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EP4638484A2 - Compositions et procédés pour améliorer des réponses immunologiques dans des cellules immunologiques modifiées - Google Patents

Compositions et procédés pour améliorer des réponses immunologiques dans des cellules immunologiques modifiées

Info

Publication number
EP4638484A2
EP4638484A2 EP23908029.4A EP23908029A EP4638484A2 EP 4638484 A2 EP4638484 A2 EP 4638484A2 EP 23908029 A EP23908029 A EP 23908029A EP 4638484 A2 EP4638484 A2 EP 4638484A2
Authority
EP
European Patent Office
Prior art keywords
amino acid
seq
cell
engineered
jun
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23908029.4A
Other languages
German (de)
English (en)
Inventor
Lixia ZHAO
Rui Chen
Si Li
Weiting DU
Hongbo Yang
Zhenbo SU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangdong Tcrcure Biopharma Technology Co Ltd
Tcrcure Biopharma Corp
Original Assignee
Guangdong Tcrcure Biopharma Technology Co Ltd
Tcrcure Biopharma Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangdong Tcrcure Biopharma Technology Co Ltd, Tcrcure Biopharma Corp filed Critical Guangdong Tcrcure Biopharma Technology Co Ltd
Publication of EP4638484A2 publication Critical patent/EP4638484A2/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • 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.
  • 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.
  • 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”.
  • 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
  • the disclosure is related to an engineered c-Jun polypeptide comprising a disrupted delta domain
  • the immune cell 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.
  • the delta domain is disrupted by a deletion within the delta domain.
  • 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.
  • 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.
  • 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.
  • the amino acid that corresponds to S63 of SEQ ID NO: 37 is Ala, Vai, He, Leu, Met, Phe, Try, or Trp.
  • 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.
  • the immune cell 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.
  • the immune cell 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.
  • the disclosure is related to a polynucleotide encoding the engineered c- Jun polypeptide described herein.
  • 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.
  • the first sequence encodes a CAR
  • 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),
  • the antigen-binding fragment binds to or recognizes alkaline phosphatase, placental type (ALPP).
  • the antigen-binding fragment comprises a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3.
  • 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
  • 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.
  • VL light chain variable region
  • 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
  • the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence.
  • 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:
  • 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.
  • 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.
  • 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.
  • scFv single-chain variable fragment
  • the first sequence encodes a TCR.
  • the TCR binds to or recognizes a peptide epitope from NY-ESO-1.
  • the TCR is an Aspire-TCR.
  • the Aspire-TCR binds to or recognizes a peptide epitope from IL13Ra2.
  • the Aspire-TCR comprises an IL13 (E13Y) ligand region.
  • the first sequence and the second sequence are connected by a third sequence encoding a linker
  • the linker comprises a self-cleaving peptide (e.g., P2A or T2A) and/or a protease recognition site (e g., furin).
  • 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.
  • 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.
  • the disclosure is related to an engineered cell expressing the engineered c-Jun polypeptide as described herein.
  • 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.
  • 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.
  • ALPP ALPP
  • LYPD3, IL13Ra2 IL13 receptor alpha 2
  • BCMA CD19, CD20, CD22, CD138, CD33, CD123, CD171, CD70,
  • 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.
  • 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.
  • 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)
  • TIL tumor infiltrating lymphocyte
  • microphage a microphage
  • NK natural killer
  • 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.
  • 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.
  • the sustaining polypeptide is an AP-1 transcription factor.
  • the sustaining polypeptide is a wildtype c- Jun or a variant thereof.
  • 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.
  • the sustaining polypeptide is a wildtype c-Jun.
  • the sustaining polypeptide is a c-Jun variant.
  • the c-Jun variant has a disrupted delta domain.
  • 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.
  • one or more amino acids in the JNK sites of the c-Jun variant are hydrophobic.
  • 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.
  • 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.
  • 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.
  • the method further comprises: stimulating the immune cell by the antigen for at least 1, 2, 3, 4, 5, or 6 times.
  • 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.
  • 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.
  • 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.
  • ALPP ALPP
  • LYPD3, IL13Ra2 IL13 receptor alpha 2
  • BCMA CD19, CD20, CD22, CD138, CD33, CD123, CD171, CD70,
  • the CAR binds to or recognizes a peptide epitope from ALPP, CD19, or IL13Ra2.
  • 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.
  • the TCR binds to or recognizes a peptide epitope from NY- ESO-1.
  • the immune cell expresses an Aspire-TCR (e.g., an Aspire- TCR that binds to IL13Ra2).
  • the method further comprises: modifying the immune cell to express an additional therapeutic agent.
  • the additional therapeutic agent is an immune checkpoint inhibitor.
  • 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.
  • the additional therapeutic agent is a cytokine or chemokine (e.g., IL 12 or IL- 7/CCL19), or a bifunctional trap fusion protein.
  • 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).
  • the immune cell is a tumor infiltrating lymphocyte (TIL), a microphage, or a natural killer (NK) cell.
  • the CAR or TCR is heterologous to the immune cell.
  • the immune cell is a cell line.
  • the immune cell is a primary cell obtained from a subject (e.g., a human subject).
  • 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.
  • the vector is a viral vector and the introducing is carried out by transduction.
  • 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.
  • 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.
  • the disease or disorder is a cancer. In some embodiments, the disease or disorder is a non-cancerous disease.
  • 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.
  • 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
  • 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.
  • VL light chain variable region
  • 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
  • the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 85, 86, and 87, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequence set forth in SEQ ID NOs: 88, 89, and 90, respectively.
  • 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.
  • 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.
  • 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.
  • the antibody or antigen-binding fragment thereof specifically binds to human ALPP.
  • the antibody or antigen-binding fragment thereof is an scFv.
  • the VH and VL are connected with a linker peptide (e g., SEQ ID NO: 25).
  • 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.
  • the disclosure is related to a chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof described herein.
