WO2022053036A1 - Downregulation of membrane-bound proteins by receptor tac technology - Google Patents

Downregulation of membrane-bound proteins by receptor tac technology Download PDF

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WO2022053036A1
WO2022053036A1 PCT/CN2021/117886 CN2021117886W WO2022053036A1 WO 2022053036 A1 WO2022053036 A1 WO 2022053036A1 CN 2021117886 W CN2021117886 W CN 2021117886W WO 2022053036 A1 WO2022053036 A1 WO 2022053036A1
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scfv
cell
domain
protein
cd3ζ
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PCT/CN2021/117886
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English (en)
French (fr)
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Li Zhou
Feng Shi
Michael Harris
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Shandong Boan Biotechnology Co Ltd
Boan Boston LLC
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Shandong Boan Biotechnology Co Ltd
Boan Boston LLC
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Priority to US18/026,063 priority Critical patent/US20230355764A1/en
Priority to JP2023516810A priority patent/JP7685044B2/ja
Priority to EP21866088.4A priority patent/EP4178986A4/en
Priority to CN202180062485.5A priority patent/CN116368152A/zh
Publication of WO2022053036A1 publication Critical patent/WO2022053036A1/en
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Definitions

  • This invention was made subject to a joint research agreement between SHANDONG BOAN BIOTECHNOLOGY CO., LTD. and Boan Boston LLC.
  • This application relates to downregulation of Membrane-Bound Proteins (MBPs) , and in particular, to downregulation of Membrane-Bound Proteins by Receptor TAC Technology.
  • MBPs Membrane-Bound Proteins
  • the present application provides novel fusion proteins, nucleic acids encoding said proteins, vectors comprising said nucleic acids, compositions comprising said nucleic acids or vectors, host cells comprising said nucleic acids, vectors or compositions or pharmaceutical compositions.
  • the present application also provides methods of reducing (down regulating) a target membrane-bound protein (MBP) level in a cell, methods of producing a cell having a reduced target membrane-bound protein level, or methods of treating a disease, or methods of reducing or preventing GvHD in a subject associated with the administration of one or more CAR T-cells to the subject.
  • MBP target membrane-bound protein
  • CAR Chimeric Antigen Receptor
  • Target recognition by these receptors is usually provided through a scFv domain on the extracellular terminal of the CAR, though alternative approaches have also been designed (Kuhn, N.F. et al. Cancer Cell 35, 473-488. e6 (2019) ) .
  • CD19 CAR-T drugs Two approved CD19 CAR-T drugs (Kymriah and Yescarta) as well as Abecma (BCMA CAR-T) are all autologous products. They are made from each individual patient’s own T cells. While autologous CD19 CAR-T demonstrated unprecedented efficacy in refractory and relapse cancer patients, they do represent significant challenges moving forward: they require complex manufacturing; there are usually significant variations in the quality of CAR-T cells due to patient variations, and this results in variations in the quality of the product and even production failure (with failure rates up to 14%) (Bersenev, A. Transfusion (2017) . doi: 10.1111/trf.
  • Allogeneic (off-the-shelf) CAR-T on the other hand will offer many advantages.
  • the cells can be batch-prepared in advance and can supply thousands of doses; the product is frozen, stored, and distributed for off-the-shelf use; the CAR-T cells will be prepared from healthy donors with better cell quality; the cells will be of uniform quality per batch; there will be minimal lead time for patients; and the cost of goods can be significantly reduced (Rafiq, S. et, al., Nature Reviews Clinical Oncology (2020) ) , (Depil, S. et al., Nature Reviews Drug Discovery (2020) ) .
  • GvHD Graft versus Host Disease
  • they can be prepared by gene editing technology such as CRISPR (clustered regularly interspaced short palindromic repeats) -mediated TCR knockdown, or they can be prepared through siRNA down regulation of TCR, or they can be prepared with cells that naturally do not trigger GvHD such as cord blood or PBMC-derived NK cells (Li, Y. et al., Cell Stem Cell (2016) ) , NK92 cell line, or gamma delta T cells etc. (Marcus, A. et al., Expert Opinion on Biological Therapy (2014) ) , (Depil, S.
  • the major challenge with gene editing technology is that there is always a risk of off-target modification.
  • the rate of off-target modification can be as high as 50% (Zhang, X.H. et al., Molecular Therapy -Nucleic Acids (2015) ) , which can lead to unwanted mutations and would pose a significant risk for patients.
  • the manufacturing process is complicated as it requires two steps of modifications: the first step is to transfect the CAR into T cells, the second step is to introduce CRISPR machinery to knockout TCR.
  • NK cells and gamma delta T cells are associated with less persistence and proliferation in vivo, which will significantly affect their potency. They are also difficult to expand in vitro (Li, Y., Cell Stem Cell (2018) ) , (Rezvani, K., et al., Molecular Therapy (2017) ) , (Shimasaki, N. et al., Nature Reviews Drug Discovery (2020) ) .
  • MBPs Membrane-Bound Proteins
  • the present application provides novel fusion proteins, nucleic acids encoding said proteins, vectors comprising said nucleic acids, compositions comprising said nucleic acids or vectors, cells (e.g. host cells) comprising said nucleic acids, vectors or compositions or pharmaceutical compositions.
  • the present application also provides methods of reducing (down regulating) a target membrane-bound protein (MBP) level in a cell, methods of producing a cell having a reduced target membrane-bound protein level, or methods of treating a disease, or methods of reducing or preventing GvHD in a subject associated with the administration of one or more CAR T-cells to the subject.
  • MBP target membrane-bound protein
  • MBPs Membrane-Bound Proteins
  • the degradation tag protein comprises ubiquitin ligase and/or domains of the cell surface receptors that mediate degradation (e.g. IL2R ⁇ sub domain, L2R ⁇ jm domain) .
  • the tagging specificity or targeting specificity (or recognition specificity) of the MBPs to be degraded by the degradation tag protein e.g.
  • ubiquitin ligase or IL2R ⁇ is mediated by an antibody (or antigen-binding fragment) of MBPs to be degraded (that is, antibody mediated substrate recognition) , or mediated by subunits or structural domains of the membrane-bound protein or complex to be degraded that have a transmembrane domain (that is, transmembrane domain mediated substrate recognition) .
  • the technical solution of the present application can be applied to any targets on a cell surface, independent of cell type.
  • the fusion protein of the application comprises:
  • a degradation tag protein that mediates degradation of a membrane-bound protein (MBP)
  • MBP membrane-bound protein
  • transmembrane-linking domain comprising:
  • a first transmembrane-linking domain which comprises a first transmembrane domain and a first linking protein
  • the first transmembrane domain is selected from the transmembrane domain of one or more subunits or structural domains of CD8 ⁇ , CD4, CD3, CD28, 4-1BB or IL2R or a variant thereof;
  • the first linking protein is selected from one or more of an antibody or antigen-binding fragment of the MBP to be degraded; and the first transmembrane domain is linked directly or indirectly to the first linking protein;
  • the fusion protein comprising the transmembrane-linking domain of above 1) further comprises a first signal peptide;
  • a second transmembrane-linking domain which comprises a transmembrane domain of the MBP to be degraded or a variant thereof.
  • the MBP is a membrane-bound receptor; more preferably a mammalian origin membrane-bound receptor, most preferably a human membrane-bound receptor;
  • the ubiquitin ligase comprises one or more subunits or structural domains of E3 ubiquitin ligase or a variant thereof;
  • the cell surface receptors that mediate degradation comprise one or more subunits or structural domains of Interleukin receptors or a variant thereof;
  • the antigen-binding fragment of MBP to be degraded in the above 1) comprises a scFv or nano antibody specifically binding to the MBP or a variant thereof;
  • the transmembrane domain of the MBP to be degraded or a variant thereof in the above 2) comprises a transmembrane domain of one or more of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ or a variant thereof;
  • the second transmembrane-linking domain of above 2) comprises an extracellular domain and a transmembrane domain of one or more of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ or a variant thereof;
  • the second transmembrane-linking domain of above 2) comprises a second signal peptide, extracellular domain and a transmembrane domain of one or more of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ or a variant thereof.
  • the membrane-bound receptor is selected from one or more subunits or structural domains of CD3, TCR, CD5, CD7, PD-L1, and CD47 or a variant thereof;
  • the E3 ubiquitin ligase is selected from the following proteins or the truncated forms thereof: C-terminus of Hsc70-interacting protein (CHIP) , CHIP. dTPR, F-box WD40-containing protein 7 (FBW7) , FBW7.2-293, von Hippel-Lindau (VHL) , VHL. 152-213, Speckle-type BTB-POZ protein (SPOP) , SPOP.
  • Ubiquitin-protein ligase E3A Ubiquitin-protein ligase E3A
  • MDM2 Mouse double minute 2 homolog
  • APC Anaphase-promoting complex
  • UBR5 LNX
  • Casitas B-lineage lymphoma-transforming sequence-like protein 1 CBLL1
  • HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 HACE1
  • HECT, C2 and WW domain containing E3 ubiquitin protein ligase 1 HECW1
  • HECT, C2 and WW domain containing E3 ubiquitin protein ligase 2 HECW2
  • HECT And RLD Domain Containing E3 Ubiquitin Protein Ligase 1 HERC1 , HERC2, HERC3, HERC4, HERC5, HERC6, HUWE1, ITCH
  • neural precursor cell expressed developmentally down-regulated protein 4 NEDD4
  • the E3 ubiquitin ligase comprises CHIP, CHIP. dTPR, FBW7.2-293, VHL. 152-213, SPOP. 167-374, SOCS2.143-198, GRAIL. IC, RNF133. IC, RNF122. rIC or RNF152. rIC; and more preferably, the CHIP, CHIP. dTPR, FBW7.2-293, VHL. 152-213, SPOP. 167-374, SOCS2.143-198, GRAIL. IC, RNF133. IC, RNF122. rIC or RNF152. rIC comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in any one of SEQ ID NOs. 21-26, 62, 79, 81 or 83 respectively;
  • the subunits or structural domains of Interleukin receptors comprise a lysosomal targeting motif of IL-2R; preferably, the lysosomal targeting motif of IL-2R comprises L2R ⁇ jm domain; more preferably, the L2R ⁇ jm domain (IL2R ⁇ jm) comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO. 58;
  • the first transmembrane domain in the above 1) is selected from a transmembrane domain of CD3, CD8 ⁇ , CD4, or a variant thereof; preferably, CD3 ⁇ transmembrane domain variant, CD8 ⁇ transmembrane domain, or CD4 transmembrane domain comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO. 89, 18 or 15 respectively;
  • the first signal peptide is selected from an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO. 19 or 20 respectively;
  • the antibody or antigen-binding fragment of the MBP in the above 1) is selected from antibody or antigen-binding fragment of CD3, CD5, CD7, PD-L1 or CD47; preferably, antigen-binding fragment of CD3 comprises SP34 scFv, OKT3 scFv, UCHT1 scFv, UCHT1. Y177T scFv, L2K scFv, F6A scFv, BMA031 scFv, or BMA031. H6L12 scFv; antigen-binding fragment of CD5 comprises ⁇ CD5.14 scFv; antigen-binding fragment of CD7 comprises ⁇ CD7.
