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WO2025147583A1 - Ligands pd-1 modifiés - Google Patents

Ligands pd-1 modifiés Download PDF

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Publication number
WO2025147583A1
WO2025147583A1 PCT/US2025/010194 US2025010194W WO2025147583A1 WO 2025147583 A1 WO2025147583 A1 WO 2025147583A1 US 2025010194 W US2025010194 W US 2025010194W WO 2025147583 A1 WO2025147583 A1 WO 2025147583A1
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seq
engineered
protein
icd
sequence
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Volker BOEHNERT
John James Dwyer
Michael MINGUENEAU
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Sail Biomedicines Inc
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Sail Biomedicines Inc
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    • 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
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/91—Fusion polypeptide containing a motif for post-translational modification containing a motif for glycosylation
    • C07K2319/912—Fusion polypeptide containing a motif for post-translational modification containing a motif for glycosylation containing a GPI (phosphatidyl-inositol glycane) anchor

Definitions

  • PD-L1 Programmed death-ligand 1
  • CD274 also known as CD274 or B7-H1
  • PD-L1 has been shown to suppress adaptive immunity by binding to its natural ligand, programmed cell death protein 1 (PD-1).
  • PD-1 is encoded by the CD279 gene and expressed on the surface of immune cells such as T cells.
  • the binding of PD-L1 to PD-1 transmits an inhibitory signal to the immune cells. This inhibitory signal dampens effector and helper T cells’ proliferation and functional activity, such as cytokine secretion and cytotoxicity.
  • PD-L1/PD-1 interaction also reduces apoptosis of regulatory T cells, further enhancing the immunosuppressive function of these cells.
  • the dysregulation of the PD-L1/PD-1 immune checkpoint pathway may lead to autoimmunity in humans.
  • low PD-1/PD-L1 expression levels can lead autoreactive T cells to target and induce apoptosis in hepatocytes, triggering a harmful autoimmune response against the liver.
  • supplemental PD-L1 expression in a vulnerable tissue is a promising strategy to inhibit autoimmune responses against the tissue, PD-L1 has a short half-life. Exogenous expression of the wildtype protein may not be sufficient to fully suppress autoimmune activity in cells.
  • the present disclosure provides an engineered programmed cell death 1 ligand (PD-L1) protein comprising an extracellular sequence of a PD-L1 and a transmembrane domain (TM) and/or an intracellular domain (ICD) that is heterologous to PD-L1, wherein the engineered PD-L1 protein has an increased half-life than the corresponding wildtype PD- L1 when expressed in a mammalian cell.
  • PD-L1 programmed cell death 1 ligand
  • the PD-L1 is human PD-L1, optionally wherein the extracellular sequence comprises SEQ ID NO:24 or an amino acid sequence at least 95% identical thereto.
  • the extracellular sequence comprises amino acids 1- 219, 1-210, or 1-200 of SEQ ID NO:24, or a variant amino acid sequence at least 95% identical to amino acids 1-219, 1-210, or 1-200 of SEQ ID NO:24.
  • the amino acid sequence at least 95% identical to SEQ ID NO:24 comprises one or more mutations relative to SEQ ID NO: 1 selected from N-to-Q mutations, I54E/Q, Y56H/F, E58F/M, R113T, M115L, S117G/A, G119K, G120V, A121W, R238A, an insertion ofW between wildtype residues A121 and D122, and a deletion between amino acids 230-238, optionally wherein the variant amino acid sequence comprises (i) an extracellular sequence of SEQ ID NO:54, 55, 56, 57, 67, 68, or 79, or (ii) SEQ ID NO:73, 74, 75, 76, 77, 78, or 80.
  • the TM domain comprises a wildtype PD-L1 TM sequence, optionally SEQ ID NO:25.
  • the TM domain comprises a TM sequence from integrin alpha-M (ITGAM), optionally SEQ ID NO:31, further optionally wherein the engineered PD-L1 protein comprises SEQ ID NO: 18, with or without the signal sequence shown in SEQ ID NO:23.
  • the TM domain comprises a TM sequence from carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), optionally SEQ ID NO:32, further optionally wherein the engineered PD-L1 protein comprises SEQ ID NO: 19, with or without the signal sequence shown in SEQ ID NO:23.
  • the TM domain comprises a TM sequence from integrin alpha-1 (ITGA1), optionally SEQ ID NO:33, further optionally wherein the engineered PD-L1 protein comprises SEQ ID NO:20, with or without the signal sequence shown in SEQ ID NO:23.
  • the ICD is a PD-L1 -derived ICD sequence, optionally wherein the ICD comprises SEQ ID NO:26, 34, 35, or 36.