  • CAR chimeric antigen receptor
  • the disclosure is related to a polynucleotide encoding the antibody or antigen-binding fragment thereof or the CAR described herein.
  • the disclosure is related to a vector comprising the polynucleotide described herein.
  • the disclosure is related to an engineered cell comprising the CAR, the polynucleotide, or the vector described herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the CAR can be stably expressed on T cell surface.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • basal activation refers to activation of immune cells in the absence of any antigens.
  • 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).
  • proinflammatory cytokines e.g., IFN-y
  • activation markers e.g., 4- IBB
  • “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.
  • 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).
  • 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.
  • 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.
  • 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.
  • increased cytolytic toxicity indicates no less than 1.2-fold increase of killing efficiency of specific target cells.
  • responsiveness 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).
  • antigen stimulation e.g., specific target cells
  • “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.
  • 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:
  • 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.
  • 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:
  • 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.
  • 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.
  • Aspire-TCR can be any Aspire-TCRs constructs described herein.
  • the term “increased” or “reduced” refers to the level change of no less than 10% compared to a reference level.
  • FIG. 1 shows schematic structures of A02. A02-cJun, A02-8H, and A02-8H eJun constructs.
  • 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).
  • 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).
  • FIGS. 3A-3B show the memory phenotyping of different ALPP CARs in human T cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 7 shows the in vivo efficacy of ALPP CAR-T cells in SKOV3-ALPP subcutaneous NSG mouse model.
  • FIG. 8 shows the in vivo efficacy of ALPP CAR-T cells in SiHa intraperitoneal NSG mouse model.
  • 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.
  • 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).
  • 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.
  • 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
  • 3rd sti indicating after 3rd re-stimulation.
  • 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).
  • FIGS. 12C-12F show effects of the various c-Jun variants on the activation of the engineered T cells.
  • FIGS. 12G-12H show effects of the various c-Jun variants on the cytolytic toxicity against target tumor cells.
  • FIGS. 13A-13B show effects of various c-Jun variants on the Aspire-TCR expression.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • FIG. 18 shows VH and VL CDR sequences in F8, H5, C9, F7, and A3 antibodies.
  • FIG. 19 shows VH and VL sequences in F8, H5, C9, F7, and A3 antibodies.
  • FIG. 20 lists sequences discussed in the disclosure.
  • 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).
  • 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.
  • the improvements may include one or more of the following aspects.
  • 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).
  • 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
  • dJun 34-47 AA can beter sustain the expression of the CAR/TCR on the engineered T cells.
  • 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.
  • the improvement may be on the aspect of the basal activation of the engineered immunological cells.
  • 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.
  • 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.
  • 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.
  • the improvement may be on the aspect of the antigen-responsiveness of the engineered immunological cells.
  • 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.
  • the improvement may be on the aspect of the expansion capability of the engineered immunological cells.
  • 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.
  • the improvement may be on the aspect of the cytolytic toxicity of the engineered immunological cells against target tumor cells.
  • 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.
  • 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.
  • a c-Jun variant expresses a wildtype c-Jun protein.
  • 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.
  • a basal activation level e.g., antigenindependent activation level
  • 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).
  • proinflammatory cytokines e.g., IFN-y
  • immune cell e.g., T cell
  • activation markers e.g., 4-1BB
  • 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.
  • the responsiveness is determined by measuring the secretion of one or more proinflammatory cytokines (e.g., IFN-y).
  • 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.
  • 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.
  • the cytolytic activity is determined by measuring target tumor cell killing efficiency, either under unstimulated conditions or repeated stimulation conditions.
  • 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).
  • 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.
  • 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.
  • the anti-ALPP CAR described herein is a murine ALPP CAR, a human ALPP CAR, or a chimeric ALPP CAR.
  • 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.
  • 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.
  • 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.
  • the immune cell is unstimulated.
  • the immune cell is stimulated by an antigen for at least 1, 2, 3, 4, 5, or 6 times.
  • the immune cell is restimulated by the same or a different antigen for at least once, at least twice, or at least three times.
  • 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.
  • 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.
  • c-Jun:c-Fos dimers prefer TRE sites, whereas c-Jun:ATF dimers prefer CRE sites.
  • 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.
  • 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.
  • AP-1 family proteins regulate a wide range of cellular and biological activities.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • one or more amino acid residues in the wildtype enzyme can be substituted, deleted, or mutated.
  • 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.
  • a variant is a fragment of the wildtype enzyme.
  • 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.
  • the eJun described herein includes any one of the eJun variants/mutants discussed in the disclosure. eJun: (SEQ ID NO: 37)
  • 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).
  • the sustaining polypeptide may be configured to realize a controlled expression in the immunological cells expressing the target CAR/TCR.
  • 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.
  • 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.
  • 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.
  • Jun c-Jun, JunB, and JunD
  • Fos c-Fos, FosB, Fral, and Fra2
  • ATF activating transcription factor
  • JDP Jun dimerization protein
  • the sustaining polypeptide described herein is wild-type “eJun” or a variant thereof.
  • 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.
  • 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.
  • the sustaining polypeptide described herein is “Jun-AA” or a variant thereof.
  • 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.
  • 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.
  • the sustaining polypeptide described herein is “Jun- 30-50” (or “dJun 30-50”) or a variant thereof.
  • 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.
  • 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.
  • 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.
  • the sustaining polypeptide described herein is “Jun-ATAD (2- 102)” or a variant thereof.
  • 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.
  • 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.
  • 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.
  • the sustaining polypeptide described herein is “Jun-Abasic (254-280)” or a variant thereof.
  • the Jun-Abasic (254-280) or the variant thereof does not include all or a portion of the basic motif of wildtype eJun protein.
  • 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.
  • 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.
  • 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.
  • the sustaining polypeptide described herein is “Jun-ALeu (280- 314)” or a variant thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • the sustaining polypeptide described herein is “Jun-AZIP(254- 314)” or a variant thereof.
  • 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.