  • antigen-binding fragment of PD-L1 comprises ⁇ PD-L1 scFv
  • antigen-binding fragment of CD47 comprises ⁇ CD47 scFv
  • TH69 scFv, ⁇ PD-L1 scFv or ⁇ CD47 scFv comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in any one of SEQ ID NOs. 2-13 respectively;
  • the second transmembrane-linking domain of above 2) comprises a transmembrane domain of CD3 ⁇ or CD3 ⁇ , preferably, an extracellular domain and a transmembrane domain of CD3 ⁇ or CD3 ⁇ , more preferably, a second signal peptide, extracellular domain and a transmembrane domain of CD3 ⁇ or CD3 ⁇ ; more preferably, the transmembrane domain (TM) of CD3 ⁇ or CD3 ⁇ comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO.
  • the extracellular domain (EM) of CD3 ⁇ or CD3 ⁇ comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO. 66, 69 respectively;
  • the second signal peptide of CD3 ⁇ or CD3 ⁇ comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO. 65, 68 respectively; more preferably, the second signal peptide, extracellular domain and a transmembrane domain of CD3 ⁇ (CD3 ⁇ .
  • CD3 ⁇ . ⁇ IC and the second signal peptide, extracellular domain and a transmembrane domain of CD3 ⁇ (CD3 ⁇ . ⁇ IC) comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO. 61, 64 respectively.
  • the fusion protein further comprises one or more of a hinge, or a P2A-GFP;
  • the hinge is selected from a CD8 ⁇ hinge or CD4 hinge; more preferably, CD8 ⁇ hinge or CD4 hinge comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO.: 16 or 14, respectively; more preferably, the hinge is adjacent to the N-terminal of the first transmembrane domain,
  • P2A-GFP comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO.: 57; more preferably, the P2A-GFP sequence is at the C-terminal of the fusion protein.
  • the fusion protein comprises from N-terminal to C-terminal:
  • the first linking protein comprises the antibody or antigen-binding fragment of the MBP to be degraded; more preferably, the antibody or antigen-binding fragment of the MBP to be degraded comprises antibody or antigen-binding fragment of CD3, CD5, CD7, PD-L1 or CD47; more preferably, antigen-binding fragment of CD3 comprises SP34 scFv, OKT3 scFv, UCHT1 scFv, UCHT1. Y177T scFv, L2K scFv, F6A scFv, BMA031 scFv, or BMA031.
  • the degradation tag protein comprises E3 ubiquitin ligase; more preferably, the E3 ubiquitin ligase comprises CHIP. dTPR, FBW7.2-293, VHL. 152-213, SPOP. 167-374, or SOCS2.143-198;
  • the first transmembrane domain comprises CD8 ⁇ transmembrane domain
  • the first linking protein comprises the antibody or antigen-binding fragment of the MBP to be degraded; more preferably, the antibody or antigen-binding fragment of the MBP to be degraded comprises the antibody or antigen-binding fragment of CD3; more preferably, antigen-binding fragment of CD3 comprises SP34 scFv;
  • the degradation tag protein comprises an E3 ubiquitin ligase; more preferably, E3 ubiquitin ligase comprises GRAIL. IC;
  • the first transmembrane domain is selected from one or more of CD3 ⁇ transmembrane domain variant, CD8 ⁇ transmembrane domain, and CD4 transmembrane domain;
  • the second transmembrane-linking domain of the above 2) comprises CD3 ⁇ . ⁇ IC or CD3 ⁇ . ⁇ IC;
  • the cell surface receptors that mediate degradation comprise IL2R ⁇ jm; or
  • the second transmembrane-linking domain of the above 2) comprises CD3 ⁇ .
  • ⁇ IC the second transmembrane-linking domain of the above 2
  • the one or more of an E3 ubiquitin ligase or a variant thereof comprises GRAIL. IC, CHIP. dTPR, RNF133. IC, RNF122. rIC, RNF152. rIC.
  • the fusion protein comprises from N-terminal to C-terminal:
  • LG114 SP-OKT3 scFv-FBW7-CD8 ⁇ hinge/TM-P2A-eGFP;
  • LG124 SP-UCHT1.
  • LG213, CD3 ⁇ ⁇ IC-CHIP. dTPR-P2A-GFP;
  • the fusion protein does not include P2A-eGFP (i.e. P2A-GFP) and/or SP.
  • the present application provides a nucleic acid, wherein the nucleic acid comprises a polynucleotide encoding a fusion protein of the present application.
  • the present application provides a vector, wherein the vector comprises a nucleic acid of the present application.
  • an expression promoter for the fusion protein comprises CAG or EF1a;
  • the CAG or EF1a comprises a nucleic acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the nucleic acid sequence shown in SEQ ID NO.: 72 or 88, respectively;
  • the expression promoter for the a) fusion protein is EF1a;
  • the expression promoter for the b) fusion protein is EF1a;
  • the expression promoter for the c) fusion protein is EF1a;
  • the expression promoter for the LG171p1 in d) fusion protein is CAG, more preferably, the expression promoter for the other fusion proteins in d) fusion protein is EF1a.
  • the present application provides a composition, the composition comprising a nucleic acid or a vector of the present application.
  • the composition comprises a first nucleic acid and a second nucleic acid; or comprising a first vector having a first nucleic acid and a second vector having a second nucleic acid; or comprising a vector having a first nucleic acid and a second nucleic acid, wherein
  • the first nucleic acid encodes the fusion protein of the present application
  • the second nucleic acid encodes a chimeric antigen receptor (CAR) comprising:
  • a cytoplasmic segment comprising one or more signaling domains, preferably comprising a 4-1BB signaling domain and a CD3 ⁇ signaling domain.
  • the fusion protein in the composition which comprises a first nucleic acid and a second nucleic acid; or comprising a first vector having a first nucleic acid and a second vector having a second nucleic acid; or comprising a vector having a first nucleic acid and a second nucleic acid, is selected from one or more of the above a) -d) fusion proteins.
  • the predetermined antigen is a tumor-related antigen.
  • the tumor-related antigen is selected from the following group: CEA, Claudin 18.2, GPC3, Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) , CD38, CD19, CD20, CD22, BCMA, CAIX, CD446, CD133, EGFR, EGFRvIII, EpCam, GD2, EphA2, Her1, Her2, ICAM-1, IL13Ra2, Mesothelin, MUC1, MUC16, NKG2D, PSCA, NY-ESO-1, MART-1, WT1, MAGE-A10, MAGE-A3, MAGE-A4, EBV, NKG2D, PD1, PD-L1, CD25, IL-2, or CD3.
  • ROR1 Receptor tyrosine kinase-like Orphan Receptor 1
  • the tumor-related antigen is CEA.
  • the present application provides a cell (e.g. host cell) , the cell comprising a nucleic acid or a vector or a composition of the present application.
  • a cell e.g. host cell
  • the cell comprising a nucleic acid or a vector or a composition of the present application.
  • the host cell is a mammalian cell, preferably a human cell.
  • the host cell is a T cell or a primary T cell, gamma delta T cell, NK cell, NKT cell, macrophage, B cell, or non-immune cell.
  • the host cell is an allogeneic cell.
  • the present application provides a pharmaceutical composition, the pharmaceutical composition comprising the fusion protein, the nucleic acid, vector, composition or cell of the present application.
  • the composition further comprises one or more pharmaceutically acceptable excipients.
  • the present application provides a method of reducing a target membrane-bound protein level in a cell, comprising introducing into a cell a nucleic acid, a vector, or a composition of the present application.
  • the cell e.g. host cell
  • the cell is a mammalian cell, preferably a human cell.
  • the host cell is a T cell or a primary T cell, gamma delta T cell, NK cell, NKT cell, macrophage, B cell, or non-immune cell.
  • the host cell is an allogeneic cell.
  • the present application provides a method of producing a cell having reduced target membrane-bound protein level, comprising introducing into a cell a nucleic acid, a vector, or a composition of the present application.
  • the cell is a mammalian cell, more preferably a human cell; more preferably, the cell is a T cell or a primary T cell, gamma delta T cell, NK cell, NKT cell, macrophage, B cell, or non-immune cell; most preferably the cell is an allogeneic cell.
  • the present application provides a method of treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of allogeneic cells having the composition of the present application or administering to a subject in need thereof a therapeutically effective amount of cells of the present application, wherein preferably, the subject has reduced Graft-versus-Host Disease (GvHD) .
  • the cell is a mammalian cell, more preferably a human cell; more preferably, the cell is a T cell or a primary T cell, gamma delta T cell, NK cell, NKT cell, macrophage, B cell, or non-immune cell; most preferably the cell is an allogeneic cell.
  • the present application provides a method of reducing or preventing GvHD in a subject associated with the administration of one or more CAR T-cells to the subject, comprising
  • the present application provides a method of downregulating membrane-bound protein (MBP) in a cell population, comprising adding to said cell population a host cell of the present application, wherein the host cell expresses fusion protein comprising scFv specifically binding to said MBP, and/or the host cell expresses fusion protein comprising a component of MBP or a fragment thereof that has a transmembrane domain.
  • MBP membrane-bound protein
  • the host cell population is a T cell population, preferably a human primary T cell population.
  • the MBP is CD3, preferably human CD3
  • the present application has the following advantages:
  • the fusion protein of the present application could reduce (down regulate) a target membrane-bound protein (MBP) level in a cell;
  • CAR T-cells of the present application could reduce or prevent GvHD in a subject.
  • Fig. 1 Cartoon for allogenic CAR-T host cells generation to avoid potential GvHD responses against recipient tissues. Due to different development/maturation experiences of T cells, donor T cells, when infused into different recipients, may potentially elicit strong immune responses, e.g. cytotoxicity, cytokine release syndrome and neurotoxicity, against foreign antigens (GvHD) . By degrading TCR/CD3 complex in donor cells, such kind of unfavorable effects could be largely eliminated.
