, CEA, CSPG4, Lewis Y antigen, Mesothelin, NKG2D, Glypican-3 (GPC-3), FAP, FRa (folate receptor alpha), EGFR, EGFR vIII, IL-l lRa (IL11 receptor alpha), and VEGFR-II, etc. It is noted that these represent only illustrating examples, and by no means impose a limitation to the scope of this disclosure. According to certain embodiments, the target CAR may comprise, from N-terminus to C -terminus, an antigen-binding fragment, a hinge, a transmembrane domain (TM), a costimulatory signaling region, and an intracellular signaling region. The antigen-binding fragment may comprise a single-chain variable fragment (scFv) comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) that specifically targets one of the aforementioned target molecules. The VH and VL domains may optionally be joined by a flexible linker. The hinge may optionally comprise a membrane-proximal region from IgG, CD8, or CD28, etc.; the transmembrane domain (TM) may optionally comprise a transmembrane region of CD4, CD8, or CD28, etc.; the costimulatory signaling region may optionally comprise a functional signaling domain from one of the following proteins: MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, CDlla / CD18, 4-1BB (CD137), B7-H3, CDS, ICAM- 1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD lid, ITGAE, CD 103, ITGAL, CD 11 a, LFA-1, ITGAM, CD 11b, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, or CD19a, etc.; and the intracellular signaling region may optionally comprise an activating cytoplasmic signaling domain capable of inducing a pnmary activation signal in a T cell, being a T cell receptor (TCR) component, and / or comprising an immunoreceptor tyrosine-based activation motif (ITAM), and optionally, the intracellular signaling region is or comprises a functional signaling domain of CD3 zeta. Details for the specific sequence information of the above different regions can be found in the international patent application WO2020263796A1, whose contents are incorporated herein by reference.

[0154] In some embodiments, the target CAR can be a CAR that specifically targets ALPP, CD19 or IL13Ra2. (1) The specific structure and sequence of ALPP CAR can reference to the international patent application WO2020263796A1 and to the specific example (Example 1, including two embodiments of ALPP CAR, “A02” and “A02-8H”, for experimental data, and several other embodiments of the ALPP CARs with different anti- ALPP scFv) provided below.

[0155] (2) The CD19 CAR may comprise a structure and sequence of antigen-binding fragment (anti-CD19 scFv), hinge domain (e.g., CD8 or CD28), transmembrane domain (e.g., CD8, CD28, or CD4), costimulatory domain (e.g., CD28 or 4-1BB) and signal domain (e g., CD3zeta or CD3epsilon). The sequence of one specific embodiment of the CD19 CAR is listed below.

[0156] Table 1.

[0157] Thus, in some embodiments, provided herein is a CD 19 CAR that includes, optionally from N-terminus to C-terminus, a leader sequence, an antigen-binding fragment (e.g., an scFv) that binds to CD19, a CD8 hinge region, a CD28 transmembrane region, a costimulatory signaling region derived from CD28, and an intracellular signaling region derived from CD35. In some embodiments, the leader sequence includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 23. In some embodiments, the scFv described herein includes, optionally from N-terminus to C-terminus, a VL having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 24, a linker peptide having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 25, and a VH having a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 26. In some embodiments, the CD8 hinge region includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27. In some embodiments, the CD28 transmembrane region includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 28. In some embodiments, the costimulatory signaling region described herein includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 29. In some embodiments, the intracellular signaling region described herein includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 30. (3) The IL13Ra2 CAR may comprise a structure and sequence of antigen-binding fragment (anti-IL13Ra2 scFv or IL13 ligand), hinge domain (e.g., CD8, CD28 or IgG4 Fc), transmembrane domain (e.g., CD8, CD28, or CD4), costimulatory domain (e.g., CD28 or 4- 1BB) and signal domain (e.g., CD3zeta or CD3epsilon). The sequence of one specific embodiment of the IL13Ra2 CAR is also listed below.

[0158] Table 2.

[0159] Thus, in some embodiments, provided herein is a IL13Ra2 CAR that includes, optionally from N-terminus to C-terminus, a leader sequence, an antigen-binding fragment (e.g., a IL 13 ligand) that binds to IL13Ra2, an IgG4 Fc hinge region, a CD4 transmembrane region, a costimulatory signaling region derived from 4- IBB, and an intracellular signaling region derived from CD33. In some embodiments, the leader sequence includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2. In some embodiments, the IL13 ligand described herein includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32. In some embodiments, the IgG4 Fc hinge region includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 33. In some embodiments, the CD4 transmembrane region includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 34. In some embodiments, the costimulatory signaling region described herein includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 35. In some embodiments, the intracellular signaling region described herein includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 36.

[0160] According to certain embodiments, the target TCR whose surface expression in the immunological cells can be better sustained by co-expressing the aforementioned polypeptide in the immunological cells can be a TCR that specifically targets one of the following target molecules: NY-ESO-1 (disclosed in W02020086158A2), EBV LMP2 (disclosed in W02020112815A1), EBV antigen (disclosed in WO2021244653A1), HPV16 E6 / E7 (disclosed in W02020036834A1 and WO2021155830A1), KRAS (disclosed in WO- 2021083363-A1 and WO2018026691 Al), H3K27M (disclosed in WO2016179326A1), WT- 1 (disclosed in WO2015077615A1), PRAME (disclosed in W02021099360A1), etc. In some embodiments, the TCR described herein binds to or recognizes a peptide epitope from NY-ESO-1. In some embodiments, the TCR has a variable alpha (Va) region that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12, and a variable beta (Vb) region that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13. Details of the NY-ESO-1 TCRs and relevant sequences can be found, e.g., in PCT Application Publication No. W02020086158A2, which is incorporated herein by reference in its entirety.

[0161] Within the scope of the disclosure, the target CAR / TCR expressed on the cell surface of the immunological cells may show a sustainability issue after transduction in vitro, i.e., the surface expression of the target CAR / TCR on the engineered T cells drops, or is not well- sustained, over time in the absence of the aforementioned sustaining polypeptide that comprises any of the above listed actuating c-Jun fragment (e.g., compared with an earlier reference timepoint, such as on Day 4, the CAR / TCR expression drops by >10% on Day 12, or drops by >20% on Day 19; see FIG. 2A and Table 5A); whereas the co-expression of the sustaining polypeptide can, compared to otherwise (i.e. in the absence situation), realize a significantly better sustaining of the expression of the target CAR / TCR on the T cells (e.g., compared with an earlier reference timepoint, such as on Day 4, there is no drop of the expression of the target CAR / TCR, or the drop is much less, for example, the drop in the presence of the sustaining polypeptide is only 50% of the drop in the absence of the sustaining polypeptide).

[0162] As used herein, the “expression” of a target CAR / TCR in the immunological cells is typically understood as the “expression of the target CAR / TCR on the cell surface of the immunological cells,” or the “surface expression of the target CAR / TCR in the immunological cells,” and within the scope of the disclosure, it may mean the percentage of the target CAR+ / TCR+cells in the whole population of the immunological cells, the surface target CAR / TCR expression on each individual cell level of the immunological cells, or both, at a certain timepoint after transduction.

[0163] It is noted that certain target CAR / TCR may not show a pronounced sustainability issue per se (i.e., after transduction, the expression doesn't change much or drops minimally over time), but its expression is relatively low in absence of the sustaining polypeptide coexpression, whereas in the presence of the sustaining poly peptide, the expression of the target CAR / TCR on the T cells can be significantly increased. These CARs / TCRs are also deemed to be within the scope of the disclosure.

[0164] Aspire TCR

[0165] In some embodiments, the TCR described herein is an Antigen-specificity redirected TCR complex (i.e. Aspire-TCR or Aspire-T). In some embodiments, the Aspire TCR substantially comprises an engineered target-recognizing subunit and / or an exogenous CD3 zeta (i.e. CD3^ or CD3z) subunit. Such engineered TCR complex is capable of specifically and efficiently recognizing specific target molecules expressed in target cells (e.g. tumor cells). When expressed in immunological cells, the engineered T cell receptor (TCR) complex can provide specific and efficient cytotoxicity to the immunological cells expressing the engineered TCR complex against the target cells.

[0166] As used herein, the term "immunological cells" can be T lymphocytes (including a(> T cells or y5 T cells) tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, or NK T cells, or any of these above cells that have been engineered (e.g. cells expressing TCR or Chimeric antigen receptor (CAR)). In the following, the T lymphocytes, or T cells, are used as illustrating yet non-limiting example for the immunological cells.

[0167] In some embodiments, an engineered CD3 zeta subunit is provided, which comprises at least one co-stimulatory region operably linked to or incorporated into a mammalian CD3 zeta or a functional portion or a functional variant thereof. The at least one co-stimulatory region is within an intracellular domain of the engineered CD3 zeta subunit, and the engineered CD3 zeta subunit is capable of being incorporated into a T cell receptor (TCR) complex when expressed in an immunological cell.

[0168] The engineered CD3 zeta subunit is configured such that at least one of the following effects is realized:

[0169] (1) the immunological cell expressing the engineered CD3 zeta subunit has a reduced activation in the absence of antigen stimulation of the TCR complex compared with when the immunological cell does not express the engineered CD3 zeta subunit;

[0170] (2) the immunological cell expressing the engineered CD3 zeta subunit has a reduced cytotoxicity against non-target cells compared with when the immunological cell does not express the engineered CD3 zeta subunit; (3) the immunological cell expressing the engineered CD3 zeta subunit has an increased activation upon stimulation of the TCR complex compared with when the immunological cell does not express the engineered CD3 zeta subunit; or

[0171] (4) the immunological cell expressing the engineered CD3 zeta subunit has an increased immunological cell response against target cells corresponding thereto compared with when the immunological cell does not express the engineered CD3 zeta subunit.

[0172] As used herein, the term "engineered CD3 zeta" is referred to as a mammalian CD3 zeta subunit-based polypeptide, or a functional portion or a functional variant thereof that still maintains the CD3 zeta functionality, i.e., to be incorporated into the TCR complex and mediate the TCR intracellular signaling. As such, according to some embodiments, the CD3 zeta comprises an amino acid sequence that has at least 80%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity to SEQ ID NO: 55.

[0173] In the engineered CD3 zeta subunit provided herein, each of the at least one costimulatory region may optionally comprise a co-stimulatory domain, or a functional portion or a functional variant thereof.

[0174] As used herein, the term "co-stimulatory domain", or "co-stimulatory signaling domain", is referred to as a specific functional portion of the engineered CD3 zeta subunit that is capable of recruiting certain intracellular signaling molecules to thereby confer the immunological cells at least one of the following capabilities including cytotoxicity, sternness (i.e. the capability to resist exhaustion), memory', persistence, etc. For example, the 4-1BB co-stimulatory domain contains binding motifs for, and therefore is capable of recruiting, tumor necrosis factor receptor-associated factors (TRAF) signaling adaptor proteins, thereby leading to increased T cell memory and persistence (12). In another example, the CD28 costimulatory domain contains binding motifs for, and thus is capable of recruiting, certain downstream signaling molecules such as phosphatidylinositol-3 -kinase (PI3K), growth factor receptor-bound protein 2 (Grb2), and lymphocyte-specific protein tyrosinekinase (Lek), thereby leading to more effective T cell killing but reduced long-term T cell persistence.

[0175] Herein, the co-stimulatory domain (or co-stimulatory signaling domain) can be from a natural costimulatory immune receptor such as CD28, 4-1BB, 0X40, CD2, CD27, CDS, ICAM-1, LFA-1, and ICOS. As such, according to some specific embodiments of the engineered CD3 zeta subunit, the at least one co-stimulatory region comprises a first co- stimulatory region, which comprises a co-stimulatory domain, or a functional portion or a functional variant thereof, of CD28, and as such, the first co-stimulatory region may comprise an amino acid sequence that has at least 80%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity to SEQ ID NO: 56. It is noted that the co-stimulatory domain may be from a non-natural source (i.e., artificially created or synthesized) and may contain engineered binding motifs for certain intracellular signaling molecules, which may be a combination of different binding motifs from different co-stimulatory domains of different immune receptors. Such sequences have been reported in K. G. Daniels et al., Science 10. 1126 / science.abq0225 (2022) and in WO2022173703 Al, whose disclosure is incorporated by reference in its entirety.

[0176] As used herein, the term "functional portion" is referred to as a portion of the co- stimulatory domain that may wholly or partially contain the functionalities of the co- stimulatory domain. For example, the functional portion may only contain one or more binding motifs for certain downstream signaling molecules of a know n co-stimulatory domain. The term "functional variant" is referred to as a sequence variant of the co- stimulatory domain, such as those containing sequence substitutions, deletions, insertions, transpositions, etc., yet the functionalities of the co-stimulatory domain are wholly or partially retained.

[0177] In the engineered CD3 zeta subunit provided herein, the at least one co-stimulatory region may have different locations relative to the CD3 zeta sequence, e.g., the intracellular signaling domain of the CD3 zeta. As used herein, the term "intracellular signaling region of the CD3 zeta" is referred to as a portion of the intracellular domain of the CD3 zeta subunit that is responsible for transducing the signal upon stimulation of the TCR complex, which ty pically include the three immunoreceptor tyrosine activation motifs (ITAMs). In some embodiments, one or more of the at least one co-stimulatory region is fused over the C- terminal of the intracellular signaling domain of the CD3 zeta. In some other embodiments, one or more of the at least one co-stimulatory region is between a transmembrane domain and the intracellular signaling domain of the CD3 zeta. In some other embodiments, a first subset of the at least one co-stimulatory region is fused over the C-terminal of the intracellular signaling domain of the CD3 zeta, and a second subset of the at least one co-stimulatory region is between a transmembrane domain and the intracellular signaling domain of the CD3 zeta.

[0178] There is no limitation to the number of the at least one co-stimulatory region in the engineered CD3 zeta subunit disclosed herein. For example, according to some embodiment, there is only one co-stimulatory region in the engineered CD3 zeta subunit, whereas according to some other embodiments, there are more than one co-stimulatory region in the engineered CD3 zeta subunit. In the latter case, the more than one co-stimulatory region may be from a same immune receptor, or from a different immune receptor.

[0179] It is noted that there is no limitation to the actual location of the co-stimulatory region within the engineered CD3z subunit, as long as the CD3z can functionally transduce the TCR signaling upon the specific antigen / MHC recognition by the engineered TCR complex. For example, the co-stimulatory region may be located between the transmembrane domain and the immunoreceptor tyrosine activation domain that contains three immunoreceptor tyrosine activation motifs (ITAMs) of the CD3z subunit.

[0180] In some embodiments, the co-stimulatory region is an intracellular signaling region, or a functional portion or functional variant thereof of a cell surface protein expressed in the T cells having a costimulatory functionality, i.e., being capable of providing co-stimulatory signals for the activation, survival, and / or proliferation of the T cells. Non-limiting examples of a co-stimulatory protein whose intracellular signaling domain or a functional portion thereof (e.g., a functional portion may comprise one or more signaling motifs) that can be used for engineering CD3 zeta subunit may include CD28, 4-1BB, LFA-1, CD4, C CD28, CD27, ICOS, HVEM, LIGHT, CD40L, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226. In some embodiments, the co-stimulatory region can be from CD28 or 4- IBB. The co-stimulatory region can optionally be fused to the CD3z without any linker, or optionally through a flexible linker having a length of 1-20 amino acid residues and comprising largely less bulky amino acid residues such as Glycine ("G") or Serine ("S").

[0181] In some embodiments, the Aspire-TCR substantially comprises an engineered targetrecognizing subunit configured such that when expressed in an immunological cell, the subunit is able to be incorporated into the Aspire-TCR complex and confer the specific targeting of the immunological cell against the target cells expressing the specific target molecules recognizable by the engineered target-recognizing subunit.

[0182] The engineered target-recognizing subunit comprises an extracellular domain, which comprises a target-recognizing region. As used herein, the term "target-recognizing region" is referred to as a portion of the engineered target-recognizing subunit within the extracellular domain thereof that can specifically recognize and bind to a target molecule (i.e., cognate binding partner) that is expressed on the target cells to thereby allow the engineered TCR complex to exert its cytotoxicity against the target cells. Herein optionally, the target-recognizing region may take an antibody scheme, and may comprise a single-chain variable fragment (scFv) or a single-domain antibody (sdAb or nanobody) that can specifically recognize and bind to an epitope of an antigen presented on the surface of the target cells that corresponds to the scFv or the sdAb (i.e., the target molecules). Under this scenario, any scFv or sdAb that can specifically target the target cell- enriched target molecules can be used as the target-recognizing region of the engineered CD3z subunit for the engineered TCR complex.

[0183] Optionally, the target-recognizing region may take a ligand scheme, and may comprise a ligand, or a functional portion or a functional variant thereof (herein, "functional" is defined as being capable of binding to the corresponding receptor), that can specifically recognize and bind to a cell-surface receptor corresponding thereto that is expressed on the surface of the target cells. Under this scenario, any ligand, or a receptor binding portion or a functional variant thereof, that can specifically target the target cell-enriched target molecules can be used as the target-recognizing region of the engineered target-recognizing subunit. Herein the ligand or a functional portion or variant thereof may be a natural ligand, but may also be a peptide that has been artificially identified or engineered.

[0184] In some embodiments, the engineered target-recognizing subunit that carries the aforementioned target-recognizing region may optionally be based on the backbone of any one of TCRa, TCRb, CD3g, CD3d, CD3e, or optionally be based on an engineered protein that can be incorporated into the TCR complex. Further optionally, the target-recognizing region can be in any location of the engineered target-recognizing subunit, as long as it is within the extracellular domain thereof. For example, the target-recognizing region can be located over the N-terminus of one of the above mentioned five subunits (i.e., TCRa, TCRb, CD3g, CD3d or CD3e). In some embodiments, the target-recognizing region (ligand) is fused with the CD3e via a GS-rich linker (i.e., "GS linker").

