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WO2025101877A1 - Plateforme pour augmenter la présentation d'antigène hla de classe i à l'aide de protéines de fusion chaperon modifiées - Google Patents

Plateforme pour augmenter la présentation d'antigène hla de classe i à l'aide de protéines de fusion chaperon modifiées Download PDF

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WO2025101877A1
WO2025101877A1 PCT/US2024/055092 US2024055092W WO2025101877A1 WO 2025101877 A1 WO2025101877 A1 WO 2025101877A1 US 2024055092 W US2024055092 W US 2024055092W WO 2025101877 A1 WO2025101877 A1 WO 2025101877A1
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hla
cfp
cell
nucleic acid
cells
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Nikolaos SGOURAKIS
Daniel Hwang
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Childrens Hospital of Philadelphia CHOP
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Childrens Hospital of Philadelphia CHOP
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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
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to engineered variants of chaperone proteins to mediate mature peptide human leukocyte antigen (pHLA) complex production.
  • Neuroblastoma is an immunologically “cold tumor” characterized by low mutational burden, leading to a paucity of neoantigens that is further compounded by aberrant MHC-1 antigen presentation.
  • diseases are highly refractory to T cell-based immunotherapy, including checkpoint blockade, often lacking suitable surface-expressed antigens that can be targeted via chimeric antigen receptors (CARs).
  • CARs chimeric antigen receptors
  • the invention relates to methods of increasing surface human leukocyte antigen (HLA) expression which may comprise fusing a nucleic acid encoding a chaperone to a nucleic acid encoding a protein of interest, forming a fused nucleic acid encoding a chaperone fusion protein (CFP), administering the nucleic acid encoding the CFP to a cell, allowing the nucleic acid to express the CFP in the cell, wherein the CFP increases HLA expression in the cell.
  • HLA human leukocyte antigen
  • the chaperone is a HLA class I chaperone.
  • the chaperone is tapasin or transporter associated with antigen processing (TAP)- binding protein related (TAPBPR).
  • TAP antigen processing
  • the tapasin or the TAPBR is fused to a transmembrane (TM) domain.
  • the TM domain is a human leukocyte antigen (HLA) TM domain.
  • the HLA is HLA-G.
  • the nucleic acid encoding the TM domain of HLA- G is SEQ ID NO: 3.
  • the nucleic acid encoding the CFP is SEQ ID NO: 4 or SEQ ID NO: 5.
  • the protein of interest is an antigen, advantageously a tumor antigen.
  • the invention also relates to an immune adjuvant produced by a T cell in a T cell based immunotherapy comprising a T cell transduced with any of the herein disclosed CFPs.
  • the T cell based immunotherapy is chimeric antigen receptor (CAR)-T, T- cell receptor (TCR)-T or tumor infiltrating lymphocyte (TIL) therapy.
  • the invention also relates to a vaccine adjuvant comprising any of the herein disclosed CFPs.
  • the vaccine is an anti-viral vaccine or a cancer vaccine.
  • the invention also relates to a multivalent molecule comprising any of the herein disclosed CFPs conjugated to a protein or molecule that mediates targeting of the CFP to a target cell.
  • the protein that mediates targeting of the CFP to a target cell is a single chain fragment variable (scFV) antibody.
  • the target cell is a tumor cell or a viral infected cell.
  • the invention also encompasses a pharmaceutical composition which may comprise any of the herein disclosed CFPs, any of the herein disclosed immune adjuvants, any of the herein disclosed vaccine adjuvants, or any of the herein disclosed multivalent molecules and a pharmaceutically acceptable carrier.
  • the invention also encompasses a method of treating or preventing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the herein disclosed CFPs, any of the herein disclosed immune adjuvants, any of the herein disclosed vaccine adjuvants, any of the herein disclosed multivalent molecules or a pharmaceutical composition which may comprise any of the herein disclosed CFPs, any of the herein disclosed immune adjuvants, any of the herein disclosed vaccine adjuvants, or any of the herein disclosed multivalent molecules and a pharmaceutically acceptable earner.
  • the invention also encompasses the use of any of the herein disclosed CFPs, any of the herein disclosed immune adjuvants, any of the herein disclosed vaccine adjuvants, any of the herein disclosed multivalent molecules or a pharmaceutical composition which may comprise any of the herein disclosed CFPs, any of the herein disclosed immune adjuvants, any of the herein disclosed vaccine adjuvants, or any of the herein disclosed multivalent molecules and a pharmaceutically acceptable carrier to increase surface human leukocyte antigen (HLA) expression.