  • 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.
  • the sustaining polypeptide described herein is “Jun A3-102” or a variant thereof.
  • 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.
  • 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.
  • 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.
  • the sustaining polypeptide described herein is “Jun A3 -122” or a variant thereof.
  • 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.
  • 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.
  • 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.
  • the sustaining polypeptide described herein is “Jun A->D 265 A270-272” (or “dJun 270-272”) or a variant thereof.
  • the Jun A->D 265 A270-272 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).
  • the Jun A->D 265 A270-272 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.
  • the first amino acid sequence and the second amino acid sequence are connected with a aspartic acid (D).
  • the Jun A->D 265 A270-272 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.
  • the sustaining polypeptide described herein is “Jun A->D 265 A256-258” or a variant thereof.
  • the Jun A->D 265 A256-258 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).
  • the Jun A->D 265 A256-258 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.
  • the Jun A->D 265 A270- 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.
  • the sustaining polypeptide described herein is “Jun A->D 265in ” or a variant thereof.
  • the Jun A->D 265in or 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.
  • the Jun A->D 265in or 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.
  • 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.
  • the Jun A->D 265 A270-272 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.
  • the sustaining polypeptide described herein is “Jun A287-331” or a variant thereof.
  • 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.
  • 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.
  • 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.
  • the sustaining polypeptide described herein is “Jun-bZIP” (or “eJun bZIP vl”) or a variant thereof.
  • 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).
  • 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.
  • the sustaining polypeptide described herein is “Jun-Al 03-209” (or “dJun 103-209”) or a variant thereof.
  • 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.
  • 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.
  • 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 .
  • the sustaining polypeptide described herein is “Jun-Al 03 -145” (or “dJun 103-145”) or a variant thereof.
  • 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.
  • 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).
  • 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.
  • the sustaining polypeptide described herein is “dJun 34-47” or a variant thereof.
  • 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.
  • 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.
  • 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.
  • the sustaining polypeptide described herein is “dJun 34-47 AA” or a variant thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • the intracellular domain may at least include a functional intracellular T cell signaling domain (e.g.,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 VEG
  • ALPP (
  • 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, S
  • the target CAR can be a CAR that specifically targets ALPP, CD19 or IL13Ra2.
  • 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.
  • 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).
  • antigen-binding fragment antigen-binding fragment
  • hinge domain e.g., CD8 or CD28
  • transmembrane domain e.g., CD8, CD28, or CD4
  • costimulatory domain e.g., CD28 or 4-1BB
  • signal domain e.g., CD3zeta or CD3epsilon
  • 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.
  • the leader sequence includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 23.
  • 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.
  • the CD8 hinge region includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 27.
  • the CD28 transmembrane region includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 28.
  • the costimulatory signaling region described herein includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 29.
  • the intracellular signaling region described herein includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 30.
  • 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).
  • 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
  • signal domain e.g., CD3zeta or CD3epsilon
  • 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.
  • the leader sequence includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2.
  • the IL13 ligand described herein includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32.
  • the IgG4 Fc hinge region includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 33.
  • the CD4 transmembrane region includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 34.
  • the costimulatory signaling region described herein includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 35.
  • the intracellular signaling region described herein includes a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 36.
  • 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.
  • NY-ESO-1 (disclosed in W02020086158A2)
  • EBV LMP2 (disclosed in W020
  • the TCR described herein binds to or recognizes a peptide epitope from NY-ESO-1.
  • 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.
  • 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.
  • 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.
  • 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.
  • CARs/TCRs are also deemed to be within the scope of the disclosure.
  • the TCR described herein is an Antigen-specificity redirected TCR complex (i.e. Aspire-TCR or Aspire-T).
  • 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).
  • target cells e.g. tumor 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.
  • 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)).
  • TILs tumor-infiltrating lymphocytes
  • NK natural killer cells
  • NK T cells e.g. cells expressing TCR or Chimeric antigen receptor (CAR)
  • T lymphocytes, or T cells are used as illustrating yet non-limiting example for the immunological cells.
  • an engineered CD3 zeta subunit 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.
  • TCR T cell receptor
  • the engineered CD3 zeta subunit is configured such that at least one of the following effects is realized:
  • 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;
  • 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
  • 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.
  • 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.
  • 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.
  • each of the at least one costimulatory region may optionally comprise a co-stimulatory domain, or a functional portion or a functional variant thereof.
  • co-stimulatory domain 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.
  • 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).
  • TNF tumor necrosis factor receptor-associated factors
  • 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.
  • PI3K phosphatidylinositol-3 -kinase
  • Grb2 growth factor receptor-bound protein 2
  • Lek lymphocyte-specific protein tyrosinekinase
  • 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.
  • a natural costimulatory immune receptor such as CD28, 4-1BB, 0X40, CD2, CD27, CDS, ICAM-1, LFA-1, and ICOS.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • ITAMs immunoreceptor tyrosine activation motifs
  • 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.
  • the number of the at least one co-stimulatory region in the engineered CD3 zeta subunit disclosed herein 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.
  • 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.
  • ITAMs immunoreceptor tyrosine activation motifs
  • 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.
  • 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").
  • G Glycine
  • S Serine
  • 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.
  • the engineered target-recognizing subunit comprises an extracellular domain, which comprises a target-recognizing region.
  • 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.
  • 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).
  • scFv single-chain variable fragment
  • sdAb or nanobody single-domain antibody
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the target-recognizing region (ligand) is fused with the CD3e via a GS-rich linker (i.e., "GS linker").
  • the engineered protein i.e. the backbone protein based on which the targetrecognizing region is fused to or incorporated into
  • the engineered protein 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.
  • 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
  • the intracellular domain may comprise the intracellular domain of the CD3z subunit.
  • 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
  • the intracellular domain may comprise the intracellular domain of the CD3z subunit.