  • the engineered PD-L1 protein comprises SEQ ID NO:2, 3, or 4, with or without the signal sequence shown in SEQ ID NO: 23.
  • the ICD comprises a heterologous polypeptide sequence fused, optionally through a peptide linker, to the C-terminus of a PD-L1 -derived ICD sequence.
  • the heterologous polypeptide is a CMTM6 sequence, optionally SEQ ID NO:47, further optionally wherein the engineered PD-L1 protein comprises SEQ ID NO:6, with or without the signal sequence shown in SEQ ID NO:23.
  • the heterologous polypeptide is a CSN5 sequence, optionally SEQ ID NO:48, further optionally wherein the engineered PD-L1 protein comprises SEQ ID NO:7, with or without the signal sequence shown in SEQ ID NO:23.
  • the ICD comprises an ITGAM-derived ICD sequence, optionally wherein the ICD comprises SEQ ID NO:37 or 38.
  • the engineered PD-L1 protein comprises SEQ ID NO:8 or 9, with or without the signal sequence shown in SEQ ID NO:23.
  • the ICD comprises a CEACAM1 -derived ICD sequence, optionally wherein the ICD comprises SEQ ID NO:39 or 40.
  • the engineered PD-L1 protein comprises SEQ ID NO: 10, 11, 67, or 68, with or without the signal sequence shown in SEQ ID NO: 23.
  • the ICD comprises an ITGA1 -derived ICD sequence, optionally wherein the ICD comprises SEQ ID NO:41 or 42.
  • the engineered PD-L1 protein comprises SEQ ID NO: 12 or 13, with or without the signal sequence shown in SEQ ID NO: 23.
  • the ICD comprises an ENPP1 -derived ICD sequence, optionally wherein the ICD comprises SEQ ID NO:43 or 44.
  • the engineered PD-L1 protein comprises SEQ ID NO: 14 or 15, with or without the signal sequence shown in SEQ ID NO: 23.
  • the ICD comprises an APMAP-derived ICD sequence, optionally wherein the ICD comprises SEQ ID NO:45 or 46.
  • the engineered PD-L1 protein comprises SEQ ID NO: 16 or 17, with or without the signal sequence shown in SEQ ID NO: 23.
  • the extracellular sequence of a PD-L1 is fused to a GPI anchor, optionally comprising SEQ ID NO:50, further optionally wherein the engineered PD- L1 protein comprises SEQ ID NO:21.
  • the extracellular sequence of a PD-L1 is fused, with or without a peptide linker, to a GPI-anchored protein, optionally NT5E.
  • the GPI-anchored protein comprises SEQ ID NO:51, optionally wherein the engineered PD-L1 protein comprises SEQ ID NO:22.
  • the engineered PD-L1 protein comprises SEQ ID NO:5, with or without the signal sequence shown in SEQ ID NO:23, or comprising SEQ ID NO:73, 74, 76, 77, or 80.
  • the engineered PD-L1 protein herein further comprises a signal sequence, wherein the signal sequence comprises a PD-Ll signal sequence, optionally SEQ ID NO:23; or a NT5E signal sequence, optionally SEQ ID NO:29.
  • the present disclosure provides a nucleic acid molecule comprising a coding sequence for the engineered PD-L1 protein herein.
  • the nucleic acid molecule is DNA, mRNA, or circular RNA.
  • a recombinant virus comprising the nucleic acid molecule herein.
  • the present disclosure provides a lipid nanoparticle comprising an mRNA or circular RNA herein.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the nucleic acid molecule, recombinant virus, or lipid nanoparticle herein, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of treating an autoimmune condition of a tissue in a patient in need thereof, comprising administering the pharmaceutical composition herein to the patient such that the engineered PD-L1 protein is expressed in the tissue.
  • a method of treating an autoimmune condition of a tissue in a patient in need thereof comprising administering the pharmaceutical composition herein to the patient such that the engineered PD-L1 protein is expressed in the tissue.
  • use of the nucleic acid molecule, recombinant virus, or lipid nanoparticle herein for the manufacture of a medicament for treating an autoimmune condition in a patient in need thereof.
  • the nucleic acid molecule, recombinant virus, lipid nanoparticle, or pharmaceutical composition herein for use in treating an autoimmune condition in a patient in need thereof.
  • the tissue is the liver and the autoimmune condition is autoimmune hepatitis.
  • the present disclosure provides a method of preventing rejection of an organ transplant in a patient in need thereof, comprising administering the pharmaceutical composition herein to the transplant such that the engineered PD-L1 protein is expressed in the transplant.