[0185] Herein, the engineered protein (i.e. the backbone protein based on which the targetrecognizing region is fused to or incorporated into) can comprise an extracellular domain that harbors the target-recognizing region, a transmembrane domain, and optionally an intracellular domain. Each of these domains may be from any of the TCR-CD3 complex subunits such as TCRa, TCRb, CD3g, CD3d, CD3e, and CD3z, or their combinations, or may be artificially engineered.

[0186] In one example of the backbone protein, the extracellular domain may comprise the extracellular domain of the CD3e subunit which is further fused to the target-recognizing region over its N-tenninus in the engineered target-recognizing subunit, the transmembrane domain may comprise the transmembrane domain of the CD3e subunit, and the intracellular domain may comprise the intracellular domain of the CD3z subunit.

[0187] In another example, the extracellular domain may comprise the extracellular domain of the CD3g subunit which is further fused to the target-recognizing region over its N- terminus, the transmembrane domain may comprise the transmembrane domain of the CD3e subunit, and the intracellular domain may comprise the intracellular domain of the CD3z subunit.

[0188] In yet another example, the extracellular domain may comprise the extracellular domain of the TCRa subunit which is further fused to the target-recognizing region over its N-terminus, the transmembrane domain may comprise the transmembrane domain of the CD3e subunit, and the intracellular domain may comprise the intracellular stimulating domain of the CD3z subunit fused with the intracellular stimulating domain of the CD3e subunit.

[0189] It is noted that there can exist further engineering to each of the extracellular domain, For example, the intracellular domain may comprise 0-10 intracellular signaling motifs (e.g. IT AMs), each from the CD3e, CD3g, CD3g, and CD3z, or as an engineered ITAM.

[0190] In some embodiments, the present disclosure provides a chimeric polypeptide (e.g., the “IL13(E13Y)-CD3e” fusion protein described herein), which comprises at least one target-recognizing region operably linked to or incorporated into one of TCR alpha, TCR beta, CD3 gamma, CD3 delta or CD3 epsilon, or a functional portion or a functional variant thereof. Herein the chimeric polypeptide is capable of being incorporated into a T cell receptor (TCR) complex when expressed in an immunological cell; each of the at least one target-recognizing region is within an extracellular domain; and the at least one targetrecognizing region comprises at least one ligand, or a fragment thereof, that binds to a cellsurface receptor expressed on a target cell of the immunological cell.

[0191] In some embodiments, the Aspire-TCR includes a ligand or a fragment thereof, that binds to a cell-surface receptor expressed on a target cell of the immune cell. In some embodiments, the ligand is selected from the group consisting of IL13 (E13Y), IL-13, IL-11, IL- 10, APRIL, GM-CSF, TPO, Adnectin, TIE, FLT3L, EPHRIN B2, CTLX, LFA-1, and FSH. In some embodiments, the ligand is IL13(E13Y), and the ligand may comprise an amino acid sequence that has at least 80%, such as 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity to SEQ ID NO: 57. In some embodiments, the at least one target- recognizing region is operably linked to or incorporated into CD3 epsilon or a functional portion or a functional variant thereof. Herein, the CD3 epsilon region comprises an amino acid sequence that has at least 80%, e.g. 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100%, sequence identity to SEQ ID NO: 58.

[0192] In some embodiments, the Aspire-TCR binds to or recognizes a peptide epitope from IL13Ra2. In some embodiments, the Aspire TCR encodes two fusion proteins: a first fusion protein connecting a IL13 (E13Y) ligand and a human CD3e component; and a second fusion protein connecting the human CD3z signaling domain (i.e., "CD3z") and the human CD28 co-stimulatory region (i.e., "CD28"). The first fusion protein and the second fusion protein can also be separated by a self-cleaving peptide (e.g., T2A or P2A). In some embodiments, the IL13 (E13Y) ligand and the human CD3e component are connected with a GS linker (SEQ ID NO: 59).

[0193] In some embodiments, the IL 13 (E13Y) ligand described herein includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57. In some embodiments, the human CD3e component descnbed herein includes an ammo acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58. In some embodiments, the human CD28 co-stimulatory region described herein includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56. In some embodiments, the human CD3z signaling domain includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55.

[0194] Co-expressing the target CARs / TCRs and the sustaining polypeptide

[0195] There is no limitation to the means of co-expressing the target CAR / TCR and the sustaining polypeptide (i.e. the polypeptide comprising the actuating c-Jun fragment) in the T cells. Optionally, the target CAR / TCR and the sustaining polypeptide can be expressed by means of a single vector (e.g. bicistronic vector) or of two distinct vectors. Each of the single vector or the two distinct vectors may include, but are not limited to, plasmid vectors, viral vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and human artificial chromosomes (HACs). Viral vectors can optionally include, but are not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, foamy virus vectors, recombinant adeno-associated viral (AAV) vectors, hybrid vectors, and plasmid transposons (e.g., sleeping beauty transposon system, and PiggyBac transposon system) or integrase based vector systems.

[0196] In certain embodiments, the target CAR / TCR and the sustaining polypeptide are coexpressed in the T cells by means of a single vector, which comprises two polynucleotide fragments respectively encoding the target CAR / TCR and the sustaining polypeptide that are separated from each other by sequences encoding a self-cleavage peptide (e.g., P2A or T2A) and / or a protease recognition site (e.g., furin).

[0197] In certain embodiments, the target CAR / TCR and the sustaining polypeptide are coexpressed in the T cells by means of distinct vectors.

[0198] Within the scope of the disclosure, the term “co-expression” or alike, can be deemed to cover any of the following scenarios: (1) the expression of the target CAR / TCR and the expression of the c-Jun containing sustaining polypeptide are simultaneous, e.g., these two polypeptides are in a single vector transduced, or are in two distinct vectors co-transduced, into the T cells; (2) the c-Jun containing sustaining polypeptide is expressed in the T cells prior to the target CAR / TCR, e.g., the T cells are infected with a retroviral vector encoding the c-Jun containing sustaining polypeptide to thereby obtain engineered T cells that are armored with the expression of the sustaining polypeptide, which then undergo further transfection of another vector encoding the target CAR / TCR to obtain engineered T cells that also express the target CAR / TCR; and (3) the target CAR / TCR is expressed in the T cells prior to the c-Jun containing sustaining polypeptide, e g., T cells that have already been engineered to express the target CAR / TCR are further engineered to express the c-Jun containing sustaining polypeptide.

[0199] According to certain embodiments, it is designed such that the engineered immunological cells realizes a controllable / regulatable expression of the c-Jun containing sustaining polypeptide to obtain a well-balanced risk / reward profile, which can be by means of suicide genes or inducible gene expression elements arranged in the vector encoding the sustaining polypeptide.

[0200] Regarding the suicide gene approach, one of the examples is iCaspase 9 system. When exposed to a synthetic dimerizing drug, the inducible caspase 9 becomes activated and leads to the rapid apoptosis of cells expressing the construct. Another example is therapeutic monoclonal antibody (mAb) mediated system. Protein overexpression allows elimination after exposure to mAb specific to the expressed protein through complement / antibody dependent cellular cytotoxicity (CDC / ADCC). Details can be found in Jones, Benjamin S., et al. "Improving the safety of cell therapy products by suicide gene transfer." Frontiers in Pharmacology 5 (2014): 254, which is incorporated herein by reference in its entirety. It is noted that the approach above shall only represent an illustrating and non-limiting example, and other suicide gene approaches may also be employed as well.

[0201] For example, it can be controlled such that: at an initial stage (Stage 1), expression of the sustaining polypeptide is turned on in the target T cells (e.g., by turning on the promoter or enhancer that regulates the expression of the sustaining polypeptide) so as to ensure that the engineered T cells have a better sustained expression of the target CAR / TCR that coexpresses therewith; and at a later stage (Stage 2), the expression of the sustaining polypeptide is turned off (e.g., by turning off the promoter or enhancer that regulate the expression of the sustaining polypeptide) to avoid the potential adverse effect of long-term c- Jun overexpression such as the unwanted c-Jun induced tumorigenesis. Alternatively, the engineered T cells are triggered to commit suicide (e.g., by inducibly expressing a suicide gene to induce apoptosis of the host cells) to totally remove the host engineered T cells from the subject receiving such cell therapy.

[0202] Anti- ALPP Antibodies and Antigen-Binding Fragments

[0203] Alkaline phosphatase, placental (ALPP), also known as placental alkaline phosphatase (PLAP) (NCBI GENE ID: 250), is a plasma membrane-localized enzyme with normal human tissue expression restricted to the placenta, cervix, and uterus. ALPP is a homodimer, membrane-associated glycoprotein enzyme. It belongs to a multigene family composed of four alkaline phosphatase isoenzymes. The enzyme functions as a homodimer and has a catalytic site containing one magnesium and two zinc ions, which are required for its enzymatic function. It plays an important role in the regulation of specific inflammatory disease processes. There are at least four distinct but related alkaline phosphatases: intestinal, placental, placental-like, and liver / bone / kidney. Placental Alkaline Phosphatase (ALPP) reacts with a membrane-bound isoenzyme (Regan and Nagao type) of ALPP occurring in the placenta during the third trimester of gestation. Placental Alkaline Phosphatase is useful in the identification of testicular germ cell tumors. Unlike germ cell tumors, ALPP-positive somatic cell tumors uniformly express epithelial membrane antigen (EMA).

[0204] Elevated ALPP expression is commonly found in ovarian, cervical, and testicular cancers. ALPP expression has also been observed in testicular seminoma, primary intracranial germinoma, epithelial ovarian carcinoma, ovarian adenocarcinoma, serous cystadenocarcinoma, undifferentiated carcinoma, dysgerminoma, uterus cancer, endometrial cancer, urothelial cancer, stomach cancer, lung cancer, pancreatic cancer, osteosarcoma, and gastric cancer. Because of its restricted expression pattern, ALPP can be considered as both a molecular marker and a therapeutic target for ALPP-positive cancers.

[0205] The disclosure relates to anti-ALPP CAR-T cell therapy for the treatment of cancer patients with ALPP-positive cancer. The present disclosure also provides antibodies or antigen binding fragments thereof that target ALPP. Specifically, the disclosure provides anti-ALPP antibodies F8, H5, C9, F7, and A3, and the humanized antibodies thereof.

[0206] The CDR sequences for F8 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 61-63, and CDRs of the light chain variable domain, SEQ ID NOs: 64-66. The CDR sequences for H5 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 67- 69, and CDRs of the light chain variable domain, SEQ ID NOs: 70-72. The CDR sequences for C9 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 73-75, and CDRs of the light chain variable domain, SEQ ID NOs: 76-78. The CDR sequences for F7 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 79-81, and CDRs of the light chain variable domain, SEQ ID NOs: 82-84. The CDR sequences for A3 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 85-87, and CDRs of the light chain variable domain, SEQ ID NOs: 88-90.

[0207] In some embodiments, the CDR is determined based on Chothia definition scheme. In some embodiments, the CDR is determined based on Kabat definition scheme. In some embodiments, the CDR is determined based on a combination of Kabat and Chothia definition scheme. In some embodiments, the CDR is determined based on IMGT definition. In some embodiments, the CDR is determined based on contact definition.

[0208] The amino acid sequences for the heavy chain variable region of the F8 antibody is set forth in SEQ ID NO: 91. The amino acid sequences for the light chain variable region of the F8 antibody is set forth in SEQ ID NO: 92. The amino acid sequences for the heavy chain variable region of the H5 antibody is set forth in SEQ ID NO: 93. The amino acid sequences for the light chain variable region of the H5 antibody is set forth in SEQ ID NO: 94. The amino acid sequences for the heavy chain variable region of the C9 antibody is set forth in SEQ ID NO: 95. The amino acid sequences for the light chain variable region of the C9 antibody is set forth in SEQ ID NO: 96. The amino acid sequences for the heavy chain variable region of the F7 antibody is set forth in SEQ ID NO: 97. The amino acid sequences for the light chain variable region of the F7 antibody is set forth in SEQ ID NO: 98. The amino acid sequences for the heavy chain variable region of the A3 antibody is set forth in SEQ ID NO: 99. The amino acid sequences for the light chain variable region of the A3 antibody is set forth in SEQ ID NO: 100. Any of these heavy chain variable region sequences (SEQ ID NOs: 91, 93, 95, 97, and 99) can be paired with any of these light chain variable region sequences (SEQ ID NOs: 92, 94, 96, 98, and 100).

[0209] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 61-63, SEQ ID NOs: 67-69, SEQ ID NOs: 73-75, SEQ ID NOs: 79-81, and SEQ ID NOs: 85-87; and / or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 64-66, SEQ ID NOs 70-72, SEQ ID NOs: 76-78, SEQ ID NOs 82-84, and SEQ ID NOs 88-90.

[0210] In some embodiments, the antibodies or antigen-binding fragments thereof can have a heavy chain vanable region (VH) comprising complementanty determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence. The antibodies or antigen-binding fragments thereof can further have a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR3 amino acid sequence. The selected VH CDRs 1 , 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in FIG. 18.

[0211] In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 61 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 62 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 63 with zero, one or two amino acid insertions, deletions, or substitutions.

[0212] In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 67 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 68 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 69 with zero, one or two amino acid insertions, deletions, or substitutions.

[0213] In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 73 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 74 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 75 with zero, one or two amino acid insertions, deletions, or substitutions.

[0214] In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 79 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 80 with zero, one or two ammo acid insertions, deletions, or substitutions; SEQ ID NO: 81 with zero, one or two amino acid insertions, deletions, or substitutions.

[0215] In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 85 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 86 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 87 with zero, one or two amino acid insertions, deletions, or substitutions.

[0216] In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 64 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 65 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 66 with zero, one or two amino acid insertions, deletions, or substitutions.

[0217] In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 70 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 71 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 72 with zero, one or two amino acid insertions, deletions, or substitutions. In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 76 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 77 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 78 with zero, one or two amino acid insertions, deletions, or substitutions.

[0218] In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 82 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 83 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 84 with zero, one or two amino acid insertions, deletions, or substitutions.

[0219] In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 88 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 89 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 90 with zero, one or two amino acid insertions, deletions, or substitutions.

[0220] The insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.

[0221] The disclosure also provides antibodies or antigen-binding fragments thereof that binds to ALPP. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 91, and the selected VL sequence is SEQ ID NO: 92. In some embodiments, the selected VH sequence is SEQ ID NO: 93, and the selected VL sequence is SEQ ID NO: 94. In some embodiments, the selected VH sequence is SEQ ID NO: 95, and the selected VL sequence is SEQ ID NO: 96. In some embodiments, the selected VH sequence is SEQ ID NO: 97, and the selected VL sequence is SEQ ID NO: 98. In some embodiments, the selected VH sequence is SEQ ID NO: 99, and the selected VL sequence is SEQ ID NO: 100.

[0222] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0223] The disclosure also provides nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin heavy chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs as shown in FIG. 18, or have sequences as shown in FIG. 19. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptides bind to ALPP.

[0224] The anti-ALPP antibodies or antigen-binding fragments thereof can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific), human antibodies, chimeric antibodies (e.g., human-mouse chimera), single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

[0225] Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody. Thus, a fragment of an antibody that binds to ALPP will retain an ability to bind to ALPP. An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site. Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. In some embodiments, the scFv described herein has a schematic structure of VH-linker peptide-VL. In some embodiments, the scFv described herein has a schematic structure of VL-linker peptide-VH. In some embodiments, the scFv can specifically bind to ALPP, and includes any of the VH / VL combination described herein. In some embodiments, the scFv includes an ammo acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 17, 18, 19, 20, or 21.

[0226] The present disclosure also provides an antibody or antigen-binding fragment thereof that cross-competes with any antibody or antigen-binding fragment as described herein. The cross-competing assay is known in the art, and is described e.g., in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp!20 exterior envelope glycoprotein." Journal of Virology 70.3 (1996): 1863-1872, which is incorporated herein reference in its entirety. In one aspect, the present disclosure also provides an antibody or antigen-binding fragment thereof that binds to the same epitope or region as any antibody or antigen-binding fragment as described herein. The epitope binning assay is known in the art, and is described e.g., in Estep et al. "High throughput solution-based measurement of antibody-antigen affinity and epitope binning." MAbs. Vol. 5. No. 2. Taylor & Francis, 2013, which is incorporated herein reference in its entirety.

[0227] Antibodies and Antigen Binding Fragments

[0228] The present disclosure provides various antibodies and antigen-binding fragments thereof derived from anti-ALPP antibodies described herein. In general, antibodies (also called immunoglobulins) are made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting examples of antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgEl, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. An antibody can comprise two identical copies of a light chain and two identical copies of a heavy chain. The heavy chains, which each contain one variable domain (or variable region, VH) and multiple constant domains (or constant regions), bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody. The light chains, which each contain one variable domain (or variable region, VL) and one constant domain (or constant region), each bind to one heavy chain via disulfide binding. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR).

[0229] These hypervariable regions, known as the complementary determining regions (CDRs), form loops that comprise the antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting the beta-sheet structure, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding region.

[0230] Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of the CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, e.g., Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839; Wu, T.T. and Kabat, E.A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68(l-3):9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2):269-94 (Jan .1998); Chothia et al.. Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of which is incorporated herein by reference in its entirety .

[0231] The CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.

[0232] In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgGl, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgGl, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are know n in the art, and are described, e.g., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions." Frontiers in immunology 5 (2014); Irani, et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecular immunology' 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

[0233] The antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, camelid). Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide. The term “antigen binding domain” or “antigen binding fragment” is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or an antigen binding fragment thereof can be, e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a singlechain antibody molecule, a multi-specific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain. Non-limiting examples of antigen binding domains include, e.g., the heavy chain and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.