  • 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
  • the intracellular domain may comprise the intracellular stimulating domain of the CD3z subunit fused with the intracellular stimulating domain of the CD3e subunit.
  • 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.
  • IT AMs intracellular signaling motifs
  • 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.
  • a chimeric polypeptide e.g., the “IL13(E13Y)-CD3e” fusion protein described herein
  • 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.
  • 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.
  • TCR T cell receptor
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the Aspire-TCR binds to or recognizes a peptide epitope from IL13Ra2.
  • 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).
  • the IL13 (E13Y) ligand and the human CD3e component are connected with a GS linker (SEQ ID NO: 59).
  • 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.
  • 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.
  • 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.
  • 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.
  • the target CAR/TCR and the sustaining polypeptide i.e. the polypeptide comprising the actuating c-Jun fragment
  • 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.
  • a 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.
  • retroviral vectors e.g., 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.
  • AAV adeno-associated viral
  • 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).
  • a self-cleavage peptide e.g., P2A or T2A
  • a protease recognition site e.g., furin
  • the target CAR/TCR and the sustaining polypeptide are coexpressed in the T cells by means of distinct vectors.
  • 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 polypeptid
  • 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.
  • iCaspase 9 system
  • 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.
  • the sustaining polypeptide 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.
  • 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.
  • 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.
  • Placental Alkaline Phosphatase 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).
  • EMA epithelial membrane antigen
  • 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.
  • 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.
  • the disclosure provides anti-ALPP antibodies F8, H5, C9, F7, and A3, and the humanized antibodies thereof.
  • the CDR sequences for F8 derived 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 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 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 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 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • VH heavy chain vanable region
  • CDRs complementanty determining regions
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • the selected VH sequence is SEQ ID NO: 91
  • the selected VL sequence is SEQ ID NO: 92.
  • the selected VH sequence is SEQ ID NO: 93
  • the selected VL sequence is SEQ ID NO: 94.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • the scFv described herein has a schematic structure of VH-linker peptide-VL.
  • the scFv described herein has a schematic structure of VL-linker peptide-VH.
  • the scFv can specifically bind to ALPP, and includes any of the VH/VL combination described herein.
  • 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.
  • 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.
  • 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.
  • antibodies also called immunoglobulins
  • antibodies 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).
  • CDRs complementary determining regions
  • 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.
  • the CDRs are important for recognizing an epitope of an antigen.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.
  • 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.
  • purified antibody preparations e.g., purified IgGi molecules
  • antibody homodimers may be formed through chemical linkage techniques known in the art.
  • 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.
  • SMCC succinimidyl 4-(maleimidomethyl)cyclohexane-l -carboxylate
  • SATA N-succinimidyl S-acethylthio-acetate
  • 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
  • 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.
  • the interface can contain at least a part of the CH3 domain of an antibody constant domain.
  • 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 .
  • Bi-specific antibodies include cross-linked or “heteroconjugate” antibodies.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • cytotoxic or cytostatic agent e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, ten
  • the antigen binding fragment can form a part of a chimeric antigen receptor (CAR).
  • the chimeric antigen receptor are fusions of single-chain variable fragments (scFv) as described herein, fused to CD3-zeta transmembrane and endodomain.
  • the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS).
  • the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency.
  • the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors as described herein.
  • 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.
  • sequences e.g., CDRs or VH/VL sequences
  • the antibody or antigen-binding fragment thereof described herein can be used to generate a bispecific antibody targeting ALPP and an addition antigen.
  • the antigen binding fragment described herein has a scFv-Fc structure.
  • an anti- ALPP scFv e.g., any of the anti- ALPP scFv molecules described herein
  • a human Fc e.g., IgGl or IgG4 Fc
  • the antibody 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 .
  • 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 .
  • kinetic association rates (kon) is greater than 1 x 10 2 /Ms, greater than 1 x 10 3 /Ms, greater than 1 x 10 4 /Ms, greater than 1 x 10 5 /Ms, or greater than 1 x 10 6 /MS. In some embodiments, kinetic association rates (kon) is less than 1 x 10 5 /Ms, less than 1 x 10 6 /MS, or less than 1 x 10 7 /Ms.
  • KD is less than 1 x 10’ 6 M, less than 1 x 10’ 7 M, less than 1 x 10’ 8 M, less than 1 x 10’ 9 M, or less than 1 x 10’ 10 M. 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.
  • KD is greater than 1 x 10’ 7 M, greater than 1 x 10’ 8 M, greater than 1 x 10’ 9 M, greater than 1 x 10’ 1(1 M, greater than 1 x 10’ 11 M, or greater than 1 x io 12 M.
  • the antibody or antigen-binding fragment thereof described herein e.g., any of the anti-ALPP scFv-Fc molecules described herein
  • 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.
  • the antibody or antigen-binding fragment thereof described herein e.g., any of the anti-ALPP scFv-Fc molecules described herein
  • the affinity of an antibody for an antigen includes, e.g., ELISA, RIA, and surface plasmon resonance (SPR).
  • the affinity of the anti-ALPP antibodies described herein is determined by the Biacore assay.
  • 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%.
  • TGLv% tumor growth inhibition percentage
  • 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.
  • TGI% is calculated using the following formula:
  • TGI (%) [l-(Ti-To)/(Vi-Vo)]xlOO%
  • 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.
  • 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.
  • the antibodies or antigen binding fragments can induce complement-dependent cytotoxicity (CDC) and/or antibody dependent cellular cytotoxicity (ADCC), and kill the tumor cell.
  • the antibodies or antigen binding fragments have a functional Fc region.
  • effector function of a functional Fc region is antibody -dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody -dependent cell-mediated cytotoxicity
  • effector function of a functional Fc region is phagocytosis.
  • effector function of a functional Fc region is ADCC and phagocytosis.
  • the Fc region is human IgGl, human IgG2, human IgG3, or human IgG4.
  • the antibody is a human IgGl antibody.
  • the antibodies or antigen binding fragments do not have a functional Fc region.