  • a method of preventing rejection of an organ transplant in a patient in need thereof comprising administering the pharmaceutical composition herein to the transplant such that the engineered PD-L1 protein is expressed in the transplant.
  • use of the nucleic acid molecule, recombinant virus, or lipid nanoparticle herein for the manufacture of a medicament for preventing rejection of an organ transplant in a patient in need thereof.
  • the nucleic acid molecule, recombinant virus, lipid nanoparticle, or pharmaceutical composition herein for use in preventing rejection of an organ transplant in a patient in need thereof.
  • FIG. 1 is a schematic showing the function of the PD-1 ligand, PD-L1, in the inhibition of aberrant immune responses and prevention of apoptosis of hepatocytes expressing PD-L1 at their cell surface.
  • FIG. 2 is a series of flow cytometry plots demonstrating the effectiveness of detecting PD-L1 expression on CHO-K1 cells using an anti-PD-Ll antibody (top) or Alexa Fluor 647-conjugated human PD-1 extracellular domain (ECD) (bottom).
  • FIGs. 3A-B are bar graphs showing the expression levels of various engineered PD-L1 fusion proteins expressed from DNA plasmids in CHO-K1 cells, as compared to wildtype (WT) PD-L1 (FIG. 3A), and the relative binding ability of various engineered PD- L1 fusion proteins to PD-1 compared to wildtype PD-L1 (FIG. 3B).
  • FIG. 4 is a set of heat maps showing the change over a 96-hour period in the percentage of PD-L1 -positive, viable CHO-K1 cells transfected with plasmid DNA encoding a PD-L1 chimera (left) and the geometric mean expression of the PD-L1 chimeras in positive cells (right).
  • FIG. 5 is a set of heat maps showing the change over a nine-day period in the percentage of PD-L1 -positive, viable OVCAR-3 cells transfected with plasmid DNA encoding a PD-L1 chimera (left) and the geometric mean expression of the PD-L1 chimeras in positive cells (right).
  • FIG. 6 is a set of heat maps showing the change over a seven-day period in the percentage of PD-L1 positive, viable CHO-K1 cells transfected with circular RNA (circRNA) encoding a PD-L1 chimera (left), the geometric mean expression of the PD-L1 chimeras in positive cells (center), and the net mean fluorescence intensity (MFI) of the PD-L1 chimeras on Day 7 (right).
  • circRNA circular RNA
  • MFI mean fluorescence intensity
  • FIG. 7 is a set of heat maps showing the change over a seven-day period in the percentage of PD-L1 positive, viable HEK293T cells transfected with circRNA encoding a PD-L1 chimera (left), the geometric mean expression of the PD-L1 chimeras in positive cells (center), and the net MFI of the PD-L1 chimera on Day 7 (right).
  • FIG. 8 is a set of graphs showing expression of PD-L1 after six hours as determined by flow cytometry (left two graphs), the change over 48 hours in the percentage of PD-L1 -positive cells (center two graphs), and the MFI of PD-L1 + cells (right two graphs) in either hepatocytes (top row of graphs) or immune cells (bottom row of graphs).
  • the cells were obtained from the liver of mice treated with mRNA encoding PD-L1.
  • the mRNA was delivered using an ionizable lipid formulation, LRN1.
  • FIG. 9 is a line graph showing the amounts of PD-L1 detected in the liver of mice by an ELISA assay.
  • the cells were obtained from the liver of mice treated with mRNA encoding PD-L1.
  • the mRNA was delivered using an ionizable lipid formulation, LRN1.
  • FIG. 10 is a set of line graphs showing the change over 120 hours in the percentage of PD-L1 -positive cells (left) and the MFI of PD-L1 + cells (right).
  • the cells were obtained from the liver of mice pre-dosed with anti-IFNaR-1 monoclonal antibody and administered circular RNA encoding PD-L1.
  • the circular RNA was delivered with an ionizable lipid formulation, LRN1.
  • FIG. 11 is a line graph showing the change over seven days in the percentage of PD-L1 -positive cells.
  • the cells were obtained from the liver of mice pre-dosed with anti- IFNaR-1 monoclonal antibody and administered circRNA encoding PD-L1 variants.
  • the circular RNA was delivered with an ionizable lipid formulation (LP-01).
  • FIG. 12 is a line graph showing the MFI and derived EC50 values of CHO cells transfected with mRNAs encoding PD-L1 molecules bearing various point mutations, as measured by flow cytometry.
  • FIG. 13 is a plot showing the PD-L1 cell surface expression (as indicated by MFI) and PD-L1 soluble levels in the cell culture supernatant of CHO cells transfected with mRNAs encoding PD-L1 molecules bearing various point mutations.