[0234] Fragments of antibodies are suitable for use in the methods described herein are also provided. The Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CHI) of the heavy chain. F(ab')2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

[0235] Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a VH connected to a VL in the same poly peptide chain (VH and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0236] Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, fomr a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0237] Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.

[0238] Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgGi molecules) spontaneously form protein aggregates containing antibody homodimers and other higher- order antibody multimers.

[0239] Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane-l -carboxylate) and SATA (N-succinimidyl S-acethylthio-acetate) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997). Antibody homodimers can be converted to Fab’2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao et al. (J. Immunol. 25:396-404, 2002).

[0240] In some embodiments, the multi-specific antibody is a bi-specific antibody. Bispecific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., ty rosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96 / 27011, which is incorporated by reference in its entirety .

[0241] Bi-specific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

[0242] Any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution). Non-limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin). The conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human).

[0243] Tn some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxyanthracin, maytansinoids such as DM-1 and DM-4, di one, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycm, epirubicin, and cyclophosphamide and analogs).

[0244] In some embodiments, the antigen binding fragment can form a part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor are fusions of single-chain variable fragments (scFv) as described herein, fused to CD3-zeta transmembrane and endodomain. In some embodiments, the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors as described herein.

[0245] In some embodiments, the scFv has one heavy chain variable domain, and one light chain variable domain. In some embodiments, the scFv has two heavy chain variable domains, and two light chain variable domains.

[0246] In some embodiments, sequences (e.g., CDRs or VH / VL sequences) of the antibody or antigen-binding fragment thereof described herein can be used to generate a bispecific antibody targeting ALPP and an addition antigen.

[0247] In some embodiments, the antigen binding fragment described herein has a scFv-Fc structure. For example, an anti- ALPP scFv (e.g., any of the anti- ALPP scFv molecules described herein) can be connected to a human Fc (e.g., IgGl or IgG4 Fc).

[0248] Antibody Characteristics

[0249] In some implementations, the antibody (or antigen-binding fragments thereof) specifically binds to ALPP (e.g., human ALPP or monkey ALPP) with a dissociation rate (koff) of less than 0.1 s’1, less than 0.01 s’1, less than 0.001 s’1, less than 0.0001 s’1, or less than 0.00001 s’1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s’1, greater than 0.001 s’1, greater than 0.0001 s’1, greater than 0.00001 s’1, or greater than 0.000001 s’1.

[0250] In some embodiments, kinetic association rates (kon) is greater than 1 x 102 / Ms, greater than 1 x 103 / Ms, greater than 1 x 104 / Ms, greater than 1 x 105 / Ms, or greater than 1 x 106 / MS. In some embodiments, kinetic association rates (kon) is less than 1 x 105 / Ms, less than 1 x 106 / MS, or less than 1 x 107 / Ms.

[0251] Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff / kon). In some embodiments, KD is less than 1 x 10’6M, less than 1 x 10’7M, less than 1 x 10’8M, less than 1 x 10’9M, or less than 1 x 10’10M. In some embodiments, the KD is less than 50nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1 x 10’7M, greater than 1 x 10’8M, greater than 1 x 10’9M, greater than 1 x 10’1(1M, greater than 1 x 10’11M, or greater than 1 x io12M. In some embodiments, the antibody or antigen-binding fragment thereof described herein (e.g., any of the anti-ALPP scFv-Fc molecules described herein) can specifically bind to ALPP expressed on the surface of cells. In some embodiments, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof described herein (e.g., any of the anti-ALPP scFv-Fc molecules described herein) can be expressed on the surface of T cells, and activate T cells (e.g., as indicated by elevated expression level of intracellular IFN- y) when the CAR-T cells are co-cultured with target cells expressing ALPP. In some embodiments, the antibody or antigen-binding fragment thereof described herein (e g., any of the anti-ALPP scFv-Fc molecules described herein) can be stably purified, e.g., without apparent protein precipitation.

[0252] General techniques for measuring the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the affinity of the anti-ALPP antibodies described herein is determined by the Biacore assay.

[0253] In some embodiments, the antibody or antigen-binding fragment thereof described herein has a tumor growth inhibition percentage (TGLv%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibody has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI% can be determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the treatment starts, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 months after the treatment starts. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula:

[0254] TGI (%) = [l-(Ti-To) / (Vi-Vo)]xlOO%

[0255] Ti is the average tumor volume in the treatment group on Day i. To is the average tumor volume in the treatment group on Day zero. Vi is the average tumor volume in the control group on Day i. Vo is the average tumor volume in the control group on Day zero.

[0256] In some embodiments, the antibodies or antigen-binding fragments thereof as described herein are ALPP antagonist. In some embodiments, the antibodies or antigenbinding fragments thereof as described herein are ALPP agonist.

[0257] In some embodiments, the antibodies or antigen binding fragments can induce complement-dependent cytotoxicity (CDC) and / or antibody dependent cellular cytotoxicity (ADCC), and kill the tumor cell. In some embodiments, the antibodies or antigen binding fragments have a functional Fc region. In some embodiments, effector function of a functional Fc region is antibody -dependent cell-mediated cytotoxicity (ADCC). In some embodiments, effector function of a functional Fc region is phagocytosis. In some embodiments, effector function of a functional Fc region is ADCC and phagocytosis.

[0258] In some embodiments, the Fc region is human IgGl, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody is a human IgGl antibody.

[0259] In some embodiments, the antibodies or antigen binding fragments do not have a functional Fc region. For example, the antibodies or antigen binding fragments are Fab, Fab’, F(ab’)2, and Fv fragments. In some embodiments, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering), or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering). In some embodiments, the Fc have a SI mutation (S239D and I332E mutations in EU numbering).

[0260] Methods of Making Anti-ALPP Antibodies

[0261] The anti-ALPP antibodies or antigen-binding fragments thereof described herein can be developed using a ChemPamer human naive scFv phage library. First, 3-5 rounds of phage panning was performed. Specifically, each round of the propagation of the phage display library included the following steps: step 1: screening of phage display libraries against targets; step 2: wash away of non-binders; step 3: elution, infection, and lytic cycle of A’, coli; and step 4: amplification of binders. During this process, reinfection of E. coli was performed. After 3-5 rounds of panning, about 1000 clones were selected (single colonies were picked) and subject to primary screening by ELISA-based binding. 100-300 top binders were screened, and subject to secondary screening by ELISA- and FACS-based binding. In particular, the FACS-based binding assays used ALPP-293T cells and SiHa cells. Next, the screened binders were subject to IgG conversion, production, and purification. Next, 20-30 top binders were subject to IgG characterization, and the top 5 binders (i.e., F8, H5, C9, F7, and A3) were selected for subsequent experiments.

[0262] An isolated fragment of human ALPP can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised in animals by multiple injections (e g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times).

[0263] The full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens. The antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of ALPP and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein. As described above, the full length sequence of human ALPP is known in the art. In some embodiments, an Fc-tagged human ALPP protein (the Fc fusion protein contains all or a portion of human ALPP) is used as the immunogen.

[0264] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequences that make up the antigen-binding site of the antibody or an antigen-bmdmg domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein, e.g., ALPP. Any combination of deletions, insertions, and / or combinations can be made to arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target. The amino acid changes introduced into the antibody or antigen-binding fragment thereof can also alter or introduce new post-translational modifications into the antibody or antigenbinding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell), or introducing new glycosylation sites.

[0265] Additional modifications to the anti-ALPP antibodies or antigen-binding fragments thereof can be made. For example, a cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have any increased half-life in vitro and / or in vivo. Homodimeric antibodies with increased half-life in vitro and / or in vivo can also be prepared using heterobifunctional cross-linkers as described, for example, in Wolff et al. Wolff et al. ("Monoclonal antibody homodimers: enhanced antitumor activity in nude mice." Cancer research 53.11 (1993): 2560-2565). Alternatively, an antibody can be engineered which has dual Fc regions.

[0266] In some embodiments, a covalent modification can be made to the anti-ALPP antibody or antigen-binding fragment thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N- or C- terminal residues.

[0267] In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody composition may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A).

[0268] ENGINEERED CELLS

[0269] The present disclosure provides engineered cells (e.g., T cells) that comprise CAR, TCR, or antigen-binding fragment thereof, or other similar antigen-binding molecules as described herein. These engineered cells can be used to treat various disorders or disease as described herein (e.g., cancers).

[0270] In some embodiments, the engineered cells (e g., immune cells) that are armored to express the sustaining polypeptide to thereby realize a better sustaining of the expression of the target CAR / TCR molecules can be of any types, and can optionally be CD3+T cells (e.g., a combination of CD4+and CD8+T cells), CD8+T cells, CD4+T cells, natural killer (NK) T cells, alpha beta T cells, gamma delta T cells, memory T cells (e.g., central memory T cells or effector memory T cells, etc.), tumor infiltrating lymphocytes, or a combination thereof. It is noted that the present disclosure shall also cover other type of immune cells that can be engineered in a similar manner, which can include macrophages, natural killer cells, etc.

[0271] In various embodiments, the cell that is engineered can be obtained from, e.g., humans and non-human animals. In various embodiments, the cell that is engineered can be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pig or any other species. Preferably, the cell is from humans, rats or mice. More preferably, the cell is obtained from humans. In various embodiments, the cell that is engineered is a blood cell. Preferably, the cell is a leukocyte (e.g., a T cell), lymphocyte or any other suitable blood cell type. In some embodiments, the cell is a peripheral blood cell. In some embodiments, the cell is a T cell, B cell or NK cell.

[0272] In some embodiments, the cell is a T cell. In some embodiments, the T cells can express a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell. The cell surface receptor can be a wild type or recombinant T cell receptor (TCR), a chimeric antigen receptor (CAR), or any other surface receptor capable of recognizing an antigenic moiety that is associated with the target cell. T cells can be obtained by various methods known in the art, e.g., in vitro culture of T cells (e.g., tumor infiltrating lymphocytes) isolated from patients. TCR gene-modified T cells can be obtained by transducing T cells (e.g., isolated from the peripheral blood of patients), with a viral vector. In some embodiments, the T cell is a TCR gene-modified T cell. In some embodiments, the T cells are CD3+T cells, CD4+T cells, CD8+T cells, or regulatory T cells. In some embodiments, the T cells are T helper type 1 T cells and T helper type 2 T cells. In some embodiments, the T cell expressing this receptor is an a -T cell. In alternate embodiments, the T cell expressing this receptor is a y5-T cell.

[0273] In some embodiments, the cell is an NK cell. In some embodiments, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for introduction of the binding molecule, e.g., TCR, can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered. In some embodiments, the cells are stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells can be primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen. In some embodiments, the stem cells are cultured with additional differentiation factors to obtain desired cell types (e.g., T cells).

[0274] Different cell types can be obtained from appropriate isolation methods. The isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0275] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0276] Also provided are methods, nucleic acids, compositions, and kits, for expressing the binding molecules, and for producing the genetically engineered cells expressing such binding molecules. The genetic engineering generally involves introduction of a nucleic acid encoding the therapeutic molecule, e.g. TCR, CAR, e.g. TCR-like CAR, polypeptides, fusion proteins, into the cell, such as by retroviral transduction, transfection, or transformation. In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and / or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical application. In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors. In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), munne embryonic stem cell virus (MESV), murine stem cell virus (MSCV), or spleen focus forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In some embodiments, the vector is a lentivirus vector. In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation. In some embodiments, recombinant nucleic acids are transferred into T cells via transposition. Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome- mediated transfection; tungsten particle-facilitated microparticle bombardment and strontium phosphate DNA co-precipitation. Many of these methods are descried e.g., in WO2019195486, which is incorporated herein by reference in its entirety.

[0277] Also provided are populations of engineered cells, compositions containing such cells and / or enriched for such cells, such as in which cells expressing the binding molecule make up at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more percent of the total cells in the composition or cells of a certain type such as T cells, CD8+or CD4+cells.

[0278] RECOMBINANT VECTORS

[0279] The present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein), host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and / or a vector comprising the polynucleotide), and the production of recombinant polypeptides or fragments thereof by recombinant techniques.

[0280] As used herein, a “vector” is any construct capable of delivering one or more polynucleotide(s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide(s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and / or a poly -A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.

[0281] A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0282] The present disclosure provides a recombinant vector compnsmg a nucleic acid construct suitable for genetically modifying a cell, which can be used for treatment of pathological disease or condition.

[0283] Any vector or vector type can be used to deliver genetic material to the cell. These vectors include but are not limited to plasmid vectors, viral vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and human artificial chromosomes (HACs). Viral vectors can include but are not limited to recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, foamy virus vectors, recombinant adeno-associated viral (AAV) vectors, hybrid vectors, and plasmid transposons (e.g., sleeping beauty transposon system, and PiggyBac transposon system) or integrase based vector systems. Other vectors that are known in the art can also be used in connection with the methods described herein.

[0284] In some embodiments, the vector is a viral vector. The viral vector can be grown in a culture medium specific for viral vector manufacturing. Any suitable growth media and / or supplements for growing viral vectors can be used in accordance with the embodiments described herein.

[0285] In some embodiments, the vector used is a recombinant retroviral vector. A retroviral vector is capable of directing the expression of a nucleic acid molecule of interest. A retrovirus is present in the RNA form in its viral capsule and forms a double-stranded DNA intermediate when it replicates in the host cell. Similarly, retroviral vectors are present in both RNA and double-stranded DNA forms. The retroviral vector also includes the DNA form which contains a recombinant DNA fragment and the RNA form containing a recombinant RNA fragment. The vectors can include at least one transcriptional promoter / enhancer, or other elements which control gene expression. Such vectors can also include a packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used. Long terminal repeats (LTRs) are identical sequences of DNA that repeat many times (e.g., hundreds or thousands of times) found at either end of retrotransposons or proviral DNA fomied by reverse transcription of retroviral RNA. They are used by viruses to insert their genetic material into the host genomes. Optionally, the vectors can also include a signal which directs polyadenylation, selectable markers such as Ampicillin resistance, Neomycin resistance, TK, hygromycin resistance, phleomycin resistance histidinol resistance, or DHFR, as well as one or more restriction sites and a translation termination sequence. For example, such vectors can include a 5' LTR, a leading sequence, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3' LTR or a portion thereof. Additionally, retroviral vector used herein can also refers to the recombinant vectors created by removal of the retroviral gag, pol, and env genes and replaced with the gene of interest.

[0286] In some embodiments, the vector can include an additional nucleic acid encoding an inhibitory protein (e g., a checkpoint inhibitor). In various embodiments, the cell expresses the genetically engineered antigen receptor and the inhibitory protein. In various embodiments, the inhibitory protein is constitutively expressed.

[0287] In some embodiments, the vector or construct can contain a single promoter that drives the expression of one or more nucleic acid molecules. In some embodiments, such promoters can be multicistronic (bicistronic or tricistronic). For example, in some embodiments, transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows coexpression of gene products (e.g. encoding an alpha chain and / or beta chain of a TCR) by a message from a single promoter. Alternatively, in some cases, a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (e.g. encoding an alpha chain and / or beta chain of a TCR) separated from one another by sequences encoding a self-cleavage peptide (e.g., P2A or T2A) or a protease recognition site (e.g., furin). The ORF thus encodes a single polyprotein, which, either during (in the case of 2A e.g., T2A) or after translation, is cleaved into the individual proteins. In some cases, the peptide, such as T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream.

[0288] Various cell lines can be used in connection with the vectors as described herein. Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lecl3 CHO cells, and FUT8 CHO cells; PER.C6® cells; and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the binding molecule. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

[0289] The term “Linker” (L) or “linker domain” or “linker region” as used herein refer to an oligo- or polypeptide region from about 1 to 100 amino acids in length, which links together any of the domains / regions. Linkers can be composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers can be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. Linkers can be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (for example P2A, T2A), 2A-like linkers or functional equivalents thereof and combinations thereof. In some embodiments, the linkers include the picomaviral 2A-like linker, CHYSEL sequences of porcine teschovirus (P2A), Thosea asigna virus (T2A) or combinations, variants and functional equivalents thereof. Other linkers will be apparent to those of skill in the art and can be used in the methods described herein.

[0290] The present disclosure also provides a nucleic acid sequence comprising a nucleotide sequence encoding any of the CARs, TCRs, antigen binding fragments thereof, and / or TCR- derivied binding molecules (including e.g., functional portions and functional variants thereof, polypeptides, or proteins described herein). “Nucleic acid” as used herein can include “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained from natural sources, which can contain natural, non-natural or altered nucleotides. Furthermore, the nucleic acid comprises complementary DNA (cDNA). It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and / or substitutions. However, it can be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and / or substitutions.

[0291] The nucleic acids as described herein can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides. In some of any such embodiments, the nucleotide sequence is codon- optimized.

[0292] The present disclosure also provides the nucleic acids comprising a nucleotide sequence complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0293] In some embodiments, in addition to administering a subject with therapeutical immunological cells (e.g., therapeutical T cells) co-expressing the target CAR / TCR and the sustaining polypeptide, one or more additional therapeutic agents can be administered to the subject.

[0294] In some embodiments, the vector can additionally include a nucleic acid sequence that encodes a checkpoint inhibitor (CPI) (e.g., an inhibitory protein). In some embodiments, the checkpoint inhibitor is e.g., any antibody or antigen binding fragment thereof as described herein. In some embodiments, the antibody or antigen binding fragments thereof can specifically bind to PD-1, PD-L1, PD-L2, 2B4 (CD244), 4-1BB, A2aR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, BTLA, butyrophilins, CD 160, CD48, CTLA4, GITR, gp49B, HHLA2, HVEM, ICOS, ILT-2, ILT-4, KIR family receptors, LAG-3, OX-40, PIR-B, SIRPalpha (CD47), TFM-4, TIGIT, TIM-1, TIM-3, TIM-4, VISTA, or combinations thereof. In some embodiments, the inhibitor)' protein is a scFv (e.g., an anti-PD-1 scFv). More details can be found, e.g., in W02020036834A1, which is incorporated herein by reference in its entirety.