  • GvHD foreign antigens
  • Fig. 2 Schematic representation of TCR/CD3 Degradation constructs and Reference constructs.
  • E3 ligases or a variant thereof functional domain of E3 ligases
  • FBW7, VHL, SPOP and CHIP. dTPR functional domain of E3 ligases
  • dTPR CHIP. dTPR
  • Cytoplasmic constructs obtained by engaging antibody or antigen-binding fragment of the CD3 to E3 ligase/ubiquitin, but have no SP sequence and no hinge/TM sequence, and expressed in cytoplasm of a cell.
  • Cytoplasmic or Membrane-anchored constructs (LG021, LG022, LG023, LG024 constructs) have no E3 ligase/ubiquitin sequence, only antibody or antigen-binding fragment of the CD3.
  • TCR/CD3 degradation constructs (LG112, LG113) were obtained by engaging antibody or antigen-binding fragment of the CD3 to E3 ligase/ubiquitin, and have both SP sequence and hinge/TM sequence.
  • Fig. 3A-3E Effect of cytoplasmic constructs (LG089, LG091, LG092, LG085) on TCR/CD3 complex in human primary T cells.
  • Human isolated primary T cells were activated in vitro using StemCell Immunocult-XF/Activator for 3 days, in the presence of human IL-2, IL-7 and IL-15. Then activated primary T cells were collected and electroporated in the presence of plasmids expressing one of the cytoplasmic constructs for TCR/CD3 degradation. Two days later, cells were harvested and stained for both CD3 (PE) and TCR ⁇ / ⁇ (APC) .
  • PE CD3
  • APC TCR ⁇ / ⁇
  • FIG. 3A shows systemic controls pmaxGFP and LS008 has good transfection efficiency in primary T cells, and there was no downregulation of TCR ⁇ / ⁇ and CD3 in either of these two constructs.
  • FACS results of Fig. 3B-3E show no obvious degradation of TCR/CD3 complex when anti-CD3 scFv (e.g. SP34 scFv or OKT3 scFv) conjugated E3 ligases, including FBW7 (Fig. 3B) , VHL (Fig. 3C) , SPOP (Fig. 3D) and hCHIP.
  • dTPR Fig. 3E
  • FIG. 4A-4D Membrane-anchored OKT3 construct (LG021, LG022, LG023, LG024) confers downregulation of TCR/CD3 complex in human primary T cells.
  • Human isolated primary T cells were activated in vitro using StemCell Immunocult-XF/Activator for 3 days, in the presence of human IL-2, IL-7 and IL-15. Then cells were collected and electroporated with 1 ⁇ g DNA plasmid expressing one of the cytoplasmic/membrane-anchored constructs for TCR/CD3 degradation. Two days later, cells were harvested and stained for both CD3 (PE) and TCR ⁇ / ⁇ (APC) .
  • PE CD3
  • APC TCR ⁇ / ⁇
  • Membrane-anchored OKT3 construct confers downregulation of TCR/CD3 complex in Jurkat cells.
  • Jurkat cells were electroporated with both PiggyBac transposase mRNA and transposon expressing the membrane-anchored OKT3 construct (LG024) .
  • the Jurkat Cells were maintained until day 57 and subjected to flow cytometry testing post staining with CD3 (PE) and TCR ⁇ / ⁇ (APC) , showing significant degradation of TCR/CD3 complex in cells with stable integration of transposon expressing the membrane-anchored OKT3 construct (LG024) (GFP positive) .
  • PE CD3
  • APC TCR ⁇ / ⁇
  • Fig. 6A-6D The membrane outside linkage of E3 ligase to OKT3 scFv construct (LG112) confers better downregulation of TCR/CD3 complex in Jurkat cells line than cytoplasmic linkage of E3 ligase to OKT3 scFv construct (LG113) .
  • Jurkat cells were electroporated with both PiggyBac transposase mRNA and transposon expressing different membrane-anchored OKT3 constructs (LG112, LG113) .
  • the Jurkat Cells were harvested on day 3 and subjected to flow cytometry analysis post staining with CD3 (PE) and TCR ⁇ / ⁇ (APC) .
  • PE CD3
  • APC TCR ⁇ / ⁇
  • FIG. 6A shows systemic controls pmaxGFP and LS008 have good transfection efficiency in Jurkat cells, and there was no downregulation of TCR ⁇ / ⁇ and CD3 in either of these two constructs.
  • Membrane-anchored OKT3 scFv construct without E3 ubiquitin ligase (LG024) shows significant downregulation of TCR-CD3 complex in Jurkat cells (Fig. 6B) .
  • LG024 E3 ubiquitin ligase
  • Fig. 7A-7D The membrane outside linkage of E3 ligase to OKT3 construct (LG112) confers better downregulation of TCR/CD3 complex in human primary T cells than cytoplasmic linkage of E3 ligase to OKT3 scFv construct (LG113) .
  • Human isolated primary T cells were activated in vitro using StemCell Immunocult-XF/Activator for 3 days, in the presence of human IL-2, IL-7 and IL-15. Then the activated primary T cells were collected and electroporated with both PiggyBac transposase mRNA and transposon expressing different membrane-anchored OKT3 constructs.
  • Fig. 7A shows systemic controls pmaxGFP and LS008 have good transfection efficiency in primary T cells, and there was no downregulation of TCR ⁇ / ⁇ and CD3 in either of these two constructs. Similar to what we found in Jurkat cell line, the degradation could be more significant when hCHIP.
  • dTPR was linked to OKT3 scFv in an outside membrane manner (proximal linkage) (LG112) (Fig. 7C) , instead of cytoplasmic manner (LG113) (Fig. 7D) . No significant changes on TCR/CD3 degradation were found when E3 ubiquitin ligase (CHIP.
  • dTPR was located in the cytoplasmic portion of the whole construct (LG113, Fig. 7D) in comparison to Membrane-anchored OKT3 scFv construct without E3 ubiquitin ligase (LG024) (Fig. 7B) .
  • Fig. 8 A-8E TCR/CD3 Degradation constructs with different E3 ligases were designed and synthesized.
  • Human isolated primary T cells were activated in vitro using StemCell Immunocult-XF/Activator for 3 days, in the presence of human IL-2, IL-7 and IL-15. Then the primary T cells were collected and electroporated with both PiggyBac transposase mRNA and transposon expressing different membrane-anchored OKT3 constructs.
  • TCR ⁇ / ⁇ Three days later, the primary T cells were harvested and stained for both CD3 (PE) and TCR ⁇ / ⁇ (APC) .
  • PE CD3
  • APC TCR ⁇ / ⁇
  • FBW7 Fig. 8B
  • VHL Fig. 8C
  • SPOP Fig. 8D
  • SOCS2 Fig. 8E
  • Fig. 9A A list of TCR/CD3 degradation constructs with different TCR-CD3 targeting scFv, including SP34, UCHT1, UCHT1. Y177T, L2K and F6A, were designed and synthesized.
  • Human isolated primary T cells were activated in vitro using StemCell Immunocult-XF/Activator for 3 days, in the presence of human IL-2, IL-7 and IL-15. Then the primary T cells were collected and electroporated with both PiggyBac transposase mRNA and transposon DNA constructs.
  • TCR/CD3 downregulation effects by SP34 Fig. 9B
  • UCHT1 Fig. 9C
  • Y177T Fig. 9D
  • L2K Fig. 9E
  • F6A Fig. 9F
  • Fig. 10A-10E E3 ligase (CHIP. dTPR) is necessary for optimized TCR/CD3 downregulation in human primary T cells.
  • Fig. 10A A list of TCR degradation constructs with TCR targeting scFv, BMA031 or BMA031. H6L12, were designed and synthesized in the presence or absence of CHIP. dTPR.
  • Human isolated primary T cells were activated in vitro using StemCell Immunocult-XF/Activator for 3 days, in the presence of human IL-2, IL-7 and IL-15. Then cells were collected and electroporated with both PiggyBac transposase mRNA and transposon DNA constructs.
  • TCR/CD3 downregulation effects by BMA031 (LG119, Fig. 10B) (LG133, Fig. 10D) or BMA031.
  • H6L12 LG120, Fig. 10C
  • Fig. 10E were tested in the presence (Fig. 10B) (Fig. 10C) or absence (Fig. 10D) (Fig. 10E) of CHIP. dTPR.
  • Fig. 11A-Fig. 11B show CD3 downregulation confers lower activation of Jurkat cells through CD3/CD28/CD2 stimulation.
  • Fig. 11A shows that CD69 (a T cell activation marker) expression levels were low for all cells at the baseline;
  • Fig. 11B shows the CD69 expression levels in either LG024 or LG112 transfected GFP+ cells were significantly less compared to T cells LS008 transfected cells at 4 hours post stimulation.
  • Fig. 12A-12C CD5 or CD7 downregulation constructs and validation.
  • Fig. 12A shows a list of CD5 or CD7 degradation constructs were designed and synthesized.
  • Fig. 12B shows ⁇ CD5.14 scFv conferred strong downregulation of CD5;
  • Fig. 12C shows ⁇ CD7.
  • TH69 scFv CD7-targeting scFv also showed significant downregulation effect in human primary T cells.
  • Fig. 13 A-13B Down regulation of PD-L1 and CD47 by E3 CHIP construct.
  • Fig. 13A shows schematic representation of E3 Constructs MLB052 and MLB053, which are specific to PD-L1 and CD47, respectively. Each construct is comprised of an N-terminal ScFv domain specific to the target antigen and a truncated version of the E3 ligase CHIP lacking the TPR domain.
  • the E3 degrader construct (MLB052 or MLB053) is anchored to the membrane by a flexible hinge and transmembrane anchor.
  • Fig. 14A-Fig. 14B Paracrine downregulation of TCR expression by E3 CHIP linked CD3-targeting construct (LG024) .
  • Fig. 14A shows Gating Strategy for co-culture assay showing paracrine downregulation of TCR expression. Gating on CellTrace Violet-positive (i.e. Cell Trace + cells, without LG024-transfection) and -negative cells (i.e. Cell Trace – cells, LG024-transfected) allows for the characterization of these distinct populations (the left column of Fig. 14A) . LG024-transfected cells (i.e.
  • Fig. 14B shows Jurkat cells electroporated with LG024 (i.e. Cell Trace – cells, LG024-transfected) were serially diluted with parental stock Jurkat cells labeled with CellTrace Violet (i.e. Cell Trace + cells, without LG024-transfection) .