  • the present disclosure provides engineered PD-L1 proteins that have improved longevity while retaining wildtype PD-Ll’s expression levels and affinity for PD-1.
  • autoimmune diseases e.g., autoimmune hepatitis
  • PD-1 and PD-L1 have been observed to express PD-1 and PD-L1 at low levels, which may contribute to T cell-mediated destruction of healthy tissues (see, e.g., Agina et al., Clin Exp Hepatol. (2019) 5(3):256-64; Jilkova et al., Cells. (2021) 10(10):2671).
  • Expressing the present PD-L1 proteins in specific tissues can repress aberrant immune responses against those tissues, thereby preserving viability of healthy cells.
  • the present PD-L1 proteins may be expressed in liver parenchymal and residential cells to alleviate autoimmune hepatitis or other immune-driven liver diseases (e.g., primary biliary cholangitis). Additionally, the present PD-L1 proteins may also be expressed, for example, in kidney cells to alleviate lupus nephritis; in the kidney, skin, or central nervous system to alleviate systemic lupus erythematosus (SLE); in the synovium to alleviate rheumatoid arthritis; in pancreatic cells to alleviate type 1 diabetes; in cells of the salivary or lacrimal glands to alleviate Sjogren’s syndrome; in the thyroid to alleviate Graves’ disease; in oligodendrocytes to alleviate multiple sclerosis; in the central or peripheral nervous system to alleviate myasthenia gravis or neuromyelitis optica; in the intestines to alleviate Crohn’s disease, ulcerative colitis or celiac disease;
  • the present PD-L1 -derived proteins can be expressed at levels approximating wildtype PD-L1 on the cell surface in several cell types and effectively bind to PD-1, indicating their potential use as a therapeutic. Additionally, these engineered proteins exhibit longer half-lives than wildtype PD-L1, suggesting their potential enhanced therapeutic efficacy compared to supplementation with wildtype PD-L1.
  • the engineered PD-L1 proteins herein are derived from PD-L1 but may contain only a partial, rather than the entire, sequence of wildtype PD-L1. These polypeptides are cell surface proteins when expressed in mammalian cells.
  • “derived from” means that the sequence is the same as or similar to the original sequence; a derived sequence may be longer or shorter than, or have the same length as, the original sequence.
  • PD-L1 refers to human PD-L1.
  • a human PD-L1 polypeptide sequence may be found at the UniProt database (Identifier No.
  • Q9NZQ7-1) may have the following sequence:
  • LGVALTFIFR LRKGRMMDVK KCGIQDTNSK KQSDTHLEET SEQ ID NO : 1
  • the extracellular region spans amino acids 1-238.
  • the extracellular region includes a signal sequence (amino acids 1-18; underlined) and two distinct extracellular domains (ECDs).
  • the first ECD is an IgV-like domain that spans amino acids 19-127 and the second ECD is an IgC-like domain that spans amino acids 133-225.
  • the transmembrane domain (TM) spans amino acids 239-259 (boldface and underlined).
  • the intracellular domain (ICD) spans amino acids 260-290.
  • the signal sequence is removed in the mature polypeptide.
  • the signal sequence, the extracellular region minus the signal sequence, the TM domain, and the ICD domain of the above sequence are assigned SEQ ID NOs:23-26, respectively.
  • the IgV-like domain and IgC-like domain sequences are assigned SEQ ID NOs:27 and 28, respectively.
  • PD-L1 is predicted to possess a matrix metalloproteinase cleavage site that spans amino acids 230-238 (boxed; SEQ ID NO:71).
  • a PD-L1 amino acid position recited herein refers to the position in SEQ ID NO: 1 or a corresponding position in a variant of SEQ ID NO: 1 (e.g., a naturally occurring polymorphic variant or a genetically engineered variant).
  • PD-L1 proteins are fusion proteins with components from different sources, those proteins also are called “PD-L1 chimeras” or “PD-L1 fusion proteins” herein.
  • polypeptides extracellular, transmembrane, and intracellular regions of the engineered PD-L1 proteins herein are described below.
  • the extracellular region of the present engineered PD-L1 polypeptides may comprise a signal sequence that triggers the translocation of the newly synthesized PD-L1 protein into the endoplasmic reticulum for trafficking and expression to the cell surface.
  • the signal sequence may be cleaved off before the protein reaches the cell membrane.
  • the signal sequence comprises PD-Ll’s wildtype signal sequence MRIFAVFIFMTYWHLLNA (SEQ ID NO:23) or a functional variant thereof.