  • HLA human leukocyte antigen
  • the invention also encompasses methods for enhancing peptide presentation and identification in tumor cells.
  • the methods comprise (a) contacting tumor cells with a nucleic acid encoding a chaperone fused to a nucleic acid encoding a protein of interest to form a fused nucleic acid encoding a chaperone fusion protein (CFP); (b) expressing the CFP in the tumor cells; (c) upregulating human leukocyte antigen (HLA) expression; (d) capturing and identifying peptides that are bound on the upregulated HLAs.
  • the HLA is HLA-I.
  • the protein of interest is an antigen, advantageously a tumor antigen.
  • FIGS. 1A-1D CFPs enhance increase HLA-A2 expression in tapasin knockout and neuroblastoma cell lines.
  • Cell lines were transduced with lentiviral CFP constructs and then assayed for HLA-A2 expression by flow cytometry.
  • FIG. 1A Expression of HLA-A2 in Expi293F Tapasin KO cells
  • FIG. IB Expression of FLAG-TAG in Expi293F Tapasin KO cells.
  • FIG. 1C top).
  • FIG. 1C bottom).
  • FIG. 3C Expression of 4-fBB on CD8 T cells after co-culture.
  • FIG. 3D Cell surface FLAG expression on EBcl cells after co-culture with CFP transduced CD8 T cells.
  • FIG. 3E MFI of CD69 on CD8 T cells after co-culture.
  • FIG. 3F Frequency of 4-1BB expression on CD8 T cells.
  • FIG. 3G Frequency of EBcl cells expressing FLAG on the cell surface after co-culture.
  • FIG. 3H Frequency of EBcl cells expressing FLAG on the cell surface after co-culture.
  • FIGS. 4A-4F T cell derived CFPs increase efficiency of T cell killing.
  • FIG. 4A NYESO-1 specific killing of SUDHL4 cells transduced to express NYESO-1. 1G4 T cells were mixed at different effector to target ratios and incubated overnight with SUDHL4 NYESO-1 cells and SUDHL4 cell transduced with empty vector.
  • NYESO-1 specific killing is calculated by subtracting the number of surviving cells in SUDHL4 NYESO-1 cells from the number of surviving cells in SUDHL4 Empty Vector cells and divided by surviving cells in SUDHL4 Empty Vector.
  • Fig. 4B Number of surviving SUDHL4 NYESO-1 cells with 1G4 T cells and CFPs Tapasin-TM or TAPBPR-TM.
  • Fig. 4C Number of surviving SUDHL4 Empty Vector cells with 1G4 T cells and CFPs Tapasin-TM or TAPBPR-TM.
  • FIG. 4D Frequency of surviving tumor cells expressing FLAG TAG.
  • MFI of4-lBB in CD8 T cells SUDHL4 NYESO-1 on top and SUDHL4 Empty 7 Vector on bottom.
  • FIGs. 5A-5C CFPs enhance the immunogenicity of neuroblastoma cells.
  • FIG. 5A Schematic of T cell killing assay using NYESO157-165 pulsed EBc-1 cells and 1G4 TCR-T cells.
  • FIG. 5B Number of surviving cells after overnight culture for different effector to target ratios is shown (right).
  • FIG. 5C T cell killing assay using EBc-1 cells transduced with full length NYESO-1 or with a control lentivirus. NYESO-1 specific lysis with a 1 :2 E:T ratio is shown. Killing is measured using a flow 7 cytometry based assay.
  • FIG. 5D Kinetics of NYESO- 1 specific killing performed as in C but measured with xCELLigence-based impedance measurements.
  • FIGs. 6A-6C Tapasin interactions stabilize MHC-I complexes on the cell surface.
  • Parental EbCl cells and those transduced with tapasin variants were treated with (FIG. 6A) ribosomal inhibitor cycloheximide, (FIG. 6B) transport inhibitor brefeldin A, and (FIG. 6C) lysosomal inhibitor bafilomycin Al.
  • FIG. 6A ribosomal inhibitor cycloheximide
  • FIG. 6B transport inhibitor brefeldin A
  • FIG. 6C lysosomal inhibitor bafilomycin Al. MFI of surface HLA-A2 is shown.
  • FIGs. 7A-7C CFP expressing T cells kill B cell lymphoma cells more efficiently.
  • SUDHL4 B cell lymphoma cells transduced with NYESO-1 or empty vector were first labeled with CellTrace Violet as an identifying marker before plating. Cells were then cocultured overnight with CD8 + T cells transduced with 1G4 +/- CFPs.
  • FIG. 7A Quantification of NYESO-1 specific killing is shown.