[0295] Optionally, the additional therapeutic agent can include one or more cytokines / chemokines, such as IL-12 (disclosed in WO2021233317A1) and IL-7 / CCL19 (disclosed in WO2017159736A1).

[0296] In some embodiments, the vector can additionally include a nucleic acid sequence that encodes a bifunctional trap fusion protein. In some embodiments, the bifunctional trap protein targets both the PD-1 and TGF-p. In some embodiments, the bifunctional trap protein targets both the PD-L1 and TGF-p. In some embodiments, the bifunctional fusion protein designed to block PD-L1 and sequester TGF-p. M7824 (MSB0011395C) comprises the extracellular domain of human TGF-P receptor II (TGFpRII) linked to the C-terminus of the human anti-PD-Ll scFv, based on the human IgGl monoclonal antibody (mAb) avelumab. In some embodiments, the bifunctional fusion protein comprises the extracellular domain of human TGF-P receptor II (TGFpRII) linked to the C-terminus of the human anti-PD-1 scFv.

[0297] In some of any such embodiments, the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that has been codon-optimized. In certain embodiments, the alpha and / or beta chain further comprises a signal peptide. In particular embodiments, the TCR or antigen-binding fragment thereof is isolated or purified or is recombinant. In some of any such embodiments, the TCR or antigen-binding fragment is recombinant. In some of any such embodiments, the TCR or antigen-binding fragment thereof is human.

[0298] The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence as described herein. In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein.

[0299] In some embodiments, the nucleic acid sequence is at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is at least or about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.

[0300] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0301] METHOD FOR PREPARATION OF ENGINEERED CELLS

[0302] The present disclosure provides a method or process for manufacturing and using the engineered cells for treatment of pathological diseases or conditions.

[0303] The cells for introduction of the binding molecule, e.g., CAR or TCR, can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered.

[0304] Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0305] In some aspects, the sample from which the cells are denved or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources. In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, or non-human primate.

[0306] In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated "flow-through" centrifuge. In some aspects, a washing step is accomplished by tangential flow filtration (TFF). In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca2+ / Mg2+free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media. In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0307] In some embodiments, the method comprises one or more steps of: e.g., isolating the T cells from a patient’s blood; transducing the population T cells with a viral vector including the nucleic acid construct encoding a genetically engineered antigen receptor; expanding the transduced cells in vitro; and / or infusing the expanded cells into the patient, where the engineered T cells will seek and destroy antigen positive tumor cells. In some embodiments, the nucleic acid construct further includes a sequence encoding an inhibitory protein. In some embodiments, these engineered T cells can block PD-1 / PD-L1 immunosuppression and strengthen the antitumor immune response. In some embodiments, the method further comprises: transfection of T cells with the viral vector containing the nucleic acid construct.

[0308] In some embodiments, the methods involve introducing any vectors described herein into a cell in vitro or ex vivo. In some embodiments, the vector is a viral vector and the introducing is carried out by transduction. In some embodiments, the methods further involve introducing into the cell one or more agent, wherein each of the one or more agent is independently capable of inducing a genetic disruption of a T cell receptor alpha constant (TRAC) gene and / or a T cell receptor beta constant (TRBC) gene. In some embodiments, the one or more agent is an inhibitory' nucleic acid (e.g., siRNA). In some embodiments, the one or more agent is a fusion protein comprising a DNA-targeting protein and a nuclease or an RNA-guided nuclease (e.g., a clustered regularly interspaced short palindromic nucleic acid (CRISPR)-associated nuclease).

[0309] The transfection of T cells may be achieved by using any standard method such as calcium phosphate, electroporation, liposomal mediated transfer, microinjection, biolistic particle delivery' system, or any other known methods by skilled artisan. In some embodiments, transfection of T cells is performed using the calcium phosphate method.

[0310] Methods of preparing engineered cells and administering these engineered cells to a subject are known in the art, and are described e.g., in US Pat. No. 10,174,098 and Draper et al. "Targeting Of HPV-16+ Epithelial Cancer Cells By Ter Gene Engineered t Cells Directed Against e6." Clinical Cancer Research 21.19 (2015): 4431-4439, both of which are incorporated by reference in their entirety.

[0311] METHODS OF TREATMENT

[0312] The methods disclosed herein can be used for various therapeutic purposes. In one aspect, the disclosure provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of a cancer. In some embodiments, the treatment can result in the reduction of in the number, severity, and / or duration of one or more symptoms of the cancer in a subject.

[0313] As used herein, the term “cancer” refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell grow th. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “tumor” as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the agents described herein are designed for treating or diagnosing a carcinoma in a subject. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells fomi recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[0314] In some embodiments, the cancer described herein is lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myeloproliferation abnormal syndromes, and sarcomas. In some embodiments, the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia. In some embodiments, the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom macroglobulinemia. In some embodiments, the sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma , and chondrosarcoma. In a specific embodiment, the tumor is non-small cell lung cancer, metastatic colorectal cancer, cervical cancer, ovarian cancer, nasopharyngeal cancer, gastric cancer, glioma.

[0315] In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer. Patients with cancer can be identified with various methods known in the art.

[0316] Furthermore, the disclosure provides methods for treating infection or infection associated conditions in a subject. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of the disease. These methods generally involve administering a therapeutically effective amount of genetic engineered cells disclosed herein to a subject in need thereof. In some embodiments, the disease or condition treated is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, immunodeficiency, Human Papilloma Virus (HPV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus.

[0317] As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the therapeutic agent and / or therapeutic compositions is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.

[0318] An effective amount can be administered in one or more administrations. By way of example, an effective amount of a composition is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and / or delay progression of a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and / or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)) in vitro. As is understood in the art, an effective may vary, depending on, inter alia, patient history as well as other factors such as the type (and / or dosage) of compositions used.

[0319] Effective amounts and schedules for administrations may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the treatment, the route of administration, the particular type of therapeutic agents and other drugs being administered to the mammal. Guidance in selecting appropriate doses can be found in the literature. In addition, a treatment does not necessarily result in the 100% or complete treatment or prevention of a disease or a condition. There are multiple treatment / prevention methods available with a varying degree of therapeutic effect which one of ordinary skill in the art recognizes as a potentially advantageous therapeutic mean.

[0320] In some aspects, the present disclosure also provides methods of diagnosing a disease / condition in a mammal, wherein the CAR, TCRs, antigen binding fragments, TCR- derived binding molecules interact with the sample(s) obtained from a subject to form a complex, wherein the sample can comprise one more cells, polypeptides, proteins, nucleic acids, antibodies, or antigen binding portions, blood, whole cells, lysates thereof, or a fraction of the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction thereof, wherein the detection of the complex is the indicative of presence of a condition in the mammal, wherein the condition is cancer, EBV infection, or EBV-positive premalignancy. Further, the detection of the complex can be in any number of way known in the art but not limited to, ELISA, Flow cytometery, Fluorescence in situ hybridization (FISH), Polymerase chain reaction (PCR), microarray, southern blotting, electrophoresis, Phage analysis, chromatography and more. Thus, the treatment methods can further include determining whether a subject can benefit from a treatment as disclosed herein, e.g., by determining whether the subject has EBV infection or EBV-associated cancer.

[0321] In any of the methods described herein, the engineered cells and, and / or at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different engineered cells (e.g., cells express different binding molecules) are administered in the same composition (e.g., a liquid composition). In some embodiments, engineered cells and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, engineered cells and the at least one additional therapeutic agent are administered in two different compositions. In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained-release oral formulation.

[0322] In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, concurrently with, or after administering the engineered cells to the subject.

[0323] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can be a checkpoint inhibitor (CPI). In some embodiments, the checkpoint inhibitor is an inh ibi lory protein, e.g., an antibody or antigen binding fragment thereof. The checkpoint inhibitor can inhibit or block one or more immune checkpoints, including e.g., PD-1, PD-LI, PD-L2, 2B4 (CD244), 4-IBB, A2aR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, BTLA, butyrophilins, CD160, CD48, CTLA4, GITR, gp49B, HHLA2, HVEM, ICOS, ILT-2, ILT-4, KIR family receptors, LAG-3, OX-40, PIR-B, SIRPalpha (CD47), TFM-4, TIGIT, TIM-1, TIM-3, TIM-4, VISTA and combinations thereof. In some embodiments, the inhibitory protein blocks PD-1 or PD-LI. In various embodiments, the inhibitory protein comprises an anti-PD-1 scFv. The inhibitory protein is capable of leading to reduced expression of PD-1 or PD-L1 and / or inhibiting upregulation of PD- 1 or PD-L1 in T cells in the population and / or physically obstructing the formation of the PD- 1 / PD-L1 complex and subsequent signal transduction. In some embodiments, the inhibitory protein blocks PD-1. In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody. In some embodiments, the additional therapeutic agent is an anti- CTLA4 antibody (e.g., ipilimumab), an anti-CD20 antibody (e.g., rituximab), an anti- EGFR antibody (e.g., cetuximab), an anti-CD319 antibody (e.g., elotuzumab), or an anti-PDl antibody (e.g., nivolumab).

[0324] In some embodiments, the additional therapeutic agent is a bifunctional trap fusion protein. Bifunctional trap proteins can target both immune checkpoints and TGF-P negative regulatory pathways. In addition to expression of immune checkpoints, the tumor microenvironment contains other immunosuppressive molecules. Of particular interest is the cytokine TGF-P (TGFB), which has multiple functions in cancer. TGF-P prevents proliferation and promotes differentiation and apoptosis of tumor cells early in tumor development. However, during tumor progression, tumor TGF-P insensitivity arises due to the loss of TGF-P receptor expression or mutation to downstream signaling elements. TGF-P then promotes tumor progression through its effects on angiogenesis, induction of epithelial- to-mesenchymal transition (EMT), and immune suppression. High TGF-P serum level and loss of TGF-P receptor (TGFPR) expression on tumors correlates with poor prognosis. TGFP- targeted therapies have demonstrated limited clinical activity. In some embodiments, the bifunctional trap protein targets both the PD-1 and TGF-p. In some embodiments, the bifunctional trap protein targets both the PD-L1 and TGF-p. In some embodiments, the bifunctional fusion protein designed to block PD-L1 and sequester TGF-p. M7824 (MSB0011395C) comprises the extracellular domain of human TGF-P receptor II (TGFpRII) linked to the C-terminus of the human anti-PD-Ll scFv, based on the human IgGl monoclonal antibody (mAb) avelumab. In some embodiments, the bifunctional fusion protein comprises the extracellular domain of human TGF-P receptor II (TGFpRII) linked to the C- terminus of the human anti-PD-1 scFv. These bifunctional trap fusion proteins are described e.g., Knudson, et al. "M7824, a novel bifunctional anti-PD-Ll / TGFp Trap fusion protein, promotes anti -tumor efficacy as monotherapy and in combination with vaccine." Oncoimmunology 7.5 (2018): el426519, which is incorporated herein by reference in its entirety . In some embodiments, the subject is treated by cells that express CAR / TCR or antigen-binding molecules as described herein and one or more bifunctional trap fusion proteins. More details can be found, e.g., in W02020118094A1, which is incorporated herein by reference in its entirety.

[0325] In one some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of anaplastic lymphoma kinase (ALK), an inhibitor of a phosphatidylinositol 3- kinase (PI3K), an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K / mTOR inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK), and an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and / or Isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-l) (IDO1) (e.g., epacadostat). In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.

[0326] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad- GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0327] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL- 10 antagonist, an IL-4 antagonist, an IL- 13 antagonist, an IL- 17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.

[0328] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject. In some embodiments, the additional therapeutic agent is selected from asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine and / or combinations thereof.

[0329] COMPOSITIONS AND FORMULATIONS

[0330] The present disclosure provides compositions (including pharmaceutical and therapeutic compositions) containing the engineered cells and populations thereof, produced by the methods disclosed herein. Also provided are methods, e.g., therapeutic methods for administrating the engineered T cells and compositions thereof to subjects, e.g., patients.

[0331] Compositions including the engineered T cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof are provided. The pharmaceutical compositions and formulations can include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.

[0332] A pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, other than an active ingredient. The pharmaceutically acceptable carrier does not interfere with the active ingredient and is nontoxic to a subject. A pharmaceutically acceptable carrier can include, but is not limited to, a buffer, excipient, stabilizer, or preservative. The pharmaceutical formulation refers to process in which different substances and / or agents are combined to produce a final medicinal product. The formulation studies involve developing a preparation of drug acceptable for patient. Additionally, a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0333] In some embodiments, the choice of carrier is determined in part by the particular cell (e.g., T cell or NK cell) and / or by the method of administration. A variety of suitable formulations are available. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0334] Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0335] The formulations can include aqueous solutions. The formulation or composition can also contain more than one active ingredient useful for a particular indication, disease, or condition being treated with the engineered cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition can further include other pharmaceutically active agents or drugs, such as checkpoint inhibitors, fusion proteins, chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine.

[0336] The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.

[0337] The cells and compositions can be administered using standard administration techniques, formulations, and / or devices. Administration of the cells can be autologous or heterologous For example, immunoresponsive T cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject after genetically modifying them in accordance with various embodiments described herein. Peripheral blood derived immunoresponsive T cells or their progeny (e g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. Usually, when administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it is generally formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0338] Formulations disclosed herein include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0339] The compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which can in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable earner, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or colors, depending upon the route of administration and the preparation desired. Standard texts can in some aspects be consulted to prepare suitable preparations.

[0340] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0341] The formulations to be used for in vivo administration are generally sterile. Sterility can be readily accomplished, e.g., by filtration through sterile filtrationmembranes.

[0342] The compositions or pharmaceutical compositions as described herein can be included in a container, pack, or dispenser together with instructions for administration.

[0343] EXAMPLES

[0344] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0345] Materials and Methods

[0346] CAR design. The anti-ALPP CARs were second-generation CARs cloned in retrovirus vector MP71. The MP71 retroviral vector construct containing two coding regions was generated using standard molecular biology techniques. The first coding region encodes a CAR that includes an anti-ALPP scFv, a CD8 hinge region, a transmembrane domain (either from CDS or CD4), a 4-1BB co-stimulatory domain, and a CD3z activation domain. The second coding region includes the c-Jun full gene sequences. The first and second coding regions are linked by a sequence encoding a 2A self-cleaving peptide (e.g., P2A).

[0347] Cell lines and media. HEK-293T, SKOV3, and SiHa cells were purchased from ATCC. Peripheral blood mononuclear cells (PBMCs) from anonymous donors were purchased from StemCell. SKOV3-ALPP cells were produced by retroviral transduction of SKOV3 cells with a vector overexpressing human ALPP protein. Cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% FBS (fetal bovine serum), RPMI supplemented with 10% FBS, or X-Vivo™ supplemented with 5% human serum A / B. 1% HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), and 1% GlutaMAX™.

[0348] Retroviral vector production. CAR retroviruses were generated by transfecting 293T cells with a ALPP CAR retrovirus construct, Gagpol and RD114 at a ratio of 4:2: 1.25 with lipofectamine™ 2000 (Thermo Fisher Scientific, Cat#: 11668500). Four hours after transfection, fresh medium was changed. 48 hours later, the medium containing viruses was collected and filtered with a 0.44 urn filter. The viruses were ready for transduction.

[0349] T cell transduction and expansion. Before retroviral transduction, PBMCs were activated with anti-human CD3 / CD28 Dynabeads® (Thermo Fisher Scientific, Cat#: 11141D) at a 3: 1 ratio (beads: T cells) for 2 days. For transduction, freshly harvested retroviral supernatant was spin-loaded onto non-tissue culture-treated 24-well plates coated with 15 pg RetroNectin® per / well (Clontech Laboratories) by centrifuging at 2,000 g for 2 hours at 32°C. Activated PBMCs were loaded onto the plates and spun at 600 g at 32°C for 20 minites. T cells were incubated at 37°C and 5% CO2. Culture medium was replenished every 2 days.

[0350] ALPP CAR staining. 0.2 x 106untransduced (UT) and transduced cells were collected for CAR level test. The cells were stained with 0. 1 pg ALPP-His protein (Novus Biologicals, Cat#: NBP2-52266) for 15 minutes at room temperature, washed once with PBS and stained with anti -His antibody for 15 minutes at room temperature. Afterwards, the cells were washed once with PBS, resuspended with PBS and ready for flow cytometry analysis.

[0351] Intracellular IFN-y staining. 0.2 x 106untransduced (UT) or ALPP CAR-T cells were co-cultured overnight with 0.4 x 106SiHa or 293T cells. Cells were then treated with Brefeldin A and Monensin for 4 hours, after which levels of intracellular IFN-y were measured by flow cytometry. Briefly, the cells were collected and cell surface staining was performed by adding antibodies and incubating at room temperature for 15 minutes. After surface staining, the cells were fixed with the Cytofix™ solution (BD, Cat#: 554714) at room temperature for 15 minutes, washed once with PBS, and permeabilized with 0.1 ml 1 x Perm / Wash buffer (BD, Cat#: 554714) for 10 minutes at room temperature. After washing once with 1 ml Perm / W sh buffer, the cells were stained with IFN-y antibody in 80 pL of Perm buffer at room temperature for 30 minutes, washed once with 1 ml of Perm buffer, and resuspended with PBS for flow cytometry analysis. Both CD8+and CD4+T cell populations were analyzed for IFN-y. Cytolytic toxicity assays. Cytolytic toxicity assays were performed in U-shape 96-well plate with four replicates for each condition. For each well, 0.03 * 106SiHa cells labeled with CellTrace™ CFSE (Thermo Fisher Scientific, Cat#: C34554) and 0.03 * 106293T cells labeled with CellTrace™ Violet (Thermo Fisher Scientific, Cat#: C34557) were mixed and co-cultured overnight with untransduced (UT) or ALPP CAR-T cells at increasing effector- to-target cell ratios. Live SiHa and 293T cells were quantified by flow cytometry by measuring the level of pre-labeled dyes, and competitive killing efficacy was calculated based on the live SiHa / 293T cell ratio.