  • the signal sequence may be derived from another cell surface protein or an artificial sequence.
  • the signal sequence may comprise a signal sequence from human 5 ’ -nucleotidase NT5E (a.k.a. CD73), MCPRAARAPATLLLALGAVLWPAAGA (SEQ ID NO:29), or a functional variant thereof.
  • the extracellular region comprises mutations in the IgV- like domain and/or the IgC-like domain sequence relative to wildtype PD-L1.
  • the mutations are made in the IgC-like domain sequence.
  • the IgC-like domain comprises an N-to-Q substitution at one, two, or all three positions corresponding to residue 192, 200, and 219 of SEQ ID NO: 1.
  • the extracellular region comprises PD-Ll’s extracellular region with N192Q, N200Q, and N219Q substitutions.
  • the extracellular region comprises SEQ ID NO:30.
  • the extracellular region of the present engineered PD-L1 polypeptide comprises a part (e.g., amino acid 1 to amino acid 210, 211, 212, 213, 214, 215, 216, 217, or 218) or the entirety (i.e., amino acids 1 to 219) of SEQ ID NO:24.
  • the extracellular region comprises an additional phenylalanine (F) at the N- terminus.
  • the ICD of the engineered PD-L1 protein may be derived from PD-L1 or another cell surface protein, or may be completely artificial (i.e., not derived from a naturally occurring protein). Where the ICD is derived from a naturally occurring protein, it may contain the wildtype ICD or a variant thereof. The variant may, for example, contain mutations (e.g., substitution) that reduce or eradicate the signal transduction function of the ICD.
  • the variant may be the “PD-L1 ICD K5R” variant (SEQ ID NO:34), which is identical to the wildtype ICD sequence except that all five lysine residues in the wildtype sequence have been substituted by arginines.
  • the variant may be the “PD-L1 ICD K263R” variant (SEQ ID NO:35), which is identical to the wildtype ICD sequence except that the first lysine residue in the wildtype sequence (corresponding to K263 in SEQ ID NO:1) has been substituted by arginine.
  • amino acid residues other than lysines e.g., 2, 3, 4, or more additional amino acid residues are substituted in the same or similar manner.
  • the variant may be the “PD-L1 ICD KtoR,DEtoR” variant (SEQ ID NO:36), which is identical to the wildtype ICD sequence except that all lysines (5), aspartic acids (3), and glutamic acid (2) residues in the wildtype sequence have been substituted by arginines; these mutations increase the net positive charge of the ICD and help to increase interaction with the membrane and resist degradation.
  • the ICD of the engineered PD-L1 protein comprises an ICD from ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1) or a variant thereof.
  • ENPP1 ectonucleotide pyrophosphatase/phosphodiesterase family member 1
  • the ICD from ENPP1 may be in a reverse orientation in the engineered protein.
  • the ICD of the engineered protein comprises SEQ ID NO:43 (reverse of a wildtype human ENPP1 ICD), or a variant thereof.
  • the variant comprises one or more substitutions (e.g., K to R) relative to the wildtype ICD sequence.
  • the variant may be the “ENPP1 ICD (reverse) KtoR” variant (SEQ ID NO:44), which is identical to SEQ ID NO:43 except that all three lysine residues in it have been substituted by arginines.
  • the ICD of the engineered PD-L1 protein comprises an ICD from adipocyte plasma membrane-associated protein (APMAP) or a variant thereof.
  • the ICD from APMAP may be in a reverse orientation in the engineered protein.
  • the ICD of the engineered protein comprises SEQ ID NO:45 (reverse of a wildtype human APMAP ICD), or a variant thereof.
  • the variant comprises one or more substitutions (e.g., K to R) relative to the wildtype ICD sequence.
  • the variant may be the “APMAP ICD (reverse) KtoR” variant (SEQ ID NO:46), which is identical to SEQ ID NO:45 except that the lysine residue in it has been substituted by arginine.
  • the ICD of the engineered PD-L1 protein may comprise an ICD sequence of another cell surface protein with a long protein half-life (i.e., longer than 15 hours).
  • additional cell surface proteins are SLC2A2, NT5E, CD47, ABCC6, STX4, FAS, and NPTN.
  • the ICD of the engineered PD-L1 protein may comprise the polypeptide sequence of a second cell surface proteins with a long protein half-life (i.e., longer than 15 hours) or of another protein selected based on useful functions (e.g., proteins know to interact with the PD-L1 protein, stabilize the PD-L1 protein, or both).
  • this polypeptide sequence may be fused to the ICD described above, with or without a peptide linker.