  • the antibodies or antigen binding fragments are Fab, Fab’, F(ab’)2, and Fv fragments.
  • the Fc region has LALA mutations (L234A and L235A mutations in EU numbering), or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering).
  • the Fc have a SI mutation (S239D and I332E mutations in EU numbering).
  • the anti-ALPP antibodies or antigen-binding fragments thereof described herein can be developed using a ChemPamer human naive scFv phage library.
  • 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.
  • 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
  • step 4 amplification of binders.
  • reinfection of E. coli was performed.
  • about 1000 clones were selected (single colonies were picked) and subject to primary screening by ELISA-based binding.
  • top binders 100-300 top binders were screened, and subject to secondary screening by ELISA- and FACS-based binding.
  • the FACS-based binding assays used ALPP-293T cells and SiHa cells.
  • the screened binders were subject to IgG conversion, production, and purification.
  • 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.
  • 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.
  • the antigenic peptide or protein is injected with at least one adjuvant.
  • 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).
  • 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.
  • the full length sequence of human ALPP is known in the art.
  • an Fc-tagged human ALPP protein (the Fc fusion protein contains all or a portion of human ALPP) is used as the immunogen.
  • 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.
  • 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.
  • 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).
  • an antibody can be engineered which has dual Fc regions.
  • 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.
  • antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • 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.
  • the Fc region of the antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A).
  • engineered cells e.g., T cells
  • CAR CAR, TCR, or antigen-binding fragment thereof, or other similar antigen-binding molecules as described herein.
  • engineered cells can be used to treat various disorders or disease as described herein (e.g., cancers).
  • the engineered cells that are armored to express the sustaining polypeptide to thereby realize a better sustaining of the expression of the target CAR/TCR molecules
  • CD3 + T cells e.g., a combination of CD4 + and CD8 + T cells
  • CD8 + T cells CD8 + T cells
  • CD4 + T cells CD4 + T cells
  • natural killer (NK) T cells e.g., alpha beta T cells, gamma delta T cells
  • memory T cells e.g., central memory T cells or effector memory T cells, etc.
  • the cell that is engineered can be obtained from, e.g., humans and non-human animals.
  • the cell that is engineered can be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pig or any other species.
  • the cell is from humans, rats or mice. More preferably, the cell is obtained from humans.
  • the cell that is engineered is a blood cell.
  • the cell is a leukocyte (e.g., a T cell), lymphocyte or any other suitable blood cell type.
  • the cell is a peripheral blood cell.
  • the cell is a T cell, B cell or NK cell.
  • the cell is a T cell.
  • 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.
  • the T cell is a TCR gene-modified T cell.
  • the T cells are CD3 + T cells, CD4 + T cells, CD8 + T cells, or regulatory T cells.
  • the T cells are T helper type 1 T cells and T helper type 2 T cells.
  • 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.
  • the cell is an NK cell.
  • 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.
  • 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.
  • 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.
  • the stem cells are cultured with additional differentiation factors to obtain desired cell types (e.g., T cells).
  • 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.
  • 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.
  • 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.
  • 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.
  • a nucleic acid encoding the therapeutic molecule e.g. TCR, CAR, e.g. TCR-like CAR, polypeptides, fusion proteins
  • 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.
  • 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).
  • SV40 simian virus 40
  • AAV adeno-associated virus
  • recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors.
  • 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).
  • LTR long terminal repeat sequence
  • retroviral vectors are derived from murine retroviruses.
  • 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.
  • the vector is a lentivirus vector.
  • recombinant nucleic acids are transferred into T cells via electroporation.
  • 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.
  • 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.
  • 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.
  • recombinant vectors e.g., an expression vectors
  • 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.
  • 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.
  • regulatory elements such as a promoter, enhancer, and/or a poly -A tail
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • LTRs 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.
  • 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.
  • selectable markers such as Ampicillin resistance, Neomycin resistance, TK, hygromycin resistance, phleomycin resistance histidinol resistance, or DHFR
  • 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.
  • 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.
  • the vector can include an additional nucleic acid encoding an inhibitory protein (e g., a checkpoint inhibitor).
  • an inhibitory protein e g., a checkpoint inhibitor.
  • the cell expresses the genetically engineered antigen receptor and the inhibitory protein.
  • the inhibitory protein is constitutively expressed.
  • the vector or construct can contain a single promoter that drives the expression of one or more nucleic acid molecules.
  • promoters can be multicistronic (bicistronic or tricistronic).
  • 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.
  • a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (e.g.
  • a self-cleavage peptide e.g., P2A or T2A
  • 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.
  • the peptide such as T2A
  • 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.
  • a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the binding molecule.
  • CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
  • Linker refers 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.
  • 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.
  • P2A porcine teschovirus
  • T2A Thosea asigna virus
  • Other linkers will be apparent to those of skill in the art and can be used in the methods described herein.
  • 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.
  • the nucleic acid comprises complementary DNA (cDNA).
  • 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.
  • nucleic acids as described herein can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art.
  • a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides.
  • the nucleotide sequence is codon- optimized.
  • 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.
  • one or more additional therapeutic agents can be administered to the subject.
  • therapeutical immunological cells e.g., therapeutical T cells
  • the vector can additionally include a nucleic acid sequence that encodes a checkpoint inhibitor (CPI) (e.g., an inhibitory protein).
  • CPI checkpoint inhibitor
  • the checkpoint inhibitor is e.g., any antibody or antigen binding fragment thereof as described herein.
  • 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.
  • 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.
  • 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).
  • cytokines/chemokines such as IL-12 (disclosed in WO2021233317A1) and IL-7/CCL19 (disclosed in WO2017159736A1).
  • the vector can additionally include a nucleic acid sequence that encodes a bifunctional trap fusion protein.
  • the bifunctional trap protein targets both the PD-1 and TGF-p.
  • the bifunctional trap protein targets both the PD-L1 and 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.