[0352] Mice (intraperitoneal (i.p.) model). Six-eight weeks old female NSG mice (Jackson Laboratories) were housed and handled in accordance with the Institutional Animal Care and Use Committee (IACUC) protocol. Specifically, 17 female, 6-week-old NSG mice were intraperitoneally implanted with 5.0 / 10'’ SiHa cells in 200 pl PBS. 40 days later (or study Day 0), the animals were placed into groups based on body weights and the presence of clinical signs indicating tumor growth, and then intraperitoneally injected with 5 / I ()fiCARpositive A02, A02 eJun, or the equivalent number of untransduced cells (7.47 x I oVmouse). The endpoint tumor weights were recorded for different groups. Animals hit study endpoints as determined by death, moribundity, a severe decrease in body condition with a body conditioning score < 2, severe abdominal distension that interfered with animals’ ability to ambulate normally, and / or a body weight gain > 20%. On Day 40, all the mice were euthanized to end the whole study.

[0353] Mice (subcutaneous (s.c.) model). Six-eight weeks old female NSG mice (Jackson Laboratories) were housed and handled in accordance with the Institutional Animal Care and Use Committee (IACUC) protocol. Specifically, 13 female, 6-week-old NSG mice were subcutaneously implanted with 5.0 x io6SKOV3-ALPP cells in 100 pl PBS. 28 days later (or study Day 0), the animals were plated into groups based on tumor size with each group having approximately the same average size (about 55 mm3) and then intravenously injected with 1.0 x 107CAR-positive A02, A02 eJun, or the equivalent number of untransduced cells (1.96 x 107 / mouse). Caliper measurements were recorded twice a week for all animals for the duration of the study. The tumor volume was calculated with the formula: width2*length / 2.

[0354] Example 1: Effect of c-Jun co-expression on the expression of anti-ALPP CARs in the target T cells Four constructs (“A02”, “A02-cJun”, “A02-8H” and “A02-8H eJun”) were developed, with their respective schematic structures shown in FIG. 1. Each construct includes a coding region encoding an anti-ALPP CAR that includes, from N-terminus to C- terminus, an anti-ALPP antigen-binding fragment, a hinge region, a transmembrane domain (TM), a costimulatory signaling region, and an intracellular signaling region. The anti-ALPP antigen-binding fragment in each construct is identical, substantially including an anti-ALPP single-chain variable fragment (scFv) that has a heavy chain variable domain (VH; SEQ ID NO: 3) and a light chain variable domain (VL; SEQ ID NO: 5). In each construct, the hinge region, the costimulatory signaling region, and the intracellular signaling region are respectively the hinge region of CD8 (SEQ ID NO: 6), a functional signaling domain of 4- 1BB (SEQ ID NO: 8), and a functional activating cytoplasmic signaling domain of CD3 zeta (CD3z or CD3(^; SEQ ID NO: 9). The transmembrane domain (TM) can be either CD8 TM (SEQ ID NO: 7) for “A02” and “A02-cJun” constructs, or CD4 TM (SEQ ID NO: 15) for “A02-8H” and “A02-8H eJun” constructs. Relevant sequences are summarized in the table below.

[0355] Table 3. Sequences of anti-ALPP CARs

[0356] Details of the A02 and A02-8H CAR sequences can also be found, e.g., in U.S. Application Publication No. 20220125845A1, which is incorporated herein by reference in its entirety.

[0357] Each of the “A02-cJun” and “A02-8H eJun” constructs further includes another coding region expressing c-Jun, which is connected with the coding region expressing the anti-ALPP CAR by a sequence encoding a P2A self-cleaving peptide. Each of the four constructs can be cloned into a pMP71 retroviral vector to produce TCR-T cells.

[0358] Other anti-ALPP CAR-encoding constructs can also be made. For example, the anti- ALPP CARs encoded thereby may alternatively adopt a different version of scFv (i.e., F8, H5, C9, F7, and A3), while still sharing substantially the same leader sequence (SEQ ID NO: 2), CD8 hinge region (SEQ ID NO: 14), CD4 TM (SEQ ID NO: 15), 4- IBB costimulatory region (SEQ ID NO: 8), and CD3z cytoplasmic signaling domain (SEQ ID NO: 9).

[0359] For example, anti-ALPP CARs were generated with sequences summarized in the table below.

[0360] Table 4. Sequences of anti-ALPP CARs

[0361] Example 2: Surface expression of ALPP CAR during long-term in vitro CAR-T cell culture

[0362] In order to investigate the effects of c-Jun co-expression on the surface expression of ALPP CAR in CAR-T cells, primary human T cells were transduced with the indicated constructs. After transduction, CAR expression was measured at different timepoints by ALPP-His protein staining, using a viable lymphocyte gating strategy.

[0363] FIG. 2A shows the percentage expression of different ALPP CARs (“A02” and “A02-cJun”) in human T cells at different timepoints after transduction (Day 4, Day 12, and Day 19), and the percentages of ALPP CAR+cells over each timepoint are summarized and compared in the table below

[0364] Table 5A.

[0365] Note: * reference day; and ** “Normalized”, calculated by the raw data at any day other than the reference day relative to the raw data at the reference day minus 1.

[0366] The table shows that upon in vitro transduction, relative to the reference (i.e., Day 4), on Day 12, the percentage of the ALPP CAR+cells reduced by 11.8% in the “w / o c-Jun” group (i.e., T cells that co-express no c-Jun), whereas the percentage of the ALPP CAR+cells reduced by only 1.1% in the “w / c-Jun” group” (i.e., T cells co-expressing c-Jun). If based on scenario (1) as described herein: the drop in the latter case (1.1%) is far below the first predetermined threshold (set as 80% in certain embodiment) of the drop in the former case (11.8%), i.e., 9.4%. Actually the drop in the latter case (1.1%) is only about 9.3% of the drop in the former case (11.8%). Thus, the c-Jun co-expression significantly improved the sustaining of the surface expression of the target ALPP CAR at this time window (Day 4-12). If based on scenario (2) as described herein: ALPP CAR dropped in the former case, but remained substantially unchanged in the latter case (because the change 1. 1% is within 5%), thus the c-Jun co-expression significantly improved the sustaining of the surface expression of the target ALPP CAR at this time window (Day 4-12).

[0367] On Day 19, the percentage of the ALPP CAR+cells reduced by 25.2% in the “w / o c- Jun” group (i.e., T cells that co-express no c-Jun), whereas the percentage of the ALPP CAR+cells reduced by only 2.3% in the “w / c-Jun” group” (i.e., T cells co-expressing c-Jun). If based on scenario (1), the drop in the latter case (2.3%) is far below the first predetermined threshold (set as 80% in certain embodiment) of the drop in the former case (25.2%), i.e., 20.2%. Actually the drop in the latter case (2.3%) is only about 9.1% of the drop in the former case (25.2%). Thus, the c-Jun co-expression significantly improved the sustaining of the surface expression of the target ALPP CAR at this time window (Day 4-19). If based on scenario (2), ALPP CAR dropped in the former case, but remained substantially unchanged in the latter case (because the change 2.3% is within 5%), thus the c-Jun co-expression significantly improved the sustaining of the surface expression of the target ALPP CAR at this time window (Day 4-19).

[0368] Therefore, regardless of scenario (1) or (2), it can be concluded that, based on the parameter of the percentage of the ALPP CAR+cells, c-Jun can significantly sustain, or provide a better sustaining of, the surface expression of ALPP CAR in human primary T cells.

[0369] In addition to the above experiment to analyze the percentage of ALPP CAR+cells, the surface ALPP CAR expression level on each individual cell was also measured, by geometric mean fluorescence intensity (MFI) analysis, as shown in the table below.

[0370] Table 5B. Geometric MFI of surface staining data | (w / c-Jun) | Normalized** | 100% | -32.7% | -66,2% | Note: * reference day; and ** “Normalized”, calculated by the raw data at any day other than the reference day relative to the raw data at the reference day minus 1.

[0371] The table shows that upon in vitro transduction, relative to the reference (i.e., Day 4), on Day 12, the surface expression level of ALPP on the individual cell reduced by 60.3% in the “w / o c-Jun” group (i.e., T cells that co-express no c-Jun), whereas the surface expression level of ALPP on the individual cell reduced by 32.7% in the “w / c-Jun” group” (i.e., T cells co-expressing c-Jun). Thus, based on scenario (1) as described herein, the drop in the latter case (32.7%) is below the first predetermined threshold (set as 80% in certain embodiment) of the drop in the former case (60.3%), i.e., 48.2%. Actually the drop in the latter case (32.7%) is only about 54.2% of the drop in the former case (60.3%). Thus, the c-Jun coexpression significantly improved the sustaining of the surface expression of the target ALPP CAR at this time window (Day 4-12).

[0372] On Day 19, the surface expression level of ALPP on the individual cell reduced by 78.9% in the “w / o c-Jun” group (i.e., T cells that co-express no c-Jun), whereas the surface expression level of ALPP on the individual cell reduced by 66.2% in the “w / c-Jun” group” (i.e., T cells co-expressing c-Jun). Thus, based on scenario (1), the drop in the latter case (66.2%) is slightly higher than the first predetermined threshold (set as 80% in certain embodiment) of the drop in the former case (78.9%), i.e., 63.1%. Yet, the drop in the latter case (66.2%) is still below (about 83.9% of) the drop in the former case (78.9%). Thus, at this time window (Day 4-19), although the drop did not reach the first predetermined threshold of 80%, the results indicate a trend that c-Jun co-expression still improved the sustaining of the surface expression of the target ALPP CAR.

[0373] Because at least one time window (Day 4-12) showed that the c-Jun co-expression significantly improved the sustaining of the surface expression of the target ALPP CAR, it can be concluded that, based on the surface ALPP expression level on each individual cell as determined by geometric MFI of surface staining, c-Jun can significantly sustain, or provide a better sustaining of, the surface expression of ALPP CAR in human primary T cells.

[0374] In parallel, FIG. 2B further shows the percentage expression of different ALPP CARs (i.e. “8H” and “8H eJun”) in human T cells at different timepoints after transduction (Day 5, Day 7 and Day 20), and the percentages of ALPP CAR+cells over each indicated timepoint are summarized and compared in the table below.

[0375] Table 6 A.

[0376] Note: * reference day; and ** “Normalized”, calculated by the raw data at any day other than the reference day relative to the raw data at the reference day minus 1.

[0377] The table shows that upon in vitro transduction, relative to the reference (i.e., Day 5), on Day 7, the percentage of the ALPP CAR+cells increased barely by 0.8% in the “8H” group (i.e., T cells that co-express no c-Jun), whereas the percentage of the ALPP CAR+cells increased by 19.4% in the “8H c-Jun” group” (i.e., T cells co-expressing c-Jun). Thus, based on scenario (3) as described herein, the increase in the latter case (19.4%) is far greater than the second predetermined threshold (set as 120%) of the increase in the former case (0.8%), i.e., 0.96%. Thus, the c-Jun co-expression significantly improved the sustaining of the expression of the target ALPP CAR at this time window (Day 5-7).

[0378] On Day 20, the percentage of the ALPP CAR+cells increased by 25.8% in the “8H” group (i.e., T cells that co-express no c-Jun), whereas the percentage of the ALPP CAR+cells increased by 60.4% in the “8H c-Jun” group” (i.e., T cells co-expressing c-Jun). Thus, based on scenario (3) as described herein, the increase in the latter case (60.4%) is far greater than the second predetermined threshold (set as 120%) of the increase in the former case (25.8%), i.e., 30.96%. Thus, the c-Jun co-expression significantly improved the sustaining of the expression of the target ALPP CAR at this time window (Day 5-20).

[0379] Therefore, it can be concluded that, based on the parameter of the percentage of the ALPP CAR+cells, c-Jun can significantly sustain, or provide a better sustaining of, the surface expression of ALPP CAR in human primary T cells.

[0380] Table 6B. Geometric MFI of surface staining data

[0381] Note: * reference day; and ** “Normalized”, calculated by the raw data at any day other than the reference day relative to the raw data at the reference day minus 1.

[0382] The table shows that upon in vitro transduction, relative to the reference (i.e., Day 5), on Day 7, the surface expression level of ALPP on the individual cell reduced by 34.7% in the “8H” group (i.e., T cells that co-express no c-Jun), whereas the surface expression of ALPP on the individual cell level reduced by 1.5% in the “8H c-Jun” (i.e., T cells coexpressing c-Jun). Thus, based on scenario (1) as described herein, the drop in the latter case (1.5%) is below the first predetermined threshold (set as 80% in certain embodiment) of the drop in the former case (34.7%), i.e., 27.8%. Actually the drop in the latter case (1.5%) is only about 4.3% of the drop in the former case (34.7%). Thus, the c-Jun co-expression significantly improved the sustaining of the surface expression of the target ALPP CAR at this time window (Day 5-7).

[0383] On Day 20, the surface expression level of ALPP on the individual cell increased by 36.4% in the “8H” group (i.e., T cells that co-express no c-Jun), whereas the surface expression level of ALPP on the individual cell increased by 117.4% in the “8H c-Jun” group (i.e., T cells co-expressing c-Jun). Thus, based on scenario (3): the increase in the latter case (117.4%) is far greater than the second predetermined threshold (set as 120%) of the increase in the former case (36.4%), i.e., 43.7%. Thus, the c-Jun co-expression significantly improved the sustaining of the surface expression of the target ALPP CAR at this time window (Day 5- 20).

[0384] Therefore, it can be concluded that, based on the surface ALPP expression level on each individual cell as determined by geometric MFI of surface staining, c-Jun can significantly sustain, or provide a better sustaining of, the surface expression of ALPP CAR in human primary T cells.

[0385] Overall, it can be concluded that c-Jun significantly sustains, or provides a better sustaining of, the surface expression of ALPP CAR in human primary T cells.

[0386] Example 3: Phenotyping analysis of ALPP CAR-T cells

[0387] Primary human T cells were transduced with the indicated constructs. 26 days post transduction, memory T cell markers (CD45RO / CCR7) were measured by flow cytometry. A viable CD3+lymphocyte gating strategy was used. Both CD4+(FIG. 3A) and CD8+(FIG. 3B) populations were analyzed. The results show that A02 eJun CAR-T cells included more effector memory cells than the A02 CAR-T cells.

[0388] Example 4: ALPP CAR-T cell activation upon antigen-specific stimulation

[0389] Human PBMCs were transduced with A02, A02 eJun, A02-8H (short as “8H”) and A02-8H eJun (short as “8H eJun”) constructs. 7 days post-transduction, 0.2 x io6 untransduced (UT), A02, A02 eJun; or UT, 8H, 8H eJun CAR-T cells were co-cultured overnight with 0.4 * 106SiHa (target tumor cells) or 293T (non-target tumor cells) cells. Cells were then treated with Brefeldin A and Monensin for 4 hours, after which intracellular IFN-y was measured by flow cytometry. Both live CD8+(FIG. 4A and FIG. 4C) and CD4+(FIG. 4B and FIG. 4D) T cell populations were analyzed.

[0390] As shown in FIGS. 4A-4B, A02 eJun CAR-T cells could be better activated upon antigen-specific stimulation than the A02 CAR-T cells. As shown in FIGS. 4C-4D, 8H eJun CAR-T cells could be better activated upon antigen-specific stimulation than the 8H CAR-T cells.

[0391] Example 5: ALPP CAR-T cell activation upon repeated antigen-specific stimulation

[0392] Human PBMCs were transduced with A02, A02 eJun; or 8H, 8H eJun. 12 days post transduction, 0.2 x 106A02, A02 eJun (FIGS. 5A-5B) or 8H, 8H eJun (FIGS. 5C-5D) CAR- T cells were co-cultured with 0.1 x 106SiHa for 3 days, then with or without fresh SiHa cells overnight. Cells were then treated with Brefeldin A and Monensin for 4 hours, after which intracellular IFN-y was measured by flow cytometry. Both live CD8+(FIG. 5A and FIG. 5C) and CD4+(FIG. 5B and FIG. 5D) T cell populations were analyzed.

[0393] As shown in FIGS. 5A-5B, with multiple antigen-specific stimulations, A02 eJun could be better restimulated, compared to A02. As shown in FIGS. 5C-5D, with multiple antigen-specific stimulations, 8H eJun could be better restimulated, compared to 8H.

[0394] Example 6: In vitro proliferation of ALPP CAR-T cells upon repeated antigen-specific stimulation

[0395] Human PBMCs were transduced with A02, A02 eJun; or 8H, 8H eJun. 12 days post transduction, 1 x 106A02, A02 eJun (FIG. 6A) or 8H, 8H eJun (FIG. 6B) CAR-T cells were co-cultured with 0.5 x 106SiHa for 3 or 4 days, then the cells were counted and 1 x 106T cells were aliquoted and co-cultured with 0.5 x io6fresh SiHa cells for another 3 or 4 days, totally 4 times co-culture for 14 days. The cells were counted every time when each fresh coculture was set up and after the final co-culture. The expansion rate was calculated based on expansion ratio after each time point.