  • the ICD of the engineered PD-L1 protein may comprise a wildtype PD-L1 ICD fused through a peptide linker to CKLF-like MARVEL transmembrane domain-containing protein 6 (CMTM6) (e.g., SEQ ID NO:47).
  • the ICD of the engineered PD-L1 protein may comprise a wildtype PD-L1 ICD fused through a peptide linker to COP9 signalosome complex subunit 5 (CSN5) (e.g., SEQ ID NO:48).
  • the peptide linker may be a flexible linker such as a GS linker.
  • a GS linker is rich in glycine and serine (more than 50% of the residues are glycine and/or serine).
  • the peptide linker comprises GGGGSGGGSGGGS (SEQ ID NO:49).
  • the engineered PD-L1 protein is anchored in the cell membrane through a glycophosphatidylinositol (GPI) anchor.
  • GPI glycophosphatidylinositol
  • the extracellular region of the protein is fused to a GPI anchor sequence, e.g., SEQ ID NO:50.
  • the present polypeptide comprises, consists of, or consists essentially of a PD-L1 protein sequence with one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more) mutations, such as substitutions.
  • the engineered PD-L1 sequence is termed “PDL1-K5R,” with five K-to-R mutations (underlined) in the ICD relative to wildtype PD-L1 :
  • LGVALTFI FR LRRGRMMDVR RCGIQDTNSR RQSDTHLEET SEQ ID NO : 2
  • the engineered PD-L1 sequence is termed “PDL1-
  • K263R contains one K-to-R mutation (underlined) in the ICD relative to wildtype PD-
  • the engineered PD-L1 sequence is termed “PDL1- KtoR,DEtoR” and contains ten amino acid substitutions relative to wildtype PD-L1, where all lysines, aspartic acids, and glutamic acids in the wildtype PD-L1 ICD have been substituted with arginines (underlined):
  • the engineered PD-L1 sequence is termed “PDL1 (3NQ)” and contains three N-to-Q mutations (underlined) in the IgC-like domain relative to wildtype PD-
  • the engineered PD-L1 sequence is termed “PDL1-CMTM6 fusion with linker,” in which a wildtype PD-L1 sequence is fused at its C-terminus to a CMTM6 polypeptide sequence (boldfaced; SEQ ID NO:47) through a peptide linker of SEQ ID NO:49 (underlined):
  • CEACAM1 in which the ICD of a wildtype PD-L1 sequence is replaced with an ICD from CEACAM1 (italic; SEQ ID NO:39): MRIFAVFI FM TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEI FYCT FRRLDPEENH TAELVIPELP LAHPPNERTH LVILGAILLC
  • the engineered PD-L1 sequence is termed “PDL1-ENPP1
  • the reversed APMAP ICD sequence is mutated, where the lysine residue is substituted by arginine (underlined), and the engineered PD-L1 sequence is called “PDL1 -APMAP -KtoR (ICD reverse)”:
  • TM in which the TM domain of a wildtype PD-L1 is replaced with a TM domain from
  • the engineered PD-L1 sequence is called “PDL1-CEACAM1
  • TM in which the TM domain of a wildtype PD-L1 is replaced with a TM domain from CEACAM1 (boldface; SEQ ID NO:32):
  • the engineered PD-L1 sequence is called “PDL1-ITGA1
  • TM in which the TM domain of a wildtype PD-L1 is replaced with a TM domain from
  • the engineered PD-L1 sequence comprises the PD-Ll’s extracellular portion that interacts with PD-1 fused to a GPI anchor.
  • the engineered PD-L1 sequence is called “PDL1-GPI” and comprises the extracellular region of a wildtype PD-L1 in which the signal sequence is replaced with that of a human NT5E (underlined; SEQ ID NO:29) and the C-terminus of the extracellular region is fused to a GPI anchor with the sequence of SEQ ID NO:50 (italic):
  • the engineered PD-L1 sequence comprises the PD-Ll’s extracellular portion that interacts with PD-1 fused to a GPI-anchorable protein such as NT5E.