  • 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.
  • the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that has been codon-optimized.
  • the alpha and/or beta chain further comprises a signal peptide.
  • the TCR or antigen-binding fragment thereof is isolated or purified or is recombinant.
  • the TCR or antigen-binding fragment is recombinant.
  • the TCR or antigen-binding fragment thereof is human.
  • 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.
  • the disclosure relates to nucleotide sequences
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present disclosure provides a method or process for manufacturing and using the engineered cells for treatment of pathological diseases or conditions.
  • the cells for introduction of the binding molecule can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • a sample such as a biological sample, e.g., one obtained from or derived from a subject.
  • 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.
  • 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.
  • 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.
  • PBMCs peripheral blood mononuclear cells
  • Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
  • 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.
  • 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.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and/or magnesium and/or many or all divalent cations.
  • a washing step is accomplished a semi-automated "flow-through" centrifuge.
  • a washing step is accomplished by tangential flow filtration (TFF).
  • the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca 2+ /Mg 2+ free PBS.
  • components of a blood cell sample are removed and the cells directly resuspended in culture media.
  • 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.
  • 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.
  • the nucleic acid construct further includes a sequence encoding an inhibitory protein.
  • these engineered T cells can block PD-1/PD-L1 immunosuppression and strengthen the antitumor immune response.
  • the method further comprises: transfection of T cells with the viral vector containing the nucleic acid construct.
  • the methods involve introducing any vectors described herein into a cell in vitro or ex vivo.
  • the vector is a viral vector and the introducing is carried out by transduction.
  • 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.
  • the one or more agent is an inhibitory' nucleic acid (e.g., siRNA).
  • 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).
  • a nuclease or an RNA-guided nuclease e.g., a clustered regularly interspaced short palindromic nucleic acid (CRISPR)-associated nuclease.
  • CRISPR clustered regularly interspaced short palindromic nucleic acid
  • 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.
  • transfection of T cells is performed using the calcium phosphate method.
  • 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.
  • the treatment can halt, slow, retard, or inhibit progression of a cancer.
  • 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.
  • 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.
  • tumor 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.
  • the agents described herein are designed for treating or diagnosing a carcinoma in a subject.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma , and chondrosarcoma.
  • the tumor is non-small cell lung cancer, metastatic colorectal cancer, cervical cancer, ovarian cancer, nasopharyngeal cancer, gastric cancer, glioma.
  • 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.
  • the disclosure provides methods for treating infection or infection associated conditions in a subject.
  • 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.
  • 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.
  • 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.
  • an effective amount can be administered in one or more administrations.
  • 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.
  • a cell e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)
  • 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.
  • 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.
  • 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.
  • the condition is cancer, EBV infection, or EBV-positive premalignancy.
  • 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.
  • 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.
  • 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).
  • at least two different engineered cells e.g., cells express different binding molecules
  • engineered cells and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition).
  • engineered cells and the at least one additional therapeutic agent are administered in two different compositions.
  • the at least one additional therapeutic agent is administered as a pill, tablet, or capsule.
  • the at least one additional therapeutic agent is administered in a sustained-release oral formulation.
  • 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.
  • one or more additional therapeutic agents can be administered to the subject.
  • the additional therapeutic agent can be a checkpoint inhibitor (CPI).
  • CPI checkpoint inhibitor
  • 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.
  • 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,
  • the inhibitory protein blocks PD-1 or PD-LI.
  • 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.
  • the inhibitory protein blocks PD-1.
  • 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.
  • 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).
  • the additional therapeutic agent is a bifunctional trap fusion protein.
  • Bifunctional trap proteins can target both immune checkpoints and TGF-P negative regulatory pathways.
  • the tumor microenvironment contains other immunosuppressive molecules.
  • TGFB cytokine TGF-P
  • TGF-P prevents proliferation and promotes differentiation and apoptosis of tumor cells early in tumor development.
  • 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.
  • EMT epithelial- to-mesenchymal transition
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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).
  • the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-l) (IDO1) (e.g., epacadostat).
  • IDO1 indoleamine 2,3-dioxygenase-l
  • 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.
  • 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, pra
  • therapeutic agents
  • 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.
  • TNF tumor necrosis factor
  • carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.
  • 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.
  • compositions including pharmaceutical and therapeutic compositions
  • methods e.g., therapeutic methods for administrating the engineered T cells and compositions thereof to subjects, e.g., patients.
  • 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.
  • the composition includes at least one additional therapeutic agent.
  • 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.
  • 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.
  • 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, arg
  • 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).
  • 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.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • 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.
  • 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.
  • chemotherapeutic agents e.g., asparaginase
  • 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.
  • the cells and compositions can be administered using standard administration techniques, formulations, and/or devices. Administration of the cells can be autologous or heterologous
  • 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
  • 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).
  • Formulations disclosed herein include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
  • the cell populations are administered parenterally.
  • parenteral includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration.
  • the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • antimicrobial preservatives 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.
  • the formulations to be used for in vivo administration are generally sterile. Sterility can be readily accomplished, e.g., by filtration through sterile filtrationmembranes.
  • compositions or pharmaceutical compositions as described herein can be included in a container, pack, or dispenser together with instructions for administration.
  • 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).
  • 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-VivoTM supplemented with 5% human serum A/B.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • RPMI fetal bovine serum
  • X-VivoTM supplemented with 5% human serum A/B.
  • HEPES 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid
  • GlutaMAXTM GlutaMAXTM.
  • 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 lipofectamineTM 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.
  • 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.
  • anti-human CD3/CD28 Dynabeads® Thermo Fisher Scientific, Cat#: 11141D
  • T cells T cells
  • 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.
  • ALPP CAR staining 0.2 x 10 6 untransduced (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.
  • Intracellular IFN-y staining 0.2 x 10 6 untransduced (UT) or ALPP CAR-T cells were co-cultured overnight with 0.4 x 10 6 SiHa 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.