  • Fig. 15A-Fig. 15D Impacts of different hinge/transmembrane domains on TCR/CD3 downregulation in Jurkat mediated by both CD3-targeting scFv (i.e. SP34) and GRAIL.
  • Fig. 15A shows construct map for LG222, LG222.1 and LG222.2
  • Fig. 15B-D shows Jurkat cells were electroporated with PiggyBac vectors expressing LG222, LG222.1 or LG222.2 in combination with PiggyBac Transposase mRNA.
  • TCR surface expression was evaluated by CD3 and TCR ⁇ / ⁇ staining.
  • Fig. 16A-Fig. 16C The impacts of different hinge/transmembrane domains on TCR/CD3 downregulation in human primary T cells mediated by both CD3-targeting scFv (i.e. SP34) and GRAIL. IC.
  • Fig. 16A-C shows human primary T cells were pre-activated and electroporated with PiggyBac vectors expressing LG222, LG222.1 or LG222.2 in combination with PiggyBac Transposase mRNA. At day 3 post electroporation, TCR surface expression was evaluated by CD3 and TCR ⁇ / ⁇ staining.
  • Fig. 17 Overview of receptor endocytosis.
  • Cell surface receptors are endocytosed via clathrin-mediated endocytosis or via clathrin-independent endocytosis, where they are internalized as intracellular vesicles. Vesicles containing the surface receptor ectodomain within their lumens are trafficked via adaptor proteins to the early endosomes for further sorting. From the early endosome, surface receptors can either be trafficked back to the cell surface through recycling endosomes, or aggregated within multivesicular bodies. Sorting back to the surface or to alternative intracellular pathways is dependent on motifs in the transmembrane and entodomains of these surface receptors.
  • receptors are processed further into late endosomes and eventually to lysosomes, where the low pH of the lumen, as well as lysosomal proteases, results in the degradation of the cell surface receptor and its associated cargo.
  • FIG. 18A Schematic diagram of the CD3 ⁇ . ⁇ IC-IL2R ⁇ jm (MLB014) and CD3 ⁇ . ⁇ IC-IL2R ⁇ jm (MLB046) chimeric proteins (i.e. fusion proteins) .
  • Our design is based on the on the extracellular and transmembrane sequences of CD3 ⁇ (MLB014) and CD3 ⁇ (MLB046) directly fused to the first 27 amino acids of the IL2R ⁇ entodomain (i.e. IL2R ⁇ jm) , which bears its lysosomal targeting motif. Inclusion of the CD3 ⁇ and CD3 ⁇ transmembrane domains allows our constructs to be incorporated into the multi-subunit TCR complex.
  • ⁇ IC-IL2R ⁇ jm and CD3 ⁇ ⁇ IC-IL2R ⁇ jm chimeric proteins (i.e. fusion proteins) causes TCR downregulation in Jurkat E6.1 NFAT-luciferase reporter cells.
  • Jurkat cells were electroporated with PiggyBac vectors expressing the CD3 fusion proteins and PiggyBac Transposase mRNA to induce expression of CD3 ⁇ .
  • ⁇ IC-IL2R ⁇ jm MLB014) or CD3 ⁇ .
  • ⁇ IC-IL2R ⁇ jm MLB046) .
  • TCR surface expression was evaluated by CD3 staining.
  • ⁇ IC-IL2R ⁇ jm and CD3 ⁇ ⁇ IC-IL2R ⁇ jm chimeric proteins causes TCR downregulation in donor-derived T cells.
  • primary T cells were electroporated with PiggyBac vector and PiggyBac Transposase mRNA to induce expression of CD3 ⁇ .
  • ⁇ IC-IL2R ⁇ jm MMB014
  • CD3 ⁇ ⁇ IC-IL2R ⁇ jm
  • MLB046 At day 5 post-electroporation, TCR expression was evaluated by staining for CD3 (upper panels) and TCR ⁇ / ⁇ (lower panels) .
  • FIG. 19 Schematic diagram of the CD3 ⁇ . ⁇ IC-GRAIL. IC (LG171) .
  • Our design is based on the extracellular and transmembrane sequences of CD3 ⁇ (i.e. CD3 ⁇ . ⁇ IC or CD3 ⁇ truncation) and intracellular domain of membrane-anchored E3 ligase GRAIL (GRAIL. IC) . Inclusion of the CD3 ⁇ transmembrane domains may allow our constructs to be incorporated into the multi-subunit TCR complex.
  • LG171 chimeric proteins causes TCR/CD3 downregulation in Jurkat cells.
  • Jurkat cells were electroporated with PiggyBac vectors expressing the CD3 fusion proteins (i.e. LG171) and PiggyBac Transposase mRNA to induce expression of CD3 ⁇ .
  • IC-GRAIL PiggyBac Transposase mRNA
  • FIG. 21 Expression of LG171 chimeric proteins causes TCR/CD3 downregulation in human primary T cells.
  • primary T cells were electroporated with PiggyBac vector LG171 and PiggyBac Transposase to induce expression of CD3 ⁇ . ⁇ IC-GRAIL.
  • IC LG171
  • TCR expression was evaluated by staining for CD3 and TCR ⁇ / ⁇ .
  • downregulation of TCR/CD3 expression were significant in GFP+fraction.
  • LG212 (without E3 ligase) could not reduce TCR expression in human primary T cells.
  • Primary T cells were electroporated with PiggyBac vector LG212 and PiggyBac Transposase.
  • TCR expression was evaluated by staining for CD3 and TCR ⁇ / ⁇ . No TCR/CD3 downregulation can be seen when only CD3 ⁇ (i.e. CD3 ⁇ . ⁇ IC) was adopted in human primary T cells.
  • Fig. 23A-23C Mutation or domain switch in GRAIL intracellular domain confer similar downregulation of TCR in LG171 as LG212 (CD3 ⁇ . ⁇ IC) .
  • ZF domain switch
  • H2N2 contains point mutation
  • Fig. 23A Both FACS (Fig. 23B) and western blot (Fig. 23C) tests indicated that functional zinc-finger domain of GRAIL is necessary for a better TCR/CD3 downregulation in LG171 (Fig. 21) .
  • LG171 chimeric proteins couldnot confer non-specific downregulation of CD5 or CD7.
  • the downregulation effect in primary T cells by LG171 was TCR/CD3 specific, as neither CD5 nor CD7 was affected at all.
  • LG171p1 with CAG promoter confers downregulation of TCR/CD3 on T cells.
  • LG171p1 fusion protein construct is shown in Fig. 25A.
  • Fig. 25A shows construct LG171p1 was generated by replacing EF1a promoter in LG171 with CAG promoter;
  • Fig. 25B shows CAG promoter confers downregulation of TCR/CD3 on Jurkat cells.
  • Jurkat cells were electroporated with PiggyBac vectors expressing LG171p1 fusion proteins and PiggyBac Transposase mRNA.
  • TCR surface expression was evaluated by CD3 and TCR ⁇ / ⁇ staining.
  • 25C shows CAG promoter confers sustainable downregulation of TCR/CD3 on human primary T cells.
  • primary T cells were pre-activated and electroporated with PiggyBac vector LG171p1 and PiggyBac Transposase.
  • TCR surface expression was evaluated by CD3 and TCR ⁇ / ⁇ staining.
  • Fig. 26 A-26B The downregulation in LG213 was mainly contributed by CD3 ⁇ truncation. CHIP. ⁇ TPR was linked to full length CD3 ⁇ or CD3 ⁇ . ⁇ IC to generate new chimeras (LG132, LG213) for TCR targeting (Fig. 26A) . FACS data for TCR levels in primary T cells transfected with LG132 or LG213. Compared to no effects in LG132 with full length of CD3 ⁇ , the downregulation in LG213 were mainly contributed by CD3 ⁇ truncation (Fig. 26B) .
  • Fig. 27A-27C Additional E3 chimeras based on CD3 ⁇ truncation and their efficacy in TCR downregulation.
  • Fig. 27A shows construct design for additional E3 ligase and their chimeras constructs based on CD3 ⁇ truncation.
  • Fig. 27B FACS data for additional E3 ligase and their chimeras constructs in TCR downregulation.
  • Fig. 27C Western blot data showing a significant decrease of TCR in additional E3 chimera constructs based on CD3 ⁇ , using ⁇ -action as the marker.
  • Fig. 28A-Fig. 28B Decreased activation in Jurkat cells transfected with LG171.
  • Fig. 28A shows decreased activation in Jurkat cells transfected with LG171 (diminished CD69 upregulation)
  • Fig. 28B shows decreased activation in Jurkat cells transfected with LG171 (diminished ppERK upregulation) .
  • Jurkat cells transfected with the indicated constructs were briefly stimulated with ⁇ CD3/CD28.
  • CD69 (Fig. 28A) and ppERK (Fig. 28B) were checked by FACS.
  • Fig. 29A-29C Decreased activation in primary T cells transfected with LG171 and LG171p1.
  • Human primary T cells transfected with the indicated constructs were stimulated with OKT3 (anti CD3 antibody) for CD69 upregulation and IFN- ⁇ cytokine secretion.
  • CD69 upregulation was checked by FACS (Fig. 29A) . 24hrs later, supernatant was collected and tested for IFN- ⁇ levels (Fig. 29B) .
  • Mock T cells (untreated T cells) or LG171p1 transfected T cells were cocultured with allogeneic mDC cells for 5 days before testing IFN- ⁇ levels using ELISA (Fig. 29C) .
  • Fig. 29A-29C Decreased activation in primary T cells transfected with LG171 and LG171p1.
  • Human primary T cells transfected with the indicated constructs were stimulated with OKT3 (anti CD3 antibody) for CD69 upregulation and IFN- ⁇ cytokine secretion
  • 29A shows decreased activation in primary T cells transfected with LG171 (CD69) .
  • Fig. 29B shows decreased activation in primary T cells transfected with LG171 and LG171p1 (IFN- ⁇ ) .
  • Fig. 29C shows decreased activation in primary T cells transfected with LG171p1 (IFN- ⁇ ) .
  • Fig. 30A-Fig. 30C Expression and killing efficiency of LG171. CldnCar. LG171. CldnCar and CldnCar fusion protein constructs are shown in Fig. 30A.
  • Fig. 30B shows TCR/CAR expression in T cells transfected with the indicated constructs.
  • Fig. 30C shows cytotoxicity assays by coculturing engineered T cells with either parental HEK cells (i.e. HEK) or CLDN18.2 overexpressing HEK cells (i.e. HEK-CLDN 18.2) for 24 hrs.