[0396] The results show that eJun armored CAR-T cells could proliferate and persist better than parental CAR-T in a longer-term culture, with or without antigen stimulation. Example 7: In vivo efficacy of ALPP CAR-T cell in subcutaneous mouse model

[0397] 13 female, 6-week-old NSG mice were subcutaneously (s.c.) implanted with 5.0 x io6SKOV3-ALPP cells in 100 pl PBS. 28 days later (or study Day 0), the animals were placed into groups based on tumor size with each group having approximately the same average size of about 55 mm3and then intravenously injected with 1.0 / I ()7CAR-positive A02, A02 eJun (“”cJun-A02”), or the equivalent number of untransduced cells (“NT”) (1.96 x 107 / mouse). Caliper measurements were recorded twice a week for all animals for the duration of the study. The tumor volume was calculated with the formula: width2*length / 2.

[0398] As shown in FIG. 7, while the A02 eJun CAR-T cells showed a significant antitumor effects compared with the control cells (i.e., NT cells), as demonstrated by the significantly lower tumor volumes over time (starting on Day 17) in the s.c. tumor model. However, the A02 CAR-T cells failed to show such an antitumor effect compared with the control cells (i.e., NT cells). Therefore, the A02 eJun CAR-T exhibited significant antitumor efficacy compared to A02 CAR-T.

[0399] If combined with the ALPP CAR surface expression data shown above (e.g. FIGS. 2A-2B and Tables 5A-5B and 6A-6B), it is contemplated that such a difference in the antitumor efficacy is correlated, and likely causally related, with whether the surface expression of ALPP CAR is sustained well or not. It is further hypothesized that this may represent a novel mechanism of tumor cell immune evasion under the CAR-T / TCR-T treatment. For example, the surface expression of CAR / TCR itself can be downregulated in the T cells over time and / or after repetitive antigen stimulation, such as the case with current ALPP CAR (i.e., A02); it can nonetheless be reversed by the co-expression of sustaining polypeptide comprising wildtype or certain mutant form of c-Jun fragment, which results in an improved sustained surface expression of ALPP CAR, in turn leading to an improved antitumor efficacy for the ALPP CAR-T therapy.

[0400] Example 8: In vivo efficacy of ALPP CAR-T cell in metastatic tumor mouse model

[0401] 17 female, 6-week-old NSG mice were intraperitoneally implanted with 5.0 x io6SiHa cells in 200 pl PBS. 40 days later (or study Day 0), the animals were placed into groups based on body weights and the presence of clinical signs indicating tumor growth, and then intraperitoneally injected with 5 x 106CAR-positive A02, A02 eJun, or the equivalent number of untransduced cells (7.47 x 106 / mouse). The endpoint tumor weights were shown for different groups. Animals hit study endpoints as determined by death, moribundity, a severe decrease in body condition with a body conditioning score < 2, severe abdominal distension that interfered with animals’ ability to ambulate normally, or a body weight gain > 20%. On Day 40, all the mice were euthanized to end the whole study.

[0402] As shown in FIG. 8, the results indicate that A02 eJun CAR-T can significantly improve antitumor efficacy in metastatic tumor model, compared to A02 CAR-T.

[0403] Example 9: Effects of c-Jun armoring to TCR-T cells

[0404] The engineered T cells expressing a TCR (e.g.„ an anti-NY-ESO-1 TCR) were tested to examine the effects of c-Jun (i.e. wildtype c-Jun or "eJun") on the TCR expression. Briefly, the non-treated T cells (i.e., "NT"), the NY-ESO-1 TCR-T cells (i.e., "NY-ESO-1 TCR"), and the NY-ESO-1 TCR-T cells armored with eJun WT (i.e., "NY-ESO-1 TCR.cJun") were expanded up to 28 days in vitro, and the expression of TCR was monitored (See FIG. 9A, where different time points were examined). The anti-NY-ESO-1 TCR described herein has a variable alpha (Va) region sequence set forth in SEQ ID NO: 12, and a variable beta (Vb) region sequence set forth in SEQ ID NO: 13.

[0405] As shown in FIG. 9A, in the absence of antigen stimulation, expression of NY-ESO-1 TCR on the engineered T cells cultured in vitro reduced over time (from about 75% on Day 6 to about 39% on Day 28), whereas the armoring of NY-ESO-1 TCR-T cells with coexpression of eJun WT can notably sustain the expression of NY-ESO-1 TCR on the engineered T cells (from about 79% on Day 6 to about 82% on Day 28). As further indicated by FIGS. 9B-9E, even though the armoring of eJun WT can effectively sustain (FIG. 9A) the TCR expression, there was no difference in terms of the expression of exhaustion-associated markers (LAG3, PD1 and Tim3) as measured by flow cytometry, indicating that armoring of TCR-T cells with c-Jun has no effect on the expression of exhaustion-associated markers. Thus, although previously it has been shown that c-Jun co-expression can reduce exhaustion of CAR-T cells, the effects of c-Jun armor on the TCR expression represent an independent effect from exhaustion.

[0406] Example 10: Effects of c-Jun variant armoring to TCR-T and CAR-T cells

[0407] A series of c-Jun variants, including wildtype c-Jun (indicated as "eJun"; SEQ ID NO: 37), and different c-Jun mutants, such as eJun bZIP vl (basic Leucine Zipper Domain; SEQ ID NO: 50), dJun 103-209 (i.e., eJun with deletion at positions 103-209; SEQ ID NO: 51), dJun 103-145 (i.e., eJun with deletion at positions 103-145; SEQ ID NO: 52), eJun AA (i.e., eJun with S63A and S73A substitutions; SEQ ID NO: 38), dJun 30-50 (i.e., eJun with deletion at positions 30-50; SEQ ID NO: 39), dJun 270-272 (i.e., eJun with deletion at positions 270-272; SEQ ID NO: 46), were tested for their respective effects on the expression of the engineered TCR / CAR (anti-NY-ESO-1 TCR / ALPP CAR), under regular culturing conditions or under antigen stimulation and re-stimulation conditions, with the latter more mimicking the in vivo condition. These eJun mutants, with respective truncation / deletion / substitutions, correspond to the alternations of different functional domains of human c-Jun (FIG. 10).

[0408] The effects of the above eJun variants over TCR and CAR were tested (see FIGS. 11A-11C). As shown in FIG. 11A, in the absence of any eJun annor (i.e., as indicated by "TCR"), the expression of the TCR reduced over time (from about 75% on Day 6 to about 40% on Day 28), yet among the various eJun variants tested, only eJun WT (i.e., "TCR.cJun"), dJun 30-50 (i.e., "TCR.dJun 30-50"), and eJun AA (i.e., "TCR.cJun AA") could rescue the reduction of the TCR expression. As further shown in FIG. 11B where the CAR-T cells were in a regular culturing condition without antigen stimulation, although the expression of the CAR increased over time (from about 50% on Day 6 to about 70% on Day 28) in the absence of any eJun armor (i.e., as indicated by "CAR"), dJun 30-50 (i.e., "CAR.dJun 30-50") could additively increase the CAR expression by about 20%. Such an additive effect on the CAR expression could also been observed even in the presence of antigen-stimulation and re-stimulation, when the armoring of CAR-T cells with dJun 30-50 could contribute additional 20-40% to the CAR expression (FIG. 11C). However, the other two eJun variants, dJun 270-272 and dJun 103-145 failed to potentiate the CAR expression in these CAR-T cells. The investigation over the activation of CAR-T cells, with the secretion of interferon-y ("IFN-y") as readouts (FIG. 11D), surprisingly indicated that the CAR expression-potentiating dJun 30-50 caused high antigen-independent activation of the CAR-T cells, i.e., high basal activation. Such an effect can be unfavored because the high level of antigen-independent activation or the high level of leaked activation may cause the unwanted off-target toxicity of such engineered CAR-T cells when administered to the subject in need thereof.

[0409] Example 11: Design of c-Jun variant armored CAR-T cells that have low basal activation and increased cytolytic toxicity against target cells Due to the unwanted basal antigen-independent activation observed for the CAR-T cells armored with dJun 30-50, efforts have been taken to explore other c-Jun variants that have better effects on the engineered CAR-T or TCR-T cells. In this example, two c-Jun variants: (1) eJun deletion mutant at positions 34-47 (indicated as "dJun 34-47"; SEQ ID NO: 53), and (2) eJun deletion mutant at 34-47 further with S63A and S73A substitutions (indicated as "dJun 34-47 AA"; SEQ ID NO: 54), were designed and tested over their respective effects on the expression of engineered CAR (e.g., an anti-ALPP CAR) in the engineered T cells, with the eJun WT in Example 9 and dJun 30-50 in Example 10 as comparison references.

[0410] Briefly, the non-treated T cells (i.e., "NT"), the ALPP CAR-T cells (i.e., "CAR"), and the ALPP CAR-T cells respectively armored with eJun WT ("CAR.cJun"), eJun deletion mutant at position 34-47 ("CAR.dJun 34-47"), eJun deletion mutant at position 34-47 further with S63A and S73A substitutions ("CAR.dJun 34-47 AA"), or eJun deletion mutant at positions 30-50 ("CAR.dJun 30-50") were expanded up to 26 days in vitro, and the expression of the ALPP CAR was monitored see FIG. 12A, where different time points were examined); and independently the above cells were repeatedly stimulated by tumor target cells, and the CAR expression was further measured (FIG. 12B). As demonstrated by FIG. 12A, in this particular case, even in the absence of antigen stimulation, the ALPP CAR expression can still be sustained very well, which notably increased from about 50% on Day 14 to about 70% on Day 26. The armoring of each of the four c-Jun variants can additively increase the expression of ALPP CAR, with the effects of eJun WT, dJun 34-47 and dJun 34- 47 AA being much more pronounced (i.e., about 20% more if estimated by the curves) than that of dJun 30-50 (i.e., about 10-15% more if estimated by the curves). In the presence of antigen stimulation and more in wvo-mimicking re-stimulation (FIG. 12B), whereas the ALPP CAR expression was increased by 1st antigen stimulation (from about 55% to about 62%), the ALPP CAR expression was notably dramatically decreased by 2nd antigen stimulation (reduced to about 10% after the 2nd stimulation), indicating that the ALPP CAR expression could be dramatically reduced or lost by antigen re-stimulation. The armoring of ALPP CAR-T cells with dJun 30-50 notably could result in only limited rescue (to about 30% after 2nd stimulation). In contrast, the armoring with other c-Jun variants (i.e., WT, dJun 34- 47 and dJun 34-47 AA) could dramatically sustain the expression of ALPP CAR on the engineered T cells (i.e., all > 70% after the 2nd stimulation). Thus, compared with the dJun 30-50, the newly designed dJun 34-47 and dJun 34-47 AA represent two better armor in terms of the effect on ALPP CAR expression, especially under the antigen-restimulation condition. It is noted that this result is somewhat unexpected, because both the deletion of positions 34-47 in dJun 34-47 and dJun 34-47 AA and the deletion of positions 30-50 are expected to similarly abrupt the functional delta domain (at positions 31-59, see FIG. 10) of c-Jun and are thus expected to have similar effects to the ALPP CAR expression.

[0411] The effects of the various c-Jun variants on the activation of the ALPP CAR-T cells were examined. Briefly, eJun WT or mutants armored CAR-T cells were stimulated (FIG. 12C) or repeated stimulated (FIG. 12D) by tumor target cells, and the CAR-T cells were cocultured with or without tumor target cells for 48 hours, after which the supernatant was collected and IFN-y ELISA was measured. The expression level of T cell activation marker 4-1BB was measured in CD8 (FIG. 12E) and CD4 (FIG. 12F) CAR-T cells without antigen stimulation. As shown in FIGS. 12C-12D, compared with the CAR-T cells armored with wildtype c-Jun, the CAR-T cells armored with either the dJun 34-47 or the dJun 34-47 AA showed favorably reduced level of basal activation (antigen-independent secretion of IFN-y) and better responsiveness. Specifically, under the unstimulation situation (FIG. 12C), in the absence of target cells, armoring of the CAR-T cells with either the dJun 34-47 or the dJun 34-47 AA exhibited only 1 / 2 basal activation compared with those armored with WT c-Jun; whereas upon antigen stimulation (i.e., in the presence of target SiHa cells), the IFN-y secretion is more responsive in CAR-T cells armored with dJun 34-47 (26 / 0.45=57.8 fold) and CAR-T cells armored with dJun 34-47 AA (21 / 0.4=52.5 fold) than in CAR-T cells armored with WT eJun (31 / 1=31 fold) group. Under the re-stimulation (FIG. 12D), in the absence of target cells, armoring of the CAR-T cells with either the dJun 34-47 or the dJun 34-47 AA exhibit only 1 / 4 basal activation compared with those armored with WT c-Jun; whereas upon antigen stimulation (i.e., in the presence of target SiHa cells), the IFN-y secretion is more responsive in CAR-T cells armored with dJun 34-47 (25 / 0.4=62.5 fold) and dJun 34-47 AA (29 / 0.5=58 fold) than in the CAR-T cells armored with WT c-Jun (36 / 1.4=25.7 fold). As further shown in FIGS. 12E-12F, in both CD4 and CD8 cells, the expression level of the T cell activation marker 4-1BB in CAR-T cells armored with dJun 34- 47 was only 1 / 3 of that of the CAR-T cells armored with WT eJun, and the 4- IBB level in the CAR-T cells armored with dJun 34-47 AA was only 1 / 4 of that of the CAR-T cells armored with WT eJun. Thus, compared to eJun WT, dJun 34-47 or dJun 34-47 AA armored CAR-T cells showed much lower basal T cell activation. The cytolytic activities of CAR-T cells against target tumor cells were further investigated. As shown in FIG. 12G, without antigen repetitive stimulation, CAR-T cells armored with dJun 34-47 or dJun 34-47 AA showed higher cytolytic toxicity compared with CAR-T cells armored with eJun WT. As shown in FIG. 12H, under antigen repeated stimulation condition, CAR-T cells armored with dJun 34-47 also showed higher cytolytic toxicity compared with CAR-T cells armored with eJun WT. Y et due to the missing of data points, no conclusion can be reached for the cytotoxic effect of dJun 34-47 AA.

[0412] Taken these above results together, at least in this ALPP CAR-T model, CAR-T cells armored with the newly designed dJun 34-47 or dJun 34-47 AA showed sustained CAR expression, exhibited relatively low basal activation and good antigen responsiveness, and had increased cytolytic activity against target cells.

[0413] Example 12: Effects of c-Jun variant armoring to Aspire-T cells

[0414] This example utilizes a different model, i.e., Antigen-specificity redirected TCR complex (i.e. Aspire-TCR or Aspire-T) model, for the examination of the effects of the various c-Jun variants, including the eJun WT, dJun 34-47 and dJun 34-47 AA, on the expression of Aspire-TCR in, and on the expansion capabilities of, such engineered Aspire-T cells. More specifically, T cells were engineered to express from a "CD3e-28z" construct, which substantially encodes two fusion proteins: a first fusion protein connecting a IL13 (EBY) ligand (SEQ ID NO: 57) and a human CD3e component (SEQ ID NO: 58); and a second fusion protein connecting the human CD3z signaling domain (i.e., "CD3z") (SEQ ID NO: 55) and the human CD28 co-stimulatory region (i.e., "CD28") (SEQ ID NO: 56). The IL 13 (EBY) ligand and the human CD3e component are connected with a GS linker (SEQ ID NO: 59). The first fusion protein and the second fusion protein are also separated by a 2A self-cleaving peptide "T2A," which can also be expressed as "IL13(E13Y)-CD3e || CD3z- CD28_CS." Such an engineered Aspire-TCR can specifically target IL-13Ra2 (i.e., IL13Ra2), which has been established as a specific cell-surface marker for gliomas and renal cell carcinomas (RCC).

[0415] Briefly, eJun WT or mutants armored IL 13 (E13Y) Aspire-T cells were expanded up to 29 days in vitro, and IL13 (EBY) Aspire-TCR expression (FIGS. 13A-13B) and the Aspire-TCR T cell expansion fold (FIG. 13C) were monitored. As demonstrated in FIG. 13A, whereas the expression of the engineered IL13 (EBY) Aspire-TCR reduced over time (from about 80% on Day 6 to about 50% on Day 29), all c-Jun variants tested, including the wildtype c-Jun, and the dJun 34-47 and dJun 34-47 AA mutants, could well sustain the expression of the engineered IL13 (EBY) Aspire-TCR (from about 65-80% on Day 6 to about 75-85% on Day 29). As further demonstrated in FIG. 13B, expression of the engineered IL 13 (EBY) Aspire-TCR in the engineered Aspire T cells reduced by about 28% in the absence of any c-Jun armor; the armoring of the IL13 (EBY) Aspire-T cells with eJun WT could sustained the IL13 (EBY) Aspire-TCR expression, causing a reduction of only < 3%; it is further noteworthy that the armoring of the IL 13 (E13Y) Aspire-T cells with the above two c-Jun deletion mutants (i.e., dJun 34-47 and dJun 34-47 AA) could even cause the expression of IL 13 (EBY) Aspire-TCR in the engineered Aspire T cells to increase by about 10%. As shown in FIG. 13C, compared with the Aspire-T cells in the absence of any c-Jun armor, the co-expression of the wild type c-Jun actually reduced the expansion of the engineered Aspire-T cells, yet noteworthily the co-expression of the two tested c-Jun mutants (i.e., dJun 34-47 and dJun 34-47 AA) can cause a much better Aspire-T cell expansion. As shown in FIG. 13D, compared with the Aspire-T cells without any armors, the Aspire-T cells armored with wild type c-Jun exhibited a high antigen-independent activation of the Aspire-T cells, i.e., high basal activation, but the two tested c-Jun mutants (i.e., dJun 34-47 and dJun 34-47 AA) favorably only displayed a slightly higher basal activation.

[0416] Taken the above results together, at least in this IL13 (EBY) Aspire-TCR-T model, the armoring of such engineered Aspire-T cells with wildtype c-Jun can sustain the expression of IL 13 (EBY) Aspire-TCR in, but may also reduce the expansion capability and cause a high basal activation of, the engineered Aspire-T cells. However, the armoring with either the dJun 34-47 or dJun 34-47 AA can even better sustain the Aspire-TCR expression in, cause a better expansion capability, and yet cause only a slight basal activation of, the engineered Aspire-T cells.