  • the cells were fixed with the CytofixTM 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 * 10 6 SiHa cells labeled with CellTraceTM CFSE (Thermo Fisher Scientific, Cat#: C34554) and 0.03 * 10 6 293T cells labeled with CellTraceTM 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.
  • 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.
  • IACUC Institutional Animal Care and Use Committee
  • 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 () fi CARpositive 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.
  • 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 io 6 SKOV3-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 mm 3 ) and then intravenously injected with 1.0 x 10 7 CAR-positive A02, A02 eJun, or the equivalent number of untransduced cells (1.96 x 10 7 /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: width 2 *length/2.
  • Example 1 Effect of c-Jun co-expression on the expression of anti-ALPP CARs in the target T cells
  • 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.
  • TM transmembrane domain
  • 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).
  • 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 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.
  • 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.
  • anti-ALPP CAR-encoding constructs can also be made.
  • 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).
  • anti-ALPP CARs were generated with sequences summarized in the table below.
  • 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
  • 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).
  • 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%.
  • 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).
  • 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%).
  • 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).
  • 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.
  • MFI geometric mean fluorescence intensity
  • 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).
  • 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%.
  • the drop in the latter case (32.7%) is only about 54.2% of the drop in the former case (60.3%).
  • the c-Jun coexpression significantly improved the sustaining of the surface expression of the target ALPP CAR at this time window (Day 4-12).
  • 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).
  • 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%.
  • 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.
  • ALPP CARs i.e. “8H” and “8H eJun”
  • 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).
  • 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%.
  • the c-Jun co-expression significantly improved the sustaining of the expression of the target ALPP CAR at this time window (Day 5-7).
  • 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).
  • 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%.
  • the c-Jun co-expression significantly improved the sustaining of the expression of the target ALPP CAR at this time window (Day 5-20).
  • c-Jun can significantly sustain, or provide a better sustaining of, the surface expression of ALPP CAR in human primary T cells.
  • 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).
  • 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%.
  • the drop in the latter case (1.5%) is only about 4.3% of the drop in the former case (34.7%).
  • 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).
  • 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).
  • 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%.
  • 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).
  • c-Jun can significantly sustain, or provide a better sustaining of, the surface expression of ALPP CAR in human primary T cells.
  • 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 io 6 untransduced (UT), A02, A02 eJun; or UT, 8H, 8H eJun CAR-T cells were co-cultured overnight with 0.4 * 10 6 SiHa (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.
  • A02 eJun CAR-T cells could be better activated upon antigen-specific stimulation than the A02 CAR-T cells.
  • A02 eJun CAR-T cells could be better activated upon antigen-specific stimulation than the A02 CAR-T cells.
  • 8H eJun CAR-T cells could be better activated upon antigen-specific stimulation than the 8H CAR-T cells.
  • Human PBMCs were transduced with A02, A02 eJun; or 8H, 8H eJun. 12 days post transduction, 0.2 x 10 6 A02, A02 eJun (FIGS. 5A-5B) or 8H, 8H eJun (FIGS. 5C-5D) CAR- T cells were co-cultured with 0.1 x 10 6 SiHa 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.
  • A02 eJun could be better restimulated, compared to A02.
  • 8H eJun could be better restimulated, compared to 8H.
  • Example 6 In vitro proliferation of ALPP CAR-T cells upon repeated antigen-specific stimulation
  • Human PBMCs were transduced with A02, A02 eJun; or 8H, 8H eJun. 12 days post transduction, 1 x 10 6 A02, A02 eJun (FIG. 6A) or 8H, 8H eJun (FIG. 6B) CAR-T cells were co-cultured with 0.5 x 10 6 SiHa for 3 or 4 days, then the cells were counted and 1 x 10 6 T cells were aliquoted and co-cultured with 0.5 x io 6 fresh 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.
  • Example 7 In vivo efficacy of ALPP CAR-T cell in subcutaneous mouse model
  • mice 13 female, 6-week-old NSG mice were subcutaneously (s.c.) implanted with 5.0 x io 6 SKOV3-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 mm 3 and then intravenously injected with 1.0 / I () 7 CAR-positive A02, A02 eJun (“”cJun-A02”), or the equivalent number of untransduced cells (“NT”) (1.96 x 10 7 /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: width 2 *length/2.
  • 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.
  • 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.
  • 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.
  • ALPP CAR i.e., A02
  • Example 8 In vivo efficacy of ALPP CAR-T cell in metastatic tumor mouse model
  • mice 17 female, 6-week-old NSG mice were intraperitoneally implanted with 5.0 x io 6 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 x 10 6 CAR-positive A02, A02 eJun, or the equivalent number of untransduced cells (7.47 x 10 6 /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.
  • the results indicate that A02 eJun CAR-T can significantly improve antitumor efficacy in metastatic tumor model, compared to A02 CAR-T.
  • Example 9 Effects of c-Jun armoring to TCR-T cells
  • the engineered T cells expressing a TCR were tested to examine the effects of c-Jun (i.e. wildtype c-Jun or "eJun") on the TCR expression.
  • c-Jun i.e. wildtype c-Jun or "eJun”
  • the non-treated T cells i.e., "NT”
  • the NY-ESO-1 TCR-T cells i.e., "NY-ESO-1 TCR”
  • the NY-ESO-1 TCR-T cells armored with eJun WT i.e., "NY-ESO-1 TCR.cJun
  • 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.
  • 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).
  • FIGS. 9B-9E even though the armoring of eJun WT can effectively sustain (FIG.
  • Example 10 Effects of c-Jun variant armoring to TCR-T and CAR-T cells
  • 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 T
  • FIGS. 11A-11C The effects of the above eJun variants over TCR and CAR were tested (see FIGS. 11A-11C).
  • TCR eJun annor
  • 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.
  • TCR.cJun eJun WT
  • dJun 30-50 i.e., "TCR.dJun 30-50
  • eJun AA i.e., "TCR.cJun AA
  • 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.