  • parental HEK cells i.e. HEK
  • CLDN18.2 overexpressing HEK cells
  • a “T cell receptor (TCR) complex” is a multimeric complex on the T-cell surface whose activation leads to the activation of the T-cell.
  • the complex comprises (i) TCR, and (ii) CD3 T-cell co-receptor.
  • the TCR comprises alpha ( ⁇ ) and beta ( ⁇ ) chains.
  • the CD3 T-cell co-receptor comprises a CD3-gamma (CD3 ⁇ ) chain, a CD3-delta (CD3 ⁇ ) chain, two CD3-epsilon (CD3 ⁇ ) chains and two zeta- ( ⁇ ) chains as accessory molecules.
  • TCRs allow for the antigen-specific activation of T-cells. Every T-cell expresses clonal TCRs which recognize a specific peptide/MHC complex during physical contact between T-cell and antigen-presenting cell-APC (via MHC class II) or any other cell type (via MHC class I) .
  • the TCR is a disulfide-linked membrane-anchored heterodimeric protein normally consisting of the highly variable alpha ( ⁇ ) and beta ( ⁇ ) chains. Each chain of the TCR comprises two extracellular domains: a variable (V) region and a constant (C) region, both of immunoglobulin superfamily (IgSF) domain forming antiparallel beta-sheets.
  • the constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide/MHC complex.
  • the variable domain of the TCR alpha-chain and the TCR beta-chain each have three hypervariable or complementarity determining regions (CDRs) that contribute to the TCR’s specificity for a particular peptide/MHC complex.
  • the variable region of the beta-chain also has an additional area of hypervariability (HV4) that does not normally contact antigen.
  • a “single-chain variable fragment (scFv) ” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids.
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker.
  • scFv antibodies are, e.g. described in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96) .
  • antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain into a functional antigen binding site and thereby provide the antigen binding property of full length antibodies.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • the molecules of the application are used to delay development of a disease or to slow the progression of a disease.
  • cancer refers to proliferative diseases, such as ovarian cancer, pancreatic cancer, colon cancer, colorectal cancer, lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the ovarian cancer, rectal cancer, cancer of the anal region
  • the present application provides novel fusion proteins with particularly advantageous properties such as reducing the membrane-bound protein (MBP) level when expressed in a host cell.
  • MBP membrane-bound protein
  • MBPs Membrane-Bound Proteins
  • degradation tag protein comprises ubiquitin ligase and/or domains of the cell surface receptors that mediate degradation (e.g. IL2R ⁇ sub domain) .
  • the tagging specificity or targeting specificity (or recognition specificity) of the MBPs to be degraded by the degradation tag protein e.g.
  • ubiquitin ligase or IL2R ⁇ is mediated by an antibody (or its antigen-binding fragment) of MBPs to be degraded (that is, antibody mediated substrate recognition) , or mediated by subunits or structural domains of the membrane-bound protein or complex to be degraded that have a transmembrane domain (that is, transmembrane domain mediated substrate recognition) .
  • the technical solution of the present application can be applied to any targets on a cell surface, independent of cell type.
  • the MBPs to be degraded can be membrane-bound receptors or receptor complex; more preferably the membrane-bound receptors can be T-cell receptors (TCRs) .
  • this application relates to the generation of allogeneic chimeric antigen receptor T cells (CAR-T) through TCR down regulation by targeted ubiquitin-mediated degradation by Receptor TAC (receptor targeting chimera) , or to the generation of a fusion protein (e.g. a fusion protein comprising a component of the membrane-bound protein to be degraded or a fragment thereof that has a transmembrane domain fused to the degradation tag protein (e.g. E3 ubiquitin ligase and/or domains of cell surface receptors that mediate degradation) ) , the degradation tag protein may be incorporated into the multi-subunit TCR complex during the formation of the TCR complex so as to cause downregulation of the TCR complex.
  • CAR-T allogeneic chimeric antigen receptor T cells
  • Receptor TAC receptor targeting chimera
  • a fusion protein e.g. a fusion protein comprising a component of the membrane-bound protein to be degraded or
  • an antibody such as an anti-CD3 antibody or antigen-binding fragment thereof was fused extracellularly with E3 ubiquitin ligase (such as the C-terminus of Hsc70-interacting protein (CHIP) ) followed by the transmembrane domain, or an E3 ubiquitin ligase or a cell surface receptor that mediates degradation was fused intracellularly to a component of the membrane-bound protein or complex to be degraded or a fragment thereof that has a transmembrane domain.
  • E3 ubiquitin ligase such as the C-terminus of Hsc70-interacting protein (CHIP)
  • E3 ubiquitin ligase or a cell surface receptor that mediates degradation was fused intracellularly to a component of the membrane-bound protein or complex to be degraded or a fragment thereof that has a transmembrane domain.
  • MBP such as TCR complex
  • Fusion protein a)
  • the present application discloses a fusion protein a) comprising: the first linking protein, the degradation tag protein, the first transmembrane domain.
  • the fusion protein comprises from N-terminal to C-terminal: the first linking protein, the degradation tag protein, the first transmembrane domain.
  • the first linking protein comprises the antibody or antigen-binding fragment of the MBP to be degraded; more preferably, the antibody or antigen-binding fragment of the MBP to be degraded comprises antibody or antigen-binding fragment of CD3, CD5, CD7, PD-L1 or CD47; more preferably, antigen-binding fragment of CD3 comprises SP34 scFv, OKT3 scFv, UCHT1 scFv, UCHT1. Y177T scFv, L2K scFv, F6A scFv, BMA031 scFv, or BMA031.
  • the degradation tag protein comprises the E3 ubiquitin ligase; more preferably, the E3 ubiquitin ligase comprises CHIP. dTPR, FBW7.2-293, VHL. 152-213, SPOP. 167-374, SOCS2.143-198;
  • the first transmembrane domain comprises CD8 ⁇ transmembrane domain.
  • the fusion protein a) further comprises a first signal peptide.
  • Linker e.g. SEQ ID No. 91
  • E3 ligase there also may be a Linker (e.g. SEQ ID No. 91) between scFv and E3 ligase.
  • Linker e.g. SEQ ID No. 91
  • CD8 ⁇ transmembrane domain CD8 TM
  • E3 ligase E3 ligase
  • Fusion protein b)
  • the present application discloses a fusion protein b) comprising: the first linking protein, the first transmembrane domain, the degradation tag protein.
  • the fusion protein comprises from N-terminal to C-terminal: the first linking protein, the first transmembrane domain, the degradation tag protein.
  • the first linking protein comprises the antibody or antigen-binding fragment of the MBP to be degraded; more preferably, the antibody or antigen-binding fragment of the MBP to be degraded comprises antibody or antigen-binding fragment of CD3; more preferably, antigen-binding fragment of CD3 comprises SP34 scFv;
  • the degradation tag protein comprises the E3 ubiquitin ligase; more preferably, E3 ubiquitin ligase comprises GRAIL. IC;
  • the first transmembrane domain is selected from one or more of CD3 ⁇ transmembrane domain variant, CD8 ⁇ transmembrane domain, and CD4 transmembrane domain.
  • the fusion protein b) further comprises a first signal peptide.
  • intracellular domain e.g. SEQ ID No. 90
  • TM domain of Fusion protein b e.g. LG222, LG222.1, LG222.2
  • the present application discloses a fusion protein c) comprising: the second transmembrane-linking domain of the above 2) , the cell surface receptors that mediate degradation.
  • the fusion protein comprises from N-terminal to C-terminal: the second transmembrane-linking domain of the above 2) , the cell surface receptors that mediate degradation.
  • the second transmembrane-linking domain of the above 2) comprises CD3 ⁇ . ⁇ IC or CD3 ⁇ . ⁇ IC;
  • the cell surface receptors that mediate degradation comprises IL2R ⁇ jm.
  • the present application discloses a fusion protein d) comprising: the second transmembrane-linking domain of the above 2) , the one or more of an E3 ubiquitin ligase or a variant thereof.
  • the fusion protein comprises from N-terminal to C-terminal: the second transmembrane-linking domain of the above 2) , the one or more of an E3 ubiquitin ligase or a variant thereof.
  • the second transmembrane-linking domain of the above 2) comprises CD3 ⁇ . ⁇ IC.
  • the one or more of an E3 ubiquitin ligase or a variant thereof comprises GRAIL. IC, CHIP. dTPR, RNF133. IC, RNF122. rIC, RNF152. rIC.
  • TH69 scFv, ⁇ PD-L1 scFv or ⁇ CD47 scFv comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in any one of SEQ ID NOs. 2-13 respectively.
  • the CHIP, CHIP. dTPR, FBW7.2-293, VHL. 152-213, SPOP. 167-374, SOCS2.143-198, GRAIL. IC, RNF133. IC, RNF122. rIC or RNF152. rIC comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in any one of SEQ ID NOs. 21-26, 62, 79, 81 or 83 respectively.
  • CD3 ⁇ transmembrane domain variant, CD8 ⁇ transmembrane domain, or CD4 transmembrane domain comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO. 89, 18 or 15 respectively.
  • the fusion protein a) further comprises a first signal peptide; preferably, the first signal peptide is selected from an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO. 19 or 20 respectively.
  • the fusion protein b) further comprises a first signal peptide; preferably, the first signal peptide is selected from an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO. 19.
  • the CD3 ⁇ . ⁇ IC and CD3 ⁇ . ⁇ IC comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO. 61, 64 respectively.
  • the transmembrane domain (TM) of CD3 ⁇ or CD3 ⁇ comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO. 67, 70 respectively;
  • the extracellular domain (EM) of CD3 ⁇ or CD3 ⁇ comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO.
  • the second signal peptide of CD3 ⁇ or CD3 ⁇ comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO. 65, 68 respectively.
  • the L2R ⁇ jm domain comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO. 58.
  • the fusion protein of the present application further comprises one or more of a hinge or a P2A-GFP.
  • the hinge is selected from a CD8 ⁇ hinge, or CD4 hinge; more preferably, CD8 ⁇ hinge or CD4 hinge comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO.: 16 or 14, respectively; more preferably, the hinge is adjacent to the N-terminal of the first transmembrane domain.
  • the P2A-GFP comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%or 100%identical to the amino acid sequence shown in SEQ ID NO.: 57; more preferably, the P2A-GFP sequence is at C-terminal of the fusion protein.