  • the engineered PD-L1 sequence is called “PDL1-NT5E fusion” and comprises the extracellular region of a wildtype PD-L1 in which the signal sequence is replaced with that of a human NT5E (underlined; SEQ ID NO:29) and the C- terminus of the extracellular region is fused to a mature sequence of NT5E (i.e., without the signal sequence; italic; SEQ ID NO:51) through a short peptide linker with the sequence of SEQ ID NO: 52 (boxed):
  • Variants of the above-described sequences are also within the present disclosure. Variants may be deletions, insertions, and/or substitutions. In some embodiments, the substitutions are conservative substitutions: for example, an aliphatic amino acid (e.g., glycine, alanine, valine, leucine, or isoleucine) may be substituted by another aliphatic amino acid; a hydroxyl or sulfur/selenium-containing amino acid (e.g., serine, cysteine, selenocysteine, threonine, or methionine) may be substituted by another hydroxyl or sulfur/selenium-containing amino acid; an aromatic amino acid (e.g., phenylalanine, tyrosine, or tryptophan) may be substituted by another aromatic amino acid; a branched chain amino acid (e.g., isoleucine, leucine, or valine) may be substituted by another branched chain amino acid;
  • a negatively charged amino acid e.g., aspartic acid or glutamic acid
  • a positively charged amino acid e.g., lysine, arginine and histidine
  • a nonpolar amino acid e.g., alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, valine, or tryptophan
  • a nonpolar amino acid e.g., alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, valine, or tryptophan
  • the engineered PD-L1 sequence comprises, consists of, or consists essentially of, one of SEQ ID NOs:2-22, 67, and 68 (with or without the signal sequence (i.e., SEQ ID NO:23)), or comprises one of SEQ ID NOs:73-78, and 80.
  • the present disclosure provides pharmaceutical compositions comprising a nucleic acid for expressing an engineered PD-L1 protein herein and a pharmaceutically acceptable carrier (e.g., water and phosphate-buffered solution).
  • a pharmaceutically acceptable carrier e.g., water and phosphate-buffered solution.
  • the pharmaceutical composition comprises a viral vector comprising the nucleic acid.
  • the pharmaceutical composition comprises an RNA (e.g., mRNA or circular RNA) encoding an engineered PD-L1, wherein the RNA is encapsulated in an LNP.
  • the pharmaceutical composition comprises a nucleic acid for expressing a polypeptide sequence comprises the sequence of PDL1-K5R, PDL1- APMAP-KtoR (ICD Reverse), PDLl-KtoR,DEtoR, PDL1-GPI, and PDL1-CEACAM1-K5R (ICD), or PDL1-APMAP (ICD reverse) or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto.
  • CH0-K1 epithelial cells were transfected with plasmid DNA encoding the various chimeric proteins.
  • CHO-K1 cells were seeded in 24-well plates at 40,000 cells per well and grown overnight at 37°C prior to transfection with 500 ng of plasmid DNA and 1.5 pL Lipofectamine2000TM (Invitrogen) per well. The cells were then incubated at 37°C in a 5% CO2 incubator.
  • This Example describes a study evaluating the half-life of the PD-L1 chimeras in transfected cells.
  • CHO-K1 cells were seeded in a 24-well plate and transfected as described in Example 1. The transfected cells were incubated at 37°C for 24 hours, 48 hours, 72 hours, and 96 hours in a 5% CO2 incubator. At each timepoint, cells were washed with PBS containing 2 mM EDTA and detached with Accutase®.
  • Detached cells were then stained for viability and PD-L1 expression and analyzed by FACS as described in Example 1, except anti-PD-Ll antibody clone 29EA3 (Biolegend) was used to detect PD-L1 at 1 :400 dilution for this and all subsequent experiments.
  • the ten chimeras were PD-L1-K5R (degradation stabilized control), PD-L1- KtoR,DEtoR, PDL1-ENPP1 (ICD reverse), PDLl-ENPPl-KtoR (ICD reverse), PDL1- CMTM6 fusion with linker, PDL1-CEACAM1 (ICD), PDL1-CEACAM1-K5R (ICD), PDL1-APMAP (ICD reverse), PDLl-APMAP-KtoR (ICD reverse), and PDL1-GPI.
  • This experiment was completed twice with CHO-K1 cells and both transfection replicates yielded consistent results.
  • OVCAR-3 ovarian adenocarcinoma cells were seeded in a 96-well plate at 30,000 cells per well and grown overnight at 37°C. The cells were then transfected with 200 ng of plasmid DNA encoding the PD-L1 fusion proteins and 0.6 pL Lipofectamine2000TM (1 :3 ratio) per well. The transfected cells were incubated at 37°C for 1 day, 5 days, or 9 days in a 5% CO2 incubator with cell culture medium replaced on Day 5 for cells that were cultured for 9 days.
  • DNA vectors carry safety risks because vector DNA may be incorporated into the host genome.
  • circRNA also termed herein “eRNATM” for “Endless RNATM”
  • eRNATM circular RNA
  • CHO-K1 cells were seeded in a 96-well plate with 35,000 cells per well and grown overnight at 37°C. The cells were then transfected with 0.2 pmol eRNA encoding the PD-L1 chimeras and 0.6 pL LipofectamineTM MessengerMAXTM Transfection Reagent (Thermo Fisher) (1 :3 ratio) per well.