  • the non-treated T cells i.e., "NT"
  • the ALPP CAR-T cells i.e., "CAR”
  • 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.
  • 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).
  • 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.
  • 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).
  • eJun WT effects of eJun WT, dJun 34-47 and dJun 34- 47 AA
  • dJun 30-50 i.e., about 10-15% more if estimated by the curves.
  • the armoring with other c-Jun variants could dramatically sustain the expression of ALPP CAR on the engineered T cells (i.e., all > 70% after the 2nd stimulation).
  • 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.
  • 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.
  • 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.
  • CAR-T cells armored with dJun 34-47 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.
  • Example 12 Effects of c-Jun variant armoring to Aspire-T cells
  • 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.
  • Aspire-TCR or Aspire-T Antigen-specificity redirected TCR complex
  • 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
  • T2A 2A self-cleaving peptide
  • CD3z- CD28_CS 2A self-cleaving peptide
  • CD3z- CD28_CS 2A self-cleaving peptide
  • CD3z- CD28_CS 2A self-cleaving peptide
  • CD3z- CD28_CS 2A self-cleaving peptide
  • E13Y armored IL 13
  • EBY Aspire-TCR expression
  • FIG. 13C Aspire-TCR T cell expansion fold
  • 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.
  • 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.
  • 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.
  • Example 13 Anti-hALPP scFv-Fc fusion proteins binding to human ALPP-expressing cells
  • 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
  • the five scFv-Fc fusion proteins exhibited specific binding to ALPP-293T and SiHa cells, respectively.
  • the five scFv-Fc fusion proteins did not bind to hALPL-293T, hALPI-293T, or parental 293T cells.
  • 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.
  • Example 14 Anti-hALPP scFv-Fc fusion proteins binding to human ALPP protein
  • 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.
  • F8 and H5 CARs 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 10 6 untransduced (UT), the F8 or H5 CAR-T cells were co-cultured overnight with 0.4 * 10 6 SiHa (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.
  • Example 17 SDS-PAGE results of scFv-Fc fusion proteins
  • scFv-Fc fusion proteins C9, F7, F8, H5, and A3 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • 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.
  • the other four scFv-Fc fusion proteins can be successfully expressed, with a single major band in both non-reduced and reduced conditions.
  • 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.
  • CAR chimeric antigen receptor
  • TCR target T cell receptor
  • 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
  • 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:
  • (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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Embodiment 1.11 The method of embodiment 1.10, wherein the target TCR is NY- ESO-1.
  • 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.
  • the immunological cells are T cells, tumor infiltrating lymphocytes, macrophages, natural killer cells, or a combination thereof.
  • 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).
  • CD3 + T cells e.g., a combination of CD4 + and CD8 + T cells
  • NK natural killer
  • alpha beta T cells e.g., alpha beta T cells
  • gamma delta T cells e.g., central memory T cells or effector memory T cells.
  • 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).
  • 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).
  • Embodiment 1.15 The method of embodiment 1.14, wherein the immunological cells are CD4 + T cells.
  • Embodiment 1.16 The method of embodiment 1.14, wherein the immunological cells are CD8 + T cells.
  • 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).
  • a selfcleavage peptide e.g., P2A or T2A
  • a protease recognition site e.g., furin
  • Embodiment 1.19 The method of embodiment 1.17, wherein the two polynucleotide fragments in the single vector have two different promoters.
  • 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.
  • 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.
  • CAR target chimeric antigen receptor
  • TCR target T cell receptor
  • Embodiment 2.2 The engineered immunological cell of embodiment 2.1, further co-expressing other agent (e.g., PD-1 and/or IL12).
  • other agent e.g., PD-1 and/or IL12
  • 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.
  • CAR target chimeric antigen receptor
  • TCR target T cell receptor
  • Embodiment 3.2 The method of embodiment 3.1, wherein the disease is cancer.
  • 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.
  • Embodiment 3.4 The method of embodiment 3.1, wherein the disease is a non-cancer disease.
  • 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).
  • other agent e.g., PD-1 and/or IL12
  • 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.
  • CAR chimeric antigen receptor
  • TCR target T cell receptor
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Embodiment 4.8 The method of any one of embodiments 4. 1-4.7, wherein the target CAR is a ALPP CAR.
  • Embodiment 4.9 The method of any one of embodiments 4. 1-4.7, wherein the target TCR is aNY-ESO-1 TCR.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • TILs tumor infiltrating lymphocytes
  • NK natural killer
  • 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.
  • 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.
  • 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.
  • Embodiment 5.4 The population of immunological cells of any one of embodiments
  • Embodiment 5.5 The population of immunological cells of any one of embodiments
  • 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.
  • 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.
  • 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.
  • 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
  • T lymphocytes T lymphocytes
  • TILs tumor infiltrating lymphocytes
  • NK. natural killer cells

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Abstract

L'invention des présentes propose des compositions et des procédés pour maintenir l'expression de surface d'un récepteur chimérique de l'antigène (CAR) cible et/ou d'un récepteur des lymphocytes T (TCR) cible dans des cellules immunologiques modifiées (par exemple, des lymphocytes T). Dans certains modes de réalisation, les procédés comprennent : la co-expression d'un polypeptide qui comprend un type sauvage ou un certain mutant de c-Jun, ou le variant fonctionnel associé dans les cellules immunologiques modifiées. L'invention propose également une composition comprenant un certain mutant c-Jun avec une région delta perturbée. Des cellules immunologiques co-exprimant le mutant c-Jun présentent une réponse immunologique améliorée, telle qu'un niveau réduit d'activation basale, une réactivité accrue, une capacité d'expansion accrue et/ou une toxicité cytolytique accrue contre des cellules cibles.
EP23908029.4A 2022-12-23 2023-06-14 Compositions et procédés pour améliorer des réponses immunologiques dans des cellules immunologiques modifiées Pending EP4638484A2 (fr)

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