  • the first transmembrane-linking domain comprises a first transmembrane domain and a first linking protein
  • the first linking protein An antibody of the membrane-bound protein or antigen-binding fragment thereof (e.g. scFv)
  • an antibody of the membrane-bound protein or antigen-binding fragment thereof can be used as a linking protein that mediates binding (or specificity binding, specificity recognition) of the degradation tag protein to the membrane-bound protein (that is, antibody mediated substrate recognition) .
  • the scFvs of the present application are those specifically binding to membrane-bound proteins (including but not limited to CD3, CD5, CD7, PD-1, PD-L1 or CD47) , preferably binding to the extracellular part of those membrane-bound proteins.
  • the scFvs are those specifically binding to a component (or subunit) of the TCR/CD3 complex.
  • the scFv specifically binds to CD3;
  • the CD3 is a mammalian origin CD3, preferably a human CD3.
  • the first transmembrane domain is the first transmembrane domain
  • the first transmembrane domain is selected from transmembrane domain of one or more subunits or structural domains of CD8 ⁇ , CD4, CD3, CD28, 4-1BB and IL2R or a variant thereof.
  • the second transmembrane-linking domain comprises a transmembrane domain of the MBP to be degraded or a variant thereof
  • a transmembrane domain of the MBP to be degraded or a variant thereof (Linking protein 2)
  • one or more subunits or structural domains (or fragments) of the membrane-bound protein to be degraded or a variant thereof that has a transmembrane domain may be used as a linking protein to mediate incorporation (or specific recognition through incorporation) of the degradation tag protein to the membrane-bound protein, for example, incorporation of the degradation tag protein into the membrane-bound protein during the formation of the membrane-bound protein (that is, transmembrane domain mediated substrate recognition) .
  • the membrane-bound protein to be degraded is a membrane-bound receptor.
  • the membrane-bound receptor is CD3, preferably a mammalian origin CD3, more preferably a human CD3.
  • the Linking protein 2 comprises a transmembrane domain of one or more of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ or a variant thereof.
  • the linking protein 2 comprises an extracellular domain and a transmembrane domain of one or more of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ or a variant thereof.
  • the linking protein 2 comprises a second signal peptide, extracellular domain and a transmembrane domain of one or more of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ or a variant thereof.
  • Degradation tag protein 1 E3 Ubiquitin ligase
  • E3 ubiquitin ligase or “E3 ligase” or “Ubiquitin Ligase” (UL) is used herein to describe a target enzyme (s) binding site of ubiquitin ligase moieties in the bifunctional compounds according to the present application.
  • E3 UL is a protein that in combination with an E2 ubiquitin-conjugating enzyme causes the attachment of ubiquitin to a lysine on a target protein; the E3 ubiquitin ligase targets specific protein substrates for degradation by the proteasome.
  • E3 ubiquitin ligase alone or in complex with an E2 ubiquitin conjugating enzyme is responsible for the transfer of ubiquitin to targeted proteins.
  • the ubiquitin ligase is involved in polyubiquitination such that a second ubiquitin is attached to the first, a third is attached to the second, and so forth.
  • Polyubiquitination marks proteins for degradation by the proteasome.
  • Mono-ubiquitinated proteins are not targeted to the proteasome for degradation, but may instead be altered in their cellular location or function, for example, via binding other proteins that have domains capable of binding ubiquitin.
  • different lysines on ubiquitin can be targeted by an E3 to make chains. The most common lysine is Lys48 on the ubiquitin chain. This is the lysine used to make polyubiquitin, which is recognized by the proteasome.
  • the E3 ubiquitin ligase is selected from the following proteins or the truncated forms thereof: GRAIL, C-terminus of Hsc70-interacting protein (CHIP) , CHIP. dTPR, F-box WD40-containing protein 7 (FBW7) , FBW7.2-293, von Hippel-Lindau (VHL) , VHL. 152-213, Speckle-type BTB-POZ protein (SPOP) , SPOP.
  • Ubiquitin-protein ligase E3A Ubiquitin-protein ligase E3A
  • MDM2 Mouse double minute 2 homolog
  • APC Anaphase-promoting complex
  • UBR5 Ubiquitin-protein ligase E3A
  • CBLL1 Casitas B-lineage lymphoma-transforming sequence-like protein 1
  • HECT And RLD Domain Containing E3 Ubiquitin Protein Ligase 1 HERC1 , HERC2, HERC3, HERC4, HERC5, HERC6, HUWE1, ITCH
  • NEDD4 neural precursor cell expressed developmentally down-regulated protein 4
  • the E3 Ubiquitin ligase is used as a degradation tag protein, and the degradation of MBP mediated by E3 Ubiquitin ligase also can be achieved extracellularly and/or intracellularly.
  • Degradation tag protein 2 one or more subunits or structural domains of cell surface receptors that mediate degradation
  • the one or more subunits or structural domains of cell surface receptors that mediate degradation may be a protein that mediates degradation (or endocytosis and degradation, degradation after endocytosis) of the MBP or a component thereof, and for example, the degradation of the MBP or a component thereof mediated by one or more subunits or structural domains of the cell surface receptors may be accomplished intracellularly by an E3 ubiquitin ligase, a lysosome or other degradation pathways, and possibly after endocytosis of the MBP or a component thereof.
  • the one or more subunits or structural domains of the cell surface receptors that mediate degradation is specifically incorporated into MBP or a component thereof by fusing to a component of the membrane-bound protein (MBP) that is used to form the MBP during the formation of the MBP.
  • MBP membrane-bound protein
  • the component of the MBP that is used to form the MBP can be one or more domains of the MBP, optionally, one or more domains of CD3 ⁇ or CD3 ⁇ .
  • the cell surface receptors that mediate degradation are usually different from the MBP to be degraded, although the cell surface receptors may also belong to a MBP.
  • the cell surface receptors that mediate degradation and are used as the degradation tag protein of the present application comprise Interleukin receptors, or a variant derived therefrom.
  • the subunits or structural domains of Interleukin receptors comprises a lysosomal targeting motif of IL-2R; preferably, the lysosomal targeting motif of IL-2R comprises L2R ⁇ jm domain.
  • Transmembrane domain includes an amino acid sequence of about 15 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 20, 25, 30, 35, 40, or 45 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95%or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zaklakla, W. N. et al. (1996) Annu. Rev. Neurosci. 19: 235-263, the contents of which are incorporated herein by reference.
  • transmembrane domain in the fusion protein is to anchor said fusion protein to the cell surface, and thus any transmembrane domain fulfilling that function can be used in the present application.
  • a transmembrane domain suitable for the present application can be selected from the following: CD8 ⁇ , CD28, 4-1BB or IL2R transmembrane domain, that is, the first transmembrane domain in above 6.
  • a transmembrane domain suitable for the present application also can be, a transmembrane domain that is a structural domain of the linking protein itself, that is, the transmembrane domain in above 7.
  • the present application provides a nucleic acid encoding the fusion proteins described herein.
  • the nucleic acid encoding the fusion proteins can be easily prepared from an amino acid sequence of the specified fusion proteins by a conventional method.
  • a nucleotide sequence encoding an amino acid sequence can be obtained from the aforementioned NCBI RefSeq IDs or accession numbers of GenBank for an amino acid sequence of each domain, and the nucleic acid of the present disclosure can be prepared using a standard molecular biological and/or chemical procedure.
  • a nucleic acid can be synthesized, and the nucleic acid of the present disclosure can be prepared by combining DNA fragments which are obtained from a cDNA library using a polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • the nucleic acid of the present disclosure can be linked to another nucleic acid so as to be expressed under control of a suitable promoter.
  • the promoter include a promoter that constitutively promotes the expression of a gene or operatively linked construct, a promoter that induces the expression of a gene or operatively linked construct by the action of a drug or the like (e.g. tetracycline or doxorubicin) .
  • the nucleic acid of the present disclosure can be also linked to, in order to attain efficient transcription of the nucleic acid, other regulatory elements that cooperate with a promoter or a transcription initiation site, for example, a nucleic acid comprising an enhancer sequence or a terminator sequence.
  • a gene that can be a marker for confirming expression of the nucleic acid may be incorporated.
  • the nucleic acid is codon-optimized nucleic acid for expression in a particular host.
  • the present application further provides a vector comprising nucleic acid encoding the fusion proteins described herein.
  • vector comprising nucleic acid encoding the fusion proteins described herein.
  • expression vector and “expression construct” or “construct” are used interchangeably, and are both defined to be a plasmid, virus, or other nucleic acid designed for protein expression in a cell.
  • the vector or construct is used to introduce a gene into a host cell whereby the vector will interact with polymerases in the cell to express the protein encoded in the vector/construct.
  • the expression vector and/or expression construct may exist in a cell extrachromosomally or integrated into the chromosome. When integrated into the chromosome the nucleic acids comprising the expression vector or expression construct will be an expression vector or expression construct.
  • the present application further provides a composition comprising at least one nucleic acid or at least one vector described herein.
  • the present application further provides a composition comprising a first nucleic acid and a second nucleic acid, comprising a first vector having a first nucleic acid and a second vector having a second nucleic acid, or comprising a vector having a first nucleic acid and a second nucleic acid, wherein
  • the first nucleic acid encodes a fusion protein described herein
  • the second nucleic acid encodes a chimeric antigen receptor (CAR) comprising:
  • a cytoplasmic segment comprising one or more signaling domains, preferably comprising a 4-1BB signaling domain and a CD3 ⁇ signaling domain.
  • the predetermined antigen is a tumor-related antigen.
  • the tumor-related antigen is selected from the following group: CEA, Claudin 18.2, GPC3, Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) , CD38, CD19, CD20, CD22, BCMA, CAIX, CD446, CD133, EGFR, EGFRvIII, EpCam, GD2, EphA2, Her1, Her2, ICAM-1, IL13Ra2, Mesothelin, MUC1, MUC16, NKG2D, PSCA, NY-ESO-1, MART-1, WT1, MAGE-A10, MAGE-A3, MAGE-A4, EBV, NKG2D, PD1, PD-L1, CD25, IL-2, and CD3.
  • ROR1 Receptor tyrosine kinase-like Orphan Receptor 1
  • the tumor-related antigen is CEA.
  • the present application provides a host cell comprising the nucleic acid or vector or composition described herein.
  • the fusion proteins described herein will be expressed on the surface of the host cells for cell therapy.
  • the host cell is an allogeneic cell.
  • the host cell is a mammalian cell, preferably a primate cell, more preferably a human cell.
  • the host cell is selected from a T cell, or a primary T cell gamma delta T cell, NK cell, NKT cell, macrophage, B cell, or non-immune cell.