[0417] Sequences of some c-Jun variants used herein are summarized in the table below.

[0418] Table 7.

[0419] Example 13: Anti-hALPP scFv-Fc fusion proteins binding to human ALPP-expressing cells

[0420] A serial dilution anti-hALPP (human ALPP) scFv-Fc fusion proteins, including C9 scFv-Fc (“C9”), F7 scFv-Fc (“F7”), F8 scFv-Fc (“F8”), H5 scFv-Fc (“H5”), and A3 scFv-Fc (“A4”), were incubated with either mammalian cells expressing human ALPP (e.g., 293T cells expressing human ALPP (hALPP-293T) or SiHa cells), or 293T cells overexpressing a human ALPP isoform (e g., human Alkaline phosphatase, Liver / Bone / Kidney type (ALPL) or human Alkaline phosphatase, Intestinal type (ALPI)). Flow cytometry was used to measure the scFv-Fc fusion proteins bound to the cell surface. Parental 293T cells were used as a control.

[0421] As shown in FIGS. 14A-14B, the five scFv-Fc fusion proteins exhibited specific binding to ALPP-293T and SiHa cells, respectively. As shown in FIGS. 14C-14E, the five scFv-Fc fusion proteins did not bind to hALPL-293T, hALPI-293T, or parental 293T cells. In particular, C9, F8, and H5 scFv-Fc fusion proteins showed the relatively higher binding to ALPP expressed on cell surface compared with F7 and A3 sc-Fv-Fc fusion proteins.

[0422] Example 14: Anti-hALPP scFv-Fc fusion proteins binding to human ALPP protein

[0423] The binding affinity of the scFv-Fc fusion proteins to human ALPP protein was determined by the Biacore assay. Specifically, a serial of 10-fold diluted His-tagged human ALPP protein were injected over the surface captured with scFv-Fc fusion proteins and binding affinity was evaluated against calibration curve. As shown in the table below, F8 scFv-Fc was determined to have the lowest KD value in the Biacore assay, indicating that F8 scFv-Fc had the highest binding affinity to the ALPP protein.

[0424] Table 8.

[0425] Example 15: Expression of different ALPP CARs in human T cells

[0426] Primary human T cells were transduced with the constructs to express the anti-ALPP CARs shown in Table 4. After transduction, CAR expression was measured at indicated time points (Day 4 and Day 11) by ALPP-His protein staining. A viable lymphocyte gating strategy was used. As shown in FIGS. 15A-15D, all CARs could be expressed on T cell surface. In particular, A3 CAR, F8 CAR, and H5 CAR were more stable as compared to C9 CAR or F7 CAR.

[0427] Example 16: Functional validation of F8 and H5 CARs by IFN-y expression assays

[0428] The ability of the F8 and H5 CARs to activate T cells were verified by IFN-y expression assays. Specifically, human PBMCs were transduced with the F8 CAR or H5 CAR. 4 days post-transduction, 0.2 x 106untransduced (UT), the F8 or H5 CAR-T cells were co-cultured overnight with 0.4 * 106SiHa (target tumor cells) or 293T cells (non-target tumor cells). Cells were then treated with Brefeldin A and Monensin for 4 hours, after which levels of intracellular IFN-y were measured by flow cytometry. Live CD8+T cell populations were analyzed. As shown in FIGS. 16A-16C, F8 CAR-T cells showed better T cell activity against ALPP-expressing tumor cells, as compared to H5 CAR-T cells.

[0429] Example 17: SDS-PAGE results of scFv-Fc fusion proteins

[0430] The expression of scFv-Fc fusion proteins C9, F7, F8, H5, and A3 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). As shown in FIGS. 17A-17B, the F7 scFv-Fc fusion protein (Lane 2 in FIG. 17A and Lane 7 in FIG. 17B) showed a low expression level. The results indicate that the F7 scFv-Fc fusion protein was unstable and easy to precipitate. By contrast, the other four scFv-Fc fusion proteins can be successfully expressed, with a single major band in both non-reduced and reduced conditions.

[0431] OTHER EMBODIMENTS

[0432] Embodiment 1.1 A method for improving the surface expression of a target chimeric antigen receptor (CAR) or a target T cell receptor (TCR) in immunological cells, comprising: co-expressing in the immunological cells a sustaining polypeptide comprising a c-Jun fragment, such that the immunological cells co-expressing the sustaining polypeptide have an improved sustaining of the surface expression of the target CAR or the target TCR compared to when the sustaining polypeptide is absent.

[0433] Embodiment 1.2 A method for improving an expression of a target chimeric antigen receptor (CAR) or a target T cell receptor (TCR) in immunological cells, comprising: co- expressing in the immunological cells a sustaining polypeptide comprising a c-Jun fragment, such that after transduction of the target CAR or the target TCR into the immunological cells, any one of the scenarios as set forth below is met: (1) surface expression of the target CAR or the target TCR drops both when the sustaining polypeptide is not co-expressed and when the sustaining polypeptide is coexpressed, wherein the drop of the expression at a later timepoint relative to an earlier reference timepoint when the sustaining polypeptide is co-expressed is less than or equal to a first predetermined threshold of the drop when the sustaining polypeptide is not co-expressed, wherein the first predetermined threshold is a percentage that is no greater than 80%, no greater than 70%, no greater than 60%, or no greater than 50%; or

[0434] (2) surface expression of the target CAR or the target TCR drops when the sustaining polypeptide is not co-expressed, but remains substantially unchanged or increases when the sustaining polypeptide is co-expressed.

[0435] Embodiment 1.3 The method of embodiment 1.2, wherein in the step of coexpressing in the immunological cells a sustaining polypeptide comprising a c-Jun fragment, the scenarios further comprise:

[0436] (3) surface expression of the target CAR or the target TCR remains substantially unchanged or increases when the sustaining polypeptide is not co-expressed, but increases when the sustaining polypeptide is co-expressed, wherein the increase of the expression at a later timepoint relative to an earlier reference timepoint when the sustaining polypeptide is co-expressed is greater than or equal to a second predetermined threshold of the increase when the sustaining polypeptide is not co-expressed, wherein the second predetermined threshold is a percentage that is no less than 120%, no less than 130%, no less than 140%, or no less than 150%.

[0437] Embodiment 1.4 The method of any one of embodiments 1. 1-1.3, wherein the expression is evaluated by the percentage of the target CAR / TCR+cells in the whole population of the immunological cells, by the expression level of the target CAR / TCR on the cell surface of the immunological cells, or by both.

[0438] Embodiment 1.5 The method of any one of embodiments 1. 1-1.4, wherein the c-Jun fragment has a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 37-54.

[0439] Embodiment 1.6 The method of embodiment 1.5, wherein the c-Jun fragment has a sequence that is at least 70% identical to any one of SEQ ID NOs: 37, 38, 39, 46, 50, 51, 52, 53, and 54.

[0440] Embodiment 1.7 The method of embodiment 1.5, wherein the c-Jun fragment has a sequence that is at least 70% identical to any one of SEQ ID NOs: 53 and 54. Embodiment 1.8 The method of any one of embodiments 1. 1-1.7, wherein the target CAR is selected from the group consisting of ALPP, LYPD3, IL13Ra2 (IL13 receptor alpha 2), BCMA, CD19, CD20, CD22, CD138, CD33, CD123, CD171, CD70, CD7, CS-1, PSMA, PSCA, R0R1, GD2, MUC1, MUC16, HER2 (ErbB2), MET, EphA2, EpCAM, CEA, CSPG4, Lewis Y antigen, Mesothelin, NKG2D, Glypican-3 (GPC-3), FAP, FRa (folate receptor alpha), EGFR, EGFR vIII, IL-1 IRa (IL11 receptor alpha), and VEGFR-II.

[0441] Embodiment 1.9 The method of embodiment 1.8, wherein the target CAR is selected from the group consisting of ALPP, CD 19, and IL13Ra2.

[0442] Embodiment 1.10 The method of any one of embodiments 1.1 -1.7, wherein the target TCR is selected from a group consisting of NY-ESO-1, EBV LMP2, EBV antigen, HPV16 E6 / E7, KRAS, H3K27M, WT-1, and PRAME.

[0443] Embodiment 1.11 The method of embodiment 1.10, wherein the target TCR is NY- ESO-1.

[0444] Embodiment 1.12 The method of any one of embodiments 1.1-1.11, wherein the immunological cells are T cells, tumor infiltrating lymphocytes, macrophages, natural killer cells, or a combination thereof.

[0445] Embodiment 1.13 The method of embodiment 1.12, wherein the immunological cells are T cells, selected from the group consisting of CD3+T cells (e.g., a combination of CD4+and CD8+T cells), natural killer (NK) T cells, alpha beta T cells, gamma delta T cells, memory T cells (e.g., central memory T cells or effector memory T cells).

[0446] Embodiment 1.14 The method of embodiment 1.13, wherein the immunological cells are T cells, selected from a group consisting of CD3+T cells (e.g., a combination of CD4+and CD8+T cells).

[0447] Embodiment 1.15 The method of embodiment 1.14, wherein the immunological cells are CD4+T cells.

[0448] Embodiment 1.16 The method of embodiment 1.14, wherein the immunological cells are CD8+T cells.

[0449] Embodiment 1.17 The method of any one of embodiment 1.1-1.16, wherein in the co-expressing in the immunological cells the sustaining polypeptide, the sustaining polypeptide and the target CAR / TCR are co-expressed by means of a single vector, the single vector comprising two polynucleotide fragments respectively encoding the target CAR / TCR and the sustaining poly peptide. Embodiment 1.18 The method of embodiment 1.17, wherein the two polynucleotide fragments in the single vector are separated from each other by sequences encoding a selfcleavage peptide (e.g., P2A or T2A) and / or a protease recognition site (e.g., furin).

[0450] Embodiment 1.19 The method of embodiment 1.17, wherein the two polynucleotide fragments in the single vector have two different promoters.

[0451] Embodiment 1.20 The method of any one of embodiment 1.1-1.16, wherein in the co-expressing in the immunological cells the sustaining polypeptide, the sustaining polypeptide and the target CAR / TCR are co-expressed by means of two distinct vectors.

[0452] Embodiment 2.1 An engineered immunological cell, co-expressing: (a) a target chimeric antigen receptor (CAR) or a target T cell receptor (TCR); and (b) a sustaining polypeptide comprising a c-Jun fragment, characterized such that when the sustaining polypeptide is co-expressed, the engineered immunological cell co-expressing the sustaining polypeptide has an improved sustaining of the expression of the target CAR or the target TCR compared to when the sustaining polypeptide is absent.

[0453] Embodiment 2.2 The engineered immunological cell of embodiment 2.1, further co- expressing other agent (e.g., PD-1 and / or IL12).

[0454] Embodiment 3.1 A method of treating a disease in a subject thereof, comprising: administering an effective amount of engineered immunological cells to the subject, wherein the engineered immunological cells co-express: (a) a target chimeric antigen receptor (CAR) or a target T cell receptor (TCR); and (b) a sustaining polypeptide comprising a c-Jun fragment, characterized such that when the sustaining polypeptide is co-expressed, the engineered immunological cell co-expressing the sustaining polypeptide has an improved sustaining of the expression of the target CAR or the target TCR compared to when the sustaining polypeptide is absent.

[0455] Embodiment 3.2 The method of embodiment 3.1, wherein the disease is cancer.

[0456] Embodiment 3.3 The method of embodiment 3.1 or 3.2, wherein the target CAR is selected from the group consisting of ALPP, CD 19, and IL13Ra2.

[0457] Embodiment 3.4 The method of embodiment 3.1, wherein the disease is a non-cancer disease.

[0458] Embodiment 3.5 The method of any one of embodiments 3. 1-3.4, wherein the engineered immunological cells further co-expresses other agent (e.g., PD-1 and / or IL12).

[0459] Embodiment 4.1 A method for improving the surface expression of a target chimeric antigen receptor (CAR) or a target T cell receptor (TCR) in immunological cells, comprising: co-expressing in the immunological cells a polypeptide comprising a c-Jun fragment, such that the immunological cells co-expressing the polypeptide have an improved sustaining of the surface expression of the target CAR or the target TCR compared to when the polypeptide is not co-expressed in the immunological cells.

[0460] Embodiment 4.2 The method of embodiment 4.1, wherein the immunological cells co-expressing the polypeptide have an improved sustaining of the surface expression of the target CAR or the target TCR under antigen re-stimulation compared to when the polypeptide is not co-expressed in the immunological cells.

[0461] Embodiment 4.3 The method of embodiment 4. 1 or 4.2, wherein the immunological cells co-expressing the polypeptide do not undergo reduced exhaustion compared to when the polypeptide is not co-expressed in the immunological cells.

[0462] Embodiment 4.4 The method of any one of embodiments 4. 1-4.3, wherein the c-Jun fragment in the polypeptide is any of a wildtype c-Jun, dJun 30-50, dJun 34-47, dJun 34-47 AA, or a functional variant or a functional portion thereof.

[0463] Embodiment 4.5 The method of embodiment 4.4, wherein the c-Jun fragment in the polypeptide is any of dJun 34-47 or dJun 34-47 AA, or a functional variant or a functional portion thereof.

[0464] Embodiment 4.6 The method of embodiment 4.5, wherein the c-Jun fragment in the polypeptide comprises dJun 34-47, having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53.

[0465] Embodiment 4.7 The method of embodiment 4 5, wherein the c-Jun fragment in the polypeptide comprises dJun 34-47 AA, having a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54.

[0466] Embodiment 4.8 The method of any one of embodiments 4. 1-4.7, wherein the target CAR is a ALPP CAR.

[0467] Embodiment 4.9 The method of any one of embodiments 4. 1-4.7, wherein the target TCR is aNY-ESO-1 TCR.

[0468] Embodiment 4.10 The method of any one of embodiments 4.1-4.7, wherein the target TCR is a IL 13 (E13Y) Aspire-TCR.

[0469] Embodiments 4.11 The method of any one of embodiments 4.1-4.10, wherein the immunological cells co-expressing the polypeptide exhibit a reduced level of basal activation compared with when the immunological cells co-express wildtype c-Jun instead of coexpressing the polypeptide. Embodiment 4.12 The method of any one of embodiments 4.1-4. 11, wherein the immunological cells co-expressing the polypeptide exhibit an increased responsiveness to antigen stimulation compared with when the immunological cells co-express wildtype c-Jun instead of co-expressing the polypeptide.

[0470] Embodiment 4.13 The method of any one of embodiments 4.1-4.12, wherein the immunological cells co-expressing the polypeptide exhibit an increased expansion capability compared to when the polypeptide is not co-expressed in the immunological cells.

[0471] Embodiment 4.14 The method of any one of embodiments 4.1-4.13, wherein the immunological cells co-expressing the polypeptide exhibit an increased cytolytic toxicity against target cells compared with when the immunological cells co-express wildtype c-Jun instead of co-expressing the polypeptide.

[0472] Embodiment 4.15 The method of any one of embodiments 4.1-4.14, wherein the immunological cells are T lymphocytes, tumor infiltrating lymphocytes (TILs), or natural killer (NK) cells.

[0473] Embodiment 5.1 A population of immunological cells, expressing a polypeptide that comprises a c-Jun fragment, wherein the population of immunological cells exhibit a reduced level of basal activation compared with when the population of immunological cells express wildtype c-Jun instead of expressing the polypeptide.

[0474] Embodiment 5.2 The population of immunological cells of embodiment 5.1, further expressing a target CAR or a target TCR, wherein the population of immunological cells exhibit an improved sustaining of the surface expression of the target CAR or the target TCR compared to when the polypeptide is not expressed in the immunological cells.

[0475] Embodiment 5.3 The population of immunological cells of embodiment 5.1 or 5.2, wherein the population of immunological cells further exhibit an increased responsiveness to antigen stimulation compared with_compared with when the population of immunological cells express wildtype c-Jun instead of expressing the polypeptide.

[0476] Embodiment 5.4 The population of immunological cells of any one of embodiments

[0477] 5. 1-5.3, wherein the population of immunological cells further exhibit an increased expansion capability compared to when the polypeptide is not co-expressed in the population of immunological cells.

[0478] Embodiment 5.5 The population of immunological cells of any one of embodiments

[0479] 5. 1-5.4, wherein the population of immunological cells further exhibit an increased cytolytic toxicity against target cells compared with when the population of immunological cells express wildtype c-Jun instead of expressing the polypeptide.

[0480] Embodiment 5.6 The population of immunological cells of any one of embodiments 5. 1-5.5, wherein the c-Jun fragment is dJun 34-47 or a functional variant or a functional portion thereof.

[0481] Embodiment 5.7 The population of immunological cells of embodiment 5.6, wherein the c-Jun fragment in the polypeptide comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53.

[0482] Embodiment 5.8 The population of immunological cells of any one of embodiments 5. 1-5.5, wherein the c-Jun fragment is dJun 34-47 AA or a functional variant or a functional portion thereof.

[0483] Embodiment 5.9 The population of immunological cells of embodiment 5.8, wherein the c-Jun fragment in the polypeptide comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54. Embodiment 5.10 The population of immunological cells of any one of embodiments

[0484] 5. 1-5.9, wherein the population of immunological cells are T lymphocytes, tumor infiltrating lymphocytes (TILs), or natural killer (NK.) cells.

Claims

WHAT IS CLAIMED IS:

1. An engineered c-Jun polypeptide comprising a disrupted delta domain, wherein when the engineered c-Jun polypeptide is expressed in an immune cell, the immune cell has at least one of the following features:(a) a sustained expression of a chimeric antigen receptor (CAR) or a T cell receptor (TCR) on the immune cell,(b) a reduced level of basal activation,(c) an increased responsiveness,(d) an increased expansion capability, or(e) an increased cytolytic toxicity; as compared to an immune cell that does not express the engineered c-Jun polypeptide.