  • the cells were cultured for 1, 2, 3, and 7 days at 37°C in a 5% CO2 incubator. For cells that were cultured for 7 days, they were split 1 : 10 on Day 3 and the culture medium was replaced on Day 5. The cells were detached, stained, and analyzed by FACS as described above.
  • MFI median fluorescent intensity
  • HEK293T cells a human embryonic kidney cell line.
  • HEK293T cells were seeded in a 96-well plate at 15,000 cells per well and grown overnight at 37°C.
  • the cells were then transfected with 35 ng eRNA encoding the PD-L1 chimeras and 0.1 pL LipofectamineTM MessengerMAXTM Transfection Reagent (2: 1 ratio) per well.
  • the cells were then incubated for 1, 3, and 7 days at 37°C in a 5% CO2 incubator. For cells that were cultured for 7 days, the culture medium was replaced on Day 5.
  • Cells were detached by pipetting. Detached cells were stained and analyzed by FACS as described above.
  • the cells were stained with anti-CD45-AF700 (BioLegend; 1 :400), anti-CD26-APC (BioLegend; 1 :200), and anti- PD-L1-PE (BioLegend; 1 :200).
  • the stained cells were washed with FACS buffer and samples were analyzed by flow cytometry.
  • CHO cells were transfected with Nl-Methylpseudouridine-modified mRNAs encoding various PD-L1 mutations shown in Table 2.
  • the PD-L1 L3C7 variant (ECD shown in SEQ ID NO: 75) has the following mutations relative to wildtype PD-L1 : I54E, Y56H, E58F, R113T, Ml 15L, SI 17G, and G119K.
  • the PD-L1 L3C7/A110W variant (ECD shown in SEQ ID NO:76) has one additional A121W mutation relative to PD-L1 L3C7.
  • PD-L1 1 lOLoop (ECD shown in SEQ ID NO: 77) differs from wildtype PD-L1 in that the former has a G120V mutation, and an insertion of W between wildtype residues A121 and D122.
  • the PD-L1 L3B3 variant (ECD shown in SEQ ID NO:78) has the following mutations relative to wildtype PD-L1 : I54Q, Y56F, E58M, R113T, Ml 15L, SI 17A, and G119K.
  • the PD-L1 Al 10W variant has an A121W mutation relative to wildtype PD-L1. (The PD-L1 Al 10W variant was so named because A121 in PD-L1 corresponds to Al 10 in PD-L2.)
  • the mutations were done in the background of CEACAM1-K5R (ICD) PD-L1.
  • K5R G 3 S (SEQ ID NO:81) and (G 3 S) 3 (SEQ ID NO:72) represent the PD-L1 chimeras where the cleavage site has been substituted by one or three G 3 S (SEQ ID NO: 81) linkers, respectively, whereas the cleavage site for K5R SCR has been scrambled using the same amino acid composition.

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Abstract

La présente divulgation concerne des polypeptides dérivés de ligands PD-1, des acides nucléiques codant pour ces polypeptides, et des procédés d'utilisation des polypeptides et des acides nucléiques à des fins thérapeutiques.
PCT/US2025/010194 2024-01-05 2025-01-03 Ligands pd-1 modifiés Pending WO2025147583A1 (fr)

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Citations (5)

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WO2019118919A1 (fr) 2017-12-15 2019-06-20 Flagship Pioneering, Inc. Compositions comprenant des polyribonucléotides circulaires et leurs utilisations
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WO2021159016A1 (fr) * 2020-02-05 2021-08-12 Diadem Biotherapeutics Inc. Synapses artificielles
WO2022245841A1 (fr) * 2021-05-17 2022-11-24 Wisconsin Alumni Research Foundation Protéine synthétique pour induire une tolérance immunitaire
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WO2019118919A1 (fr) 2017-12-15 2019-06-20 Flagship Pioneering, Inc. Compositions comprenant des polyribonucléotides circulaires et leurs utilisations
WO2020257710A1 (fr) * 2019-06-21 2020-12-24 Entelexo Biotherapeutics Inc. Plateformes, compositions et méthodes d'administration de composés thérapeutiques
WO2021159016A1 (fr) * 2020-02-05 2021-08-12 Diadem Biotherapeutics Inc. Synapses artificielles
WO2022245841A1 (fr) * 2021-05-17 2022-11-24 Wisconsin Alumni Research Foundation Protéine synthétique pour induire une tolérance immunitaire
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