  • compositions of the present application comprise a fusion protein-expressing cell, preferably a fusion protein and a CAR-expressing cell, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such compositions may comprise buffers such as neutral-buffered saline, phosphate-buffered saline and the like; carbohydrates such as glucose, mannose, sucrose, dextrans, or mannitol; proteins, polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide) ; and preservatives.
  • Compositions of the present application are in one aspect formulated for intravenous administration.
  • compositions of the present application may be administered in a manner appropriate to the disease to be treated (or prevented) .
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined through clinical trials.
  • Suitable pharmaceutically acceptable excipients are well known to a person skilled in the art.
  • examples of pharmaceutically acceptable excipients include phosphate-buffered saline (e.g. 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCI, pH 7.4) , an aqueous solution containing a mineral acid salt such as a hydrochloride, a hydrobromide, a phosphate, or a sulfate, saline, a solution of glycol or ethanol, and a salt of an organic acid such as an acetate, a propionate, a malonate or a benzoate.
  • phosphate-buffered saline e.g. 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCI, pH 7.4
  • an aqueous solution containing a mineral acid salt such as a hydrochloride, a hydrobromide, a phosphate, or a sulfate
  • an adjuvant such as a wetting agent or an emulsifier, and a pH buffering agent can also be used.
  • the pharmaceutically acceptable excipients described in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991) (which is incorporated herein by reference in its entirety for all purposes) can be appropriately used.
  • the composition of the present application can be formulated into a known form suitable for parenteral administration, for example, injection or infusion.
  • the composition of the present application may comprise formulation additives such as a suspending agent, a preservative, a stabilizer and/or a dispersant, and a preservation agent for extending a validity term during storage.
  • the present application provides a method of treating disease in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition described herein.
  • the disease is cancer.
  • the cancer is hematological malignancy or solid tumor.
  • the cancer is ovarian cancer, pancreatic cancer, colon cancer, colorectal cancer, lymphoma, esophageal cancer, lung cancer, ovarian cancer, hepatic cancer, head-neck cancer, or cancer of the gallbladder.
  • the present application provides a method of producing a cell having reduced target membrane-bound protein level, comprising introducing into a cell a nucleic acid, a vector, or a composition described herein.
  • the present application provides a method of reducing or preventing GvHD in a subject associated with the administration of one or more CAR T-cells to the subject, comprising:
  • the present application provides a method of downregulating membrane-bound protein (MBP) in a cell population, comprising adding to said cell population a host cell of the application, wherein the host cell expresses fusion protein comprising scFv specifically binding to said MBP, and/or the host cell expresses a fusion protein comprising a component of the MBP or a fragment thereof that has a transmembrane domain.
  • MBP membrane-bound protein
  • the host cell population is a T cell population, preferably a human primary T cell population.
  • the MBP is CD3, preferably human CD3.
  • P2A-GFP i.e. P2A-eGFP
  • **Italics indicate P2A sequence.
  • LG114 SP-OKT3 scFv-FBW7-CD8 ⁇ hinge/TM-P2A-eGFP;
  • LG124 SP-UCHT1.
  • Linker e.g. SEQ ID No. 91
  • E3 ligase there also may be a Linker (e.g. SEQ ID No. 91) between scFv and E3 ligase.
  • Linker e.g. SEQ ID No. 91
  • E3 ligase there also may be a Linker (e.g. SEQ ID No. 91) between CD8 TM and E3 ligase.
  • an intracellular domain e.g. SEQ ID No. 90
  • TM domain attached to the C-terminal of TM domain of LG222, LG222.1, LG222.2.
  • LG213, CD3 ⁇ LG213, CD3 ⁇ . ⁇ IC-CHIP. DTPR-P2A-GFP
  • Human isolated primary T cells were activated in vitro using StemCell Immunocult-XF/Activator (StemCell Technologies) for 3 days, in the presence of human IL-2, IL-7 and IL-15. Then the activated primary T cells were collected and electroporated with pBluescript II SK (+) -PB Donor plasmid expressing one of the cytoplasmic constructs (LG089, LG091, LG092, LG085 constructs) for TCR/CD3 degradation. Two days later, the primary T cells were harvested and stained for both CD3 (PE) and TCR ⁇ / ⁇ (APC) (Biolegend) .
  • PE CD3
  • APC TCR ⁇ / ⁇
  • pmaxGFP plasmid only express GFP
  • LS008 indicated a good transfection efficiency in primary T cells, and there was no downregulation of TCR ⁇ / ⁇ and CD3 in either of these two constructs (Fig. 3A) .
  • SP34 scFv and OKT3 scFv were adopted to facilitate engaging the TCR/CD3 complex with E3 ligases, including FBW7 (Fig. 3B) , VHL (Fig. 3C) , SPOP (Fig. 3D) and hCHIP.
  • dTPR indicating a potential mistargeting effect in the current system.
  • Human isolated primary T cells were activated in vitro using StemCell Immunocult-XF/Activator for 3 days, in the presence of human IL-2, IL-7 and IL-15. Then the activated primary T cells were collected and electroporated with plasmid expressing one of the cytoplasmic/membrane-anchored constructs (LG021, LG022, LG023, LG024) for TCR/CD3 degradation. Two days later, the primary T cells were harvested and stained for both CD3 (PE) and TCR ⁇ / ⁇ (APC) .
  • PE CD3
  • APC TCR ⁇ / ⁇
  • the Jurkat cells were electroporated with both PiggyBac transposase mRNA and transposon expressing the membrane-anchored OKT3 construct (LG024) .
  • the Jurkat Cells were maintained until day 57 and subjected to flow cytometry testing post staining with CD3 (PE) and TCR ⁇ / ⁇ (APC) , showing a significant degradation of TCR/CD3 complex in cells with stable integration of transposon expressing the membrane-anchored OKT3 construct (LG024) (>85%in GFP positive, Fig. 5) .
  • Fig. 6A shows systemic controls pmaxGFP and LS008 have good transfection efficiency in Jurkat cells, and there was no downregulation of TCR ⁇ / ⁇ and CD3 in either of these two constructs.
  • FIG. 6B shows Membrane-anchored OKT3 scFv construct without E3 ubiquitin ligase (LG024) shows significant downregulation of TCR-CD3 complex in Jurkat cells.
  • LG024 Membrane-anchored OKT3 scFv construct without E3 ubiquitin ligase
  • dTPR was linked to OKT3 scFv in an outside membrane manner (proximal linkage) (LG112) (Fig. 6C) , instead of cytoplasmic manner (LG113) (Fig. 6D) , even stronger downregulation of TCR/CD3 complex was obtained.
  • Human isolated primary T cells were activated in vitro using StemCell Immunocult-XF/Activator for 3 days, in the presence of human IL-2, IL-7 and IL-15. Then the primary T cells were collected and electroporated with both PiggyBac transposase mRNA and transposon expressing different membrane-anchored OKT3 constructs. Three days later, the primary T cells were harvested and stained for both CD3 (PE) and TCR ⁇ / ⁇ (APC) .
  • Fig. 7A shows systemic controls pmaxGFP and LS008 have good transfection efficiency in primary T cells, and there was no downregulation of TCR ⁇ / ⁇ and CD3 in either of these two constructs.
  • FIG. 8A A list of constructs with different E3 ligases, including FBW7, VHL, SPOP and SOCS2, linked to membrane-anchored OKT3 scFv were designed and synthesized (Fig. 8A) .
  • Human isolated primary T cells were activated in vitro using StemCell Immunocult-XF/Activator for 3 days, in the presence of human IL-2, IL-7 and IL-15. Then the primary T cells were collected and electroporated with plasmid expressing one of the membrane-anchored constructs for TCR/CD3 degradation. Three days later, the primary T cells were harvested and stained for both CD3 (PE) and TCR ⁇ / ⁇ (APC) .
  • PE CD3
  • APC TCR ⁇ / ⁇
  • E3 ligases including FBW7 (Fig. 8B) , VHL (Fig. 8C) and SOCS2 (Fig. 8E) , could elicit mild downregulation of TCR/CD3.
  • SPOP may confer much stronger downregulation of TCR/CD3 in human primary T cells (Fig. 8D) .
  • TCR/CD3 degradation constructs with different CD3 targeting scFv, including SP34, UCHT1, UCHT1. Y177T, L2K and F6A, were designed and synthesized (Fig. 9A) .
  • Human isolated primary T cells were activated in vitro using StemCell Immunocult-XF/Activator for 3 days, in the presence of human IL-2, IL-7 and IL-15. Then the primary T cells were collected and electroporated with both PiggyBac transposase mRNA and transposon DNA constructs. Three days later, the primary T cells were harvested and stained for both CD3 (PE) and TCR ⁇ / ⁇ (APC) . Results showed that, by directly linking to CHIP.
  • PE CD3
  • APC TCR ⁇ / ⁇
  • Example 7 E3 ligase (CHIP. dTPR) is necessary for optimized TCR/CD3 downregulation in human primary T cells.
  • TCR degradation constructs with TCR targeting scFv, BMA031 or BMA031. H6L12, were designed and synthesized in the presence or absence of CHIP.
  • dTPR Fig. 10A
  • Human isolated primary T cells were activated in vitro using StemCell Immunocult-XF/Activator for 3 days, in the presence of human IL-2, IL-7 and IL-15. Then cells were collected and electroporated with both PiggyBac transposase mRNA and transposon DNA constructs. Three days later, the primary T cells were harvested and stained for both CD3 (PE) and TCR ⁇ / ⁇ (APC) . Results showed that, in the presence of CHIP.
  • PE CD3
  • APC TCR ⁇ / ⁇
  • Example 8 CD3 downregulation confers lower activation through CD3/CD28 stimulation in Jurkat cells.
  • FIG. 11A-B is an activation marker that is upregulated within hours of T cell activation
  • CD69 expression was evaluated using a PE-conjugated anti-CD69 primary antibody (BioLegend, San Diego, CA) . Results showed that CD69 (a T cell activation marker) expression levels were low for all cells at the baseline (Fig. 11A) . However, the CD69 expression levels in either LG024 or LG112 transfected GFP+ cells were significantly less compared to T cells from LS008 transfected cells post stimulation (Fig. 11B) . This finding indicated that TCR/CD3 downregulation conferred lower responsiveness to activation through CD3/CD28/CD2 stimulation for T cells.
  • FIG. 12A A list of CD5 or CD7 degradation constructs were designed and synthesized (Fig. 12A) .