2. The engineered c-Jun polypeptide of claim 1, wherein the delta domain is disrupted by a deletion within the delta domain.

3. The engineered c-Jun polypeptide of claim 1 or 2, wherein the deletion is within a sequence that corresponds to amino acids 31-59 of SEQ ID NO: 37.

4. The engineered c-Jun polypeptide of claim 1 or 2, wherein a sequence corresponds to amino acids 30-50 of SEQ ID NO: 37 is deleted.

5. The engineered c-Jun polypeptide of any one of claims 1, 2, and 4, comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 39.

6. The engineered c-Jun polypeptide of claim 1 or 2, wherein a sequence corresponds to amino acids 34-47 of SEQ ID NO: 37 is deleted.7 The engineered c-Jun polypeptide of any one of claims 1, 2, and 6, comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53.

8. The engineered c-Jun polypeptide of any one of claims 1-7, wherein the engineered c-Jun polypeptide comprises one or more of the following:(a) the amino acid that corresponds to S63 of SEQ ID NO: 37 is hydrophobic; and(b) the amino acid that corresponds to S73 of SEQ ID NO: 37 is hydrophobic.

9. The engineered c-Jun polypeptide of claim 8, wherein the amino acid that corresponds to S63 of SEQ ID NO: 37 is Ala, Vai, He, Leu, Met, Phe, Try, or Trp.

10. The engineered c-Jun polypeptide of claim 8 or 9, wherein the amino acid that corresponds to S73 of SEQ ID NO: 37 is Ala, Vai, He, Leu, Met, Phe, Try, or Trp.

11. The engineered c-Jun polypeptide of any one of claims 1 -4, 6, and 8-10, wherein the amino acid that corresponds to S63 of SEQ ID NO: 37 is Ala, and the amino acid that corresponds to S73 of SEQ ID NO: 37 is Ala.

12. The engineered c-Jun polypeptide of any one of claims 1-4, 6, and 8-11, comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 54.

13. The engineered c-Jun polypeptide of any one of claims 1-12, wherein when the engineered c-Jun polypeptide is expressed in an immune cell, the immune cell has a sustained expression of a CAR or a TCR on the immune cell as compared to an immune cell that does not express the engineered c-Jun polypeptide.

14. The engineered c-Jun polypeptide of any one of claims 1-13, wherein when the engineered c-Jun polypeptide is expressed in an immune cell, the immune cell has a reduced level of basal activation as compared to an immune cell that does not express the engineered c-Jun polypeptide.

15. The engineered c-Jun polypeptide of any one of claims 1-14, wherein when the engineered c-Jun polypeptide is expressed in an immune cell, the immune cell has an increased responsiveness as compared to an immune cell that does not express the engineered c-Jun polypeptide.

16. The engineered c-Jun polypeptide of any one of claims 1-15, wherein when the engineered c-Jun polypeptide is expressed in an immune cell, the immune cell has an increased expansion capability as compared to an immune cell that does not express the engineered c-Jun polypeptide.

17. The engineered c-Jun polypeptide of any one of claims 1-16, wherein when the engineered c-Jun polypeptide is expressed in an immune cell, the immune cell has an increased cytolytic toxicity as compared to an immune cell that does not express the engineered c-Jun polypeptide.

18. A polynucleotide encoding the engineered c-Jun polypeptide of any one of claims 1- 17.

19. A polynucleotide comprising:(a) a first sequence encoding a CAR or a TCR; and(b) a second sequence encoding the engineered c-Jun polypeptide of any one of claims 1 - 17.

20. The polynucleotide of claim 19, wherein the first sequence encodes a CAR, wherein the CAR comprises, from N-terminus to C-terminus:(a) a leader sequence;(b) an antigen-binding fragment;(c) a hinge region comprising a membrane-proximal region from IgG, CD8, or CD28;(d) a transmembrane region comprising a transmembrane region of CD4, CD8, or CD28;(e) a costimulatory region comprising a functional signaling domain from MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, CDlla / CD18, 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6,CD49f, ITGAD, CD lid, ITGAE, CD 103, ITGAL, CD 11 a, LFA-1, ITGAM, CD 11b, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, or CD 19a; and(f an intracellular region comprising a functional signaling domain of CD3321. The polynucleotide of claim 20, wherein the antigen-binding fragment binds to or recognizes alkaline phosphatase, placental type (ALPP).

22. The polynucleotide of claim 21, wherein the antigen-binding fragment comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1 , 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 61, 62, and 63, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 64, 65, and 66, respectively;(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequencesare set forth in SEQ ID NOs: 70, 71, and 72, respectively;(3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 73, 74, and 75, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 76, 77, and 78, respectively; and(4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 85, 86, and 87, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 88, 89, and 90, respectively.

23. The polynucleotide of claim 21 or 22, wherein the antigen-binding fragment comprises a VH comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a VL comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:(1) the selected VH sequence is SEQ ID NO: 91, and the selected VL sequence is SEQ ID NO: 92;(2) the selected VH sequence is SEQ ID NO: 93, and the selected VL sequence is SEQ ID NO: 94;(3) the selected VH sequence is SEQ ID NO: 95, and the selected VL sequence is SEQ ID NO: 96; and(4) the selected VH sequence is SEQ ID NO: 99, and the selected VL sequence is SEQ ID NO: 1 0.

24. The polynucleotide of any one of claims 20-23, wherein the antigen-binding fragment is a single-chain variable fragment (scFv), e.g., an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 17, 18, 19, or 21.

25. The polynucleotide of claim 19, wherein the first sequence encodes a TCR.

26. The polynucleotide of claim 25, wherein the TCR binds to or recognizes a peptide epitope from NY-ESO-1.

27. The polynucleotide of claim 25, wherein the TCR is an Aspire-TCR.

28. The polynucleotide of claim 27, wherein the Aspire-TCR binds to or recognizes a peptide epitope from IL13Ra2.

29. The polynucleotide of claim 28, wherein and the Aspire-TCR comprises an IL 13 (E13Y) ligand region.

30. The polynucleotide of any one of claims 19-29, wherein the first sequence and the second sequence are connected by a third sequence encoding a linker, wherein the linker comprises a self-cleaving peptide (e.g., P2A or T2A) and / or a protease recognition site (e.g., furin).

31. A vector comprising the polynucleotide of any one of claims 18-30.

32. An engineered cell comprising the polynucleotide of any one of claims 18-30, or the vector of claim 31.

33. An engineered cell comprising a first vector comprising a polynucleotide encoding a CAR or a TCR, and a second vector comprising a polynucleotide encoding the engineered c-Jun polypeptide of any one of claims 1-17.

34. An engineered cell expressing the engineered c-Jun polypeptide of any one of claims 1-17.

35. The engineered cell of claim 34, further expressing a CAR or a TCR that binds to or recognizes a peptide epitope from an antigen on a target cell.

36. The engineered cell of claim 33 or 35, wherein the engineered cell expresses a CAR that binds to or recognizes a peptide epitope from ALPP, LYPD3, IL13Ra2 (IL13 receptor alpha 2), BCMA, CD19, CD20, CD22, CD138, CD33, CD123, CD171, CD70, CD7, CS- 1, PSMA, PSCA, ROR1, GD2, MUC1, MUC16, HER2 (ErbB2), MET, EphA2, EpCAM, CEA, CSPG4, Lewis Y antigen, Mesothelin, NKG2D, Glypican-3 (GPC-3), FAP, FRa (folate receptor alpha), EGFR, EGFR vIII, IL-1 IRa (IL11 receptor alpha), or VEGFR-II.Il l37. The engineered cell of claim 33 or 35, wherein the engineered cell expresses a TCR that binds to or recognizes a peptide epitope from NY-ESO-1, EBV LMP2, EBV antigen, HPV16 E6 / E7, KRAS, H3K27M, WT-1, or PRAME.

38. The engineered cell of claim 33 or 35, wherein the engineered cell expresses an Aspire-TCR that binds to or recognizes a peptide epitope from IL13Ra2.

39. The engineered cell of any one of claims 32-38, wherein the engineered cell is a T cell (e.g., a CD3+T cell, a CD4+T cell, a CD8+T cell, a natural killer (NK) T cell, an alpha beta T cell, a gamma delta T cell, or a memory T cell (e.g. a central memory T cell or an effector memory T cell)), a tumor infiltrating lymphocyte (TIL), a microphage, or a natural killer (NK) cell.

40. A method for improving the surface expression of a CAR or a TCR in an immune cell, comprising modifying the immune cell to express:(a) a CAR or a TCR that binds or recognizes a peptide epitope from an antigen; and(b) a sustaining polypeptide, wherein the immune cell has an improved sustaining of the expression of the CAR or TCR as compared to an immune cell that does not express the sustaining polypeptide.

41. The method of claim 40, wherein the sustaining polypeptide is an AP-1 transcription factor.

42. The method of claim 40 or 41, wherein the sustaining polypeptide is a wildtype c-Jun or a variant thereof.

43. The method of any one of claims 40-42, wherein sustaining polypeptide is Jun (e g., c-Jun, JunB, or JunD), Fos (e.g., c-Fos, FosB, Fral, and Fra2), activating transcription factor (ATF), Jun dimerization protein (JDP), or a variant thereof.

44. The method of any one of claims 40-43, wherein the sustaining polypeptide is a wildtype c-Jun.

45. The method of any one of claims 40-44, wherein the sustaining polypeptide is a c-Jun variant.

46. The method of claim 45, wherein the c-Jun variant has a disrupted delta domain.

47. The method of claim 45 or 46, wherein the c-Jun variant does not comprise one or more amino acids that correspond to all or a portion of the delta domain in a wildtype c- Jun.

48. The method of any one of claims 45-47, wherein one or more amino acids in the JNK sites of the c-Jun variant are hydrophobic.

49. The method of claim 44 or 45, wherein the c-Jun or the variant thereof comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to any one of SEQ ID NOs: 37-54.

50. The method of claim 44 or 45, wherein the c-Jun or the variant thereof comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to any one of SEQ ID NOs: 37, 38, 39, 46, 50, 51, 52, 53, and 54.

51. The method of claim 44 or 45, wherein the c-Jun or the variant thereof comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to SEQ ID NO: 53 or SEQ ID NO: 54.

52. The method of any one of claims 40-51, wherein the method further comprises: stimulating the immune cell by the antigen for at least 1, 2, 3, 4, 5, or 6 times.

53. The method of any one of claims 40-52, wherein the immune cell has an improved immunological response (e.g., a reduced level of basal activation; an increased responsiveness; an increased expansion capability, and / or an increased cytolytic toxicity) as compared to an immune cell that does not express the sustaining polypeptide.

54. The method of any one of claims 40-53, wherein the expression of the sustaining polypeptide has no effect on the expression of one or more exhaustion-associated markers (e.g., LAG3, PD1 and / or Tim3) in the immune cell.

55. The method of any one of claims 40-54, wherein the immune cell expresses a CAR that binds to or recognizes a peptide epitope from ALPP, LYPD3, IL13Ra2 (IL13 receptor alpha 2), BCMA, CD19, CD20, CD22, CD138, CD33, CD123, CD171, CD70, CD7, CS- 1, PSMA, PSCA, ROR1, GD2, MUC1, MUC16, HER2 (ErbB2), MET, EphA2, EpCAM, CEA, CSPG4, Lewis Y antigen, Mesothelin, NKG2D, Glypican-3 (GPC-3), FAP, FRa (folate receptor alpha), EGFR, EGFR vIII, IL-1 IRa (IL11 receptor alpha), or VEGFR-II.

56. The method of claim 55, wherein the CAR binds to or recognizes a peptide epitope from ALPP, CD 19, or IL13Ra2.

57. The method of any one claims 40-54, wherein the immune cell expresses a TCR that binds to or recognizes a peptide epitope from NY-ESO-1, EBV LMP2, EBV antigen, HPV16 E6 / E7, KRAS, H3K27M, WT-1, or PRAME.

58. The method of claim 57, wherein the TCR binds to or recognizes a peptide epitope from NY-ESO-1 .

59. The method of any one of claims 40-54, wherein the immune cell expresses an Aspire-TCR (e.g., an Aspire-TCR that binds to IL13Ra2).

60. The method of any one of claims 40-59, wherein the method further comprises: modifying the immune cell to express an additional therapeutic agent.

61. The method of claim 60, wherein the additional therapeutic agent is an immune checkpoint inhibitor.

62. The method of claim 61, wherein the checkpoint inhibitor can inhibit or block PD-1,PD-L1, PD-L2, 2B4 (CD244), 4-1BB, A2aR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6,BTLA, butyrophilins, CD 160, CD48, CTLA4, GITR, gp49B, HHLA2, HVEM, ICOS, ILT-2, ILT-4, KIR family receptors, LAG-3, OX-40, PIR-B, SIRPalpha (CD47), TFM-4, TIGIT, TIM-1, TIM-3, TIM-4, VISTA, or combinations thereof.

63. The method of claim 60, wherein the additional therapeutic agent is a cytokine or chemokine (e.g., IL12 or IL-7 / CCL19), or a bifunctional trap fusion protein.

64. The method of any one of claims 40-63, wherein the immune cell is a T cell (e g., a CD3+T cell, a CD4+T cell, a CD8+T cell, a natural killer (NK) T cell, an alpha beta T cell, a gamma delta T cell, or a memory T cell (e.g. a central memory T cell or an effector memory T cell).

65. The method of any one of claims 40-63, wherein the immune cell is a tumor infiltrating lymphocyte (TIL), a microphage, or a natural killer (NK) cell.

66. The method of any one of claims 40-65, wherein the CAR or TCR is heterologous to the immune cell.

67. The method of any one of claims 40-66, wherein the immune cell is a cell line.

68. The method of any one of claims 40-66, wherein the immune cell is a primary cell obtained from a subject (e.g., a human subject).

69. A method for producing the engineered cell, comprising introducing the vector of claim 31 into a cell in vitro or ex vivo.

70. The method of claim 69, wherein the vector is a viral vector and the introducing is carried out by transduction.

71. A method of treating a disease or disorder, comprising administering the engineered cell of any one of claims 32-39 to a subject having the disease or disorder.

72. A method of treating a disease or disorder in a subject, the method comprisingadministering to the subject in need thereof,(a) an engineered T cell, comprising: a nucleic acid encoding a CAR or a TCR; and(b) the engineered c-Jun polypeptide of any one of claims 1-17.

73. The method of claim 71 or 72, wherein the disease or disorder is a cancer.

74. The method of claim 71 or 72, wherein the disease or disorder is a non-cancerous disease.

75. An antibody or antigen-binding fragment thereof that specifically binds to ALPP, comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 61, 62, and 63, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 64, 65, and 66, respectively;(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 70, 71, and 72, respectively;(3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ IDNOs: 73, 74, and 75, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 76, 77, and 78, respectively; and(4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 85, 86, and 87, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 88, 89, and 90, respectively.

76. The antibody or antigen-binding fragment thereof of claim 75, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 61, 62, and 63, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequence set forth in SEQ ID NOs: 64, 65, and 66, respectively.

77. The antibody or antigen-binding fragment thereof of claim 75, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequence set forth in SEQ ID NOs: 70, 71, and 72, respectively.

78. The antibody or antigen-binding fragment thereof of claim 75, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 73, 74, and 75, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequence set forth in SEQ ID NOs: 76, 77, and 78, respectively.

79. The antibody or antigen-binding fragment thereof of claim 75, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 85, 86, and 87, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequence set forth in SEQ ID NOs: 88, 89, and 90, respectively.

80. The antibody or antigen-binding fragment thereof of any one of claims 75-79, wherein the VH consists of or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 91, 93, 95, or 99; and the VL consists of or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 92, 94, 96, or 100.

81. The antibody or antigen-binding fragment thereof of claim 80, wherein the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 91 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 92.

82. The antibody or antigen-binding fragment thereof of claim 80, wherein the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 93 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 94.

83. The antibody or antigen-binding fragment thereof of claim 80, wherein the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 95 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 96.

84. The antibody or antigen-binding fragment thereof of claim 80, wherein the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 99 and the VL comprises an ammo acid sequence having at least 90% identity to SEQ ID NO: 100.

85. The antibody or antigen-binding fragment thereof of any one of claims 75-84, wherein the antibody or antigen-binding fragment thereof specifically binds to human ALPP.

86. The antibody or antigen-binding fragment thereof of any one of claims 75-85, wherein the antibody or antigen-binding fragment thereof is an scFv.

87. The antibody or antigen-binding fragment thereof of any one of claims 75-86, wherein the VH and VL are connected with a linker peptide (e.g., SEQ ID NO: 25).

88. The antibody or antigen-binding fragment thereof of claim 86 or 87, wherein the scFv comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 17, 18, 19, or 21.

89. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof of any one of claims 75-88.

90. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 75-88, or the CAR of claim 89.

91. A vector comprising the polynucleotide of claim 90.

92. An engineered cell comprising the CAR of claim 89, the polynucleotide of claim 90, or the vector of claim 91.

93. The CAR of claim 89, further comprising: a leader sequence, a hinge region derived from CD8, a transmembrane region derived from CD4, a costimulatory region derived from 4- 1BB, and / or an intracellular signaling region derived from CD3^.

94. The CAR of claim 89 or 93, wherein the antibody or antigen-binding fragment thereof is an scFv that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 17, 18, or 21, wherein the CAR can be stably expressed on T cell surface.

95. The CAR of claim 89 or 93, wherein the antibody or antigen-binding fragment thereof is an scFv that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to SEQ ID NO: 17 or 18, wherein the CAR, when expressed on the surface of T cells, can activate the T cells (e.g., by inducing IFN-y expression) that are co-cultured with ALPP-expressing tumor cells.

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