  • Human isolated primary T cells were activated in vitro using StemCell Immunocult-XF/Activator for 3 days, in the presence of human IL-2, IL-7 and IL-15. Then the primary T cells were collected and electroporated with both PiggyBac transposase mRNA and transposon DNA constructs. Four days later, cells were harvested and stained for both CD5 (PE) and CD7 (APC) expressions (Fig. 12B-Fig. 12C) . Results showed that ⁇ CD5.14 scFv conferred strong downregulation of CD5 (Fig. 12B) .
  • ⁇ CD7. TH69 scFv CD7-targeting scFv
  • Fig. 12C A list of CD5 or CD7 degradation constructs were designed and synthesized.
  • E3 ligase constructs were used to modify expression of surface molecules in Jurkat cells (Fig. 13B) .
  • the EF1 ⁇ promoter was used to drive expression of an antigen-specific ScFv-E3 ligase chimeric fusion protein.
  • a P2A sequence was used to drive simultaneous GFP expression for the identification and characterization of cells expressing the E3 Ligase system (Fig. 13A) .
  • Jurkat cells were electroporated with PiggyBac vectors expressing MLB052 or MLB053 and Piggy Bac transposase mRNA. Following stable integration of the transposon into the Jurkat cell genome, degradation of PD-L1 and CD47 was evaluated by flow cytometry at day 5 post-electroporation.
  • Gating on GFP-positive cells i.e., those expressing the E3 ligase construct, showed a marginal downregulation of PD-L1 surface expression (i.e. a small left shift of the black line marked area to the dashed line marked area in the upper panel of right column of Fig. 13B) .
  • PD-L1 surface expression i.e. a small left shift of the black line marked area to the dashed line marked area in the upper panel of right column of Fig. 13B
  • –5%CO 2 in RPMI media supplemented with 5mM HEPES and 10%FBS –Jurkat cells do not strongly express PD-L1.
  • An identical gating strategy on Jurkat cells expressing MLB053 showed strong downregulation of CD47 expression in those cells expressing the E3 ligase construct (i.e.
  • Fig 14A provides an overview of the gating strategy showing delineation between the CellTrace Violet-positive parental cells (i.e. Cell Trace + cells, without LG024-transfection) and CellTrace Violet-negative E3 ligase-transfected cells (i.e. Cell Trace - cells, LG024-transfected) .
  • the CellTrace Violet negative population i.e. Cell Trace – cells, LG024-transfected
  • was further subdivided into a GFP-positive population expressing the E3 ligase construct (the right filled area in the middle column of Fig. 14A) and a GFP-negative population (the left filled area in the middle column of Fig.
  • E3 ligase-positive cells i.e. GFP-positive population, 77.8%
  • E3 ligase-negative cells i.e. GFP-negative population, 22.2%
  • TCR expression as determined by CD3 staining, was low across the entire cell population (i.e. the upper panel of right column of Fig. 14A) .
  • Figure 14B shows, as the percentage of E3-ligase positive cells was reduced through serial dilutions with parental Jurkat cells, from approximately 78%positive to 11%positive, we observed an increase in TCR surface expression in the CellTrace Violet-positive parental cell population.
  • TCR expression on adjacent cells is inversely related to the number of E3-ligase expressing cells within the population.
  • TCR downregulation in E3 ligase-expressing cells is also dependent on the expression profile of neighboring cells, with more complete knockdown when the fraction of E3 ligase-expressing cells is higher. Diluting the number of E3 ligase positive cells also rescued the phenotype of CellTrace Violet-negative, GFP-negative cells within the transfected cell population, as seen by the increase of TCR positive cells in this population.
  • Example 12 Hinge/transmembrane domain (TM) choice affected the downregulation of TCR/CD3 complex mediated through both SP34 and GRAIL. IC.
  • IC E3 functional domain
  • CD3-targeting scFv, SP34 was adopted and linked to GRAIL.
  • IC through different hinge/transmembrane domains as listed in Fig. 15A. All three constructs, LG222, LG222.1 and LG222.2 were tested in both Jurkat and human primary T cells.
  • LG222, LG222.1 and LG222.2 were tested in both Jurkat cells.
  • LG222, LG222.1 and LG222.2 were tested in human primary T cells.
  • Pre-activated primary T cells electroporated for expressing LG222, LG222.1 or LG222.2 construct were harvested at day 3 post-electroporation and TCR/CD3 expression was evaluated using both ⁇ CD3 and ⁇ TCR ⁇ / ⁇ antibodies (Fig. 16A-C) .
  • Fig. 16A-C TCR/CD3 expression was evaluated using both ⁇ CD3 and ⁇ TCR ⁇ / ⁇ antibodies
  • Fig. 16A-C Similarly as Jurkat cells, significant downregulation of TCR/CD3 expression was achieved by construct using CD3 ⁇ .
  • ⁇ IC domain Fig. 16A, LG222
  • Using CD8a hinge/TM domain only conferred TCR/CD3 downregulation in a small fraction of GFP + cells (Fig. 16B, LG222.1) .
  • multiple repeats of construct LG222.2 did not achieve good GFP expressions to draw a solid conclusion on TCR/CD3 downregulation (Fig. 16C, LG222.2) .
  • Example 14 Expression of LG171/ (CD3 ⁇ . ⁇ IC/GRAIL. IC) fusion protein dramatically promotes TCR/CD3 complex expression downregulation in both Jurkat cells and human primary T cells
  • LG171/ (CD3 ⁇ . ⁇ IC/GRAIL. IC) fusion protein construct is shown in Fig. 19.
  • TCR expression was evaluated by staining for CD3 and TCR ⁇ / ⁇ . We could not see any TCR/CD3 downregulation when only truncated CD3 ⁇ (i.e. CD3 ⁇ . ⁇ IC) was adopted in human primary T cells (Fig. 22) .
  • LG171 chimeric proteins could not confer non-specific downregulation of CD5 or CD7
  • CAG promoter is a good alternative conferring significant and sustainable downregulation of TCR/CD3 complex in both Jurkat cells and human primary T cells
  • LG171p1 was designed (Fig. 25A) and tested in both Jurkat and human primary T cells.
  • Fig. 25B At day 3 post-electroporation with LG171p1/ (CAG/CD3 ⁇ . ⁇ IC/GRAIL. IC) fusion protein construct (Fig. 25B) , Jurkat cells were harvested and stained for TCR expression using ⁇ CD3 and ⁇ TCR ⁇ / ⁇ antibodies. Cells expressing the construct were identified based on GFP co-expression on the fusion protein constructs. Gating on the GFP positive population indicated that there was a majority shift into undetectable TCR surface expression (upper left area in the third and fourth panel from left to right of Fig 25B) in Jurkat. These data suggest that CAG promoter could be a good alternative for EF1a in Jurkat conferring significant gene expression level.
  • Pre-activated primary T cells electroporated for expressing LG171p1 construct were harvested at day 3 post-electroporation and TCR/CD3 expression was evaluated using both ⁇ CD3 and ⁇ TCR ⁇ / ⁇ antibodies (Fig. 25C) .
  • downregulations of TCR/CD3 expression were significant in GFP+ fraction (upper left in each panel of the third and fourth column from left to right of Fig 25C) .
  • Example 15 TCR/CD3 Degradation constructs with different E3 ligases (CD3 ⁇ . ⁇ IC with different E3 ligases other than GRAIL. IC) .
  • E3 ligases including RNF133, RNF122 and RNF152, were also tested in a similar design as LG171 (Fig. 27A) . Both FACS and western blot showed consistent results that TCR downregulated when E3 ligase functional domains were linked to CD3 ⁇ truncation (Fig. 27 B-C) .
  • Example 16 Decreased activation in cells transfected with LG171 (CD69, ppERK, IFN- ⁇ )
  • Pre-activated primary T cells were transfected with indicated constructs. Several days later, T cells were re-stimulated with OKT3 (anti CD3 antibody) for CD69 upregulation and IFN- ⁇ cytokine secretion. Similar to what we found in Jurkat, human primary T cells transfected with either LG171 or LG171p1 (CAG promoter) showed significant lower CD69 upregulation (Fig. 29A) and IFN- ⁇ secretion (Fig. 29B) . When PHA-L (Fig.
  • Fig. 29C MLR assay was employed (Fig. 29C) .
  • mock T cells or T cells transfected with LG171p1 were cocultured with in-vitro derived allogeneic mature DC cells for 5 days. Then IFN- ⁇ levels within supernatant were tested by ELISA. Similarly, LG171p1 dramatically reduced IFN- ⁇ secretion when tested with mature DC from two different donors.
  • CldnCar and LG171. CldnCar fusion protein construct are shown in Fig. 30A.
  • CldnCar i.e. LG171/claudin18.2 CAR joint expression construct
  • TCR downregulation is detected by ⁇ CD3 ⁇ (anti-CD3 antibody, for CD3 expression) and CAR expression is detected by ⁇ F (ab’) 2 staining (for scFv expression) ; the results are shown in Fig. 30B.
  • T cells expressing the ReceptorTAC/CAR construct were gated on GFP expression (the first peak from top to bottom in both left and right panels of Fig. 30B) . From the left panel of Fig. 30B, it can be seen that, T cells transfected with LG171. CldnCar (the lowest peak) and LG171 (the second peak from top to bottom) lack of TCR expression compared to T cells transfected with CldnCar (the third peak from top to bottom) . From the right panel of Fig. 30B, it can be seen that, T cells transfected with LG171 lack of scFv expression (second peak from top to bottom) , while T cells transfected with LG171. CldnCar have normal scFv expression (the lowest peak) compared to T cells transfected with CldnCar (the third peak from top to bottom) .
  • Fig. 30C From the left panel of Fig. 30C, it can be seen that, no cytotoxicity was detected against the claudin18.2-negative parental HEK cell line (i.e. HEK) . From the right panel of Fig. 30C, it can be seen that, T cells expressing the LG171/claudin18.2 CAR joint expression construct (i.e. LG171. CldnCar) showed antigen-specific cytotoxicity identical to cells expressing the claudin18.2 CAR(i.e. CldnCar) control plasmid (i.e. HEK-CLDN 18.2) . Antigen-specific cell lysis increased with an increasing E: T ratio. T cells expressing LG171 alone showed minimal cytotoxicity in response to target cells expressing claudin18.2, indicating that this effect was indeed CAR-mediated.
  • LG171/claudin18.2 CAR joint expression construct i.e. LG171.
  • CldnCar showed antigen-specific cytotoxicity identical to cells

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