  • FIG. 7B Expression of 4-1BB on CD8 T cells after coculture.
  • FIG. 7C Cell surface FLAG expression on SUDHL4 NYESO-1 cells after co-culture with CFP transduced CD8 T cells.
  • CFPs enhance immunoprecipitation of folded HLA-I molecules for immunopeptidomics applications.
  • Parental EBcl cells and those transduced with tapasin WT, Tapasin-TM CFP, and negative control tapasin-TN6-TM were lysed and samples collected prior to immunoprecipitation as well as following immunoprecipitation with anti- HLA-A2 antibody BB7.2.
  • SDS-PAGE and native gel electrophoresis was performed prior to western blot probing for B2M or vinculin as loading control.
  • Tapasin-TM and TAPBPR-TM.
  • the features of Tapasin-TM (SEQ ID NO: 4) and TAPBPR-TM (SEQ ID NO: 5) include the signal peptide of HLA (underlined: SEQ ID NO: 1), FLAG Tag (bolded; SEQ ID NO: 2), and short peptide linker GGS (italicized) at the N’-terminus of both proteins, and the transmembrane (TM) domain of HLA-G (bold and italicized; SEQ ID NO: 3) and a stop codon (indicated by
  • Tapasin, a member of the PLC, and a related protein TAPBPR are HLA class I chaperones that are frequently mutated or dysregulated in cancers [Shionoya, Y., et al.. Loss of tapasin in human lung and colon cancer cells and escape from tumor-associated antigenspecific CTL recognition. Oncoimmunology, 2017. 6(2): p. el274476.
  • TAPBPR is an auxiliary chaperone that is not restricted to the PLC but instead actively participates in quality control through the reglycosylation of sub optimally loaded MHC molecules by recruiting them to UGGT1 [Neerincx. A., et al., TAPBPR bridges UDP-glucose:gly coprotein glucosyltransferase 1 onto MHC class I to provide quality control in the antigen presentation pathway. Elife, 2017. 6. PMC5441866 PMID: 28425917], These critical functions of HLA-I chaperones in regulating antigen presentation, and by extension immunosurveillance, are frequently disrupted across cancer types to evade immune detection [Shionoya.
  • the term “multivalent” refers to an engineered molecule that incorporates two or more biologically active segments.
  • the protein fragments forming the multivalent molecule optionally may be linked through a linker or a spacer which attaches the constituent parts and permits each to function independently.
  • the linker or a spacer allow separate domains to fold independent of each other.
  • the linker is a protein linker which may be alanine, glycine, proline or serine-based linkers or a combination thereof and may be as short as 8 amino acids to as long as 50 amino acids in length.
  • CFPs are conjugated to another protein that mediates targeting of the CFP to the target cell (e.g. tumor or viral infected cells).
  • target cell e.g. tumor or viral infected cells
  • scFv single chain fragment variable antibodies
  • the scFv mediates targeting of the CFP to the target cell (e.g. tumor or viral infected cells), allowing for target cell specific upregulation of HL A.
  • the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding any of the peptides or proteins of the invention.
  • the nucleic acid may be cDNA.
  • Such a nucleic acid molecule can be synthesized in accordance with methods known in the art. Due to the degeneracy of the genetic code, one of ordinary skill in the art will appreciate that nucleic acid molecules of different nucleotide sequence can encode the same amino acid sequence.
  • the invention provides a vector comprising a nucleic acid sequence according to the third aspect of the invention.
  • the vector may include, in addition to a nucleic acid sequence encoding only a peptide of the invention, one or more additional nucleic acid sequences encoding one or more additional peptides. Such additional peptides may, once expressed, be fused to the N-terminus or the C-terminus of the peptide of the invention.
  • the vector includes a nucleic acid sequence encoding a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly -histidine tag.
  • a peptide or protein tag such as, for example, a biotinylation site, a FLAG-tag, a MYC-tag, an HA-tag, a GST-tag, a Strep-tag or a poly -histidine tag.
  • Suitable vectors are known in the art as is vector construction, including the selection of promoters and other regulatory elements, such as enhancer elements.
  • the vector utilized in the context of the present invention desirably comprises sequences appropriate for introduction into cells.
  • the vector may be an expression vector, a vector in which the coding sequence of the polypeptide is under the control of its own cis-acting regulatory elements, a vector designed to facilitate gene integration or gene replacement in host cells, and the like.
  • a “vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. In general, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single-chain, single-stranded, double-stranded, or partially doublestranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides know n in the art.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector wherein virally- derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g.
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.'’
  • Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • the vector is a lentivector.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • cancer comprises leukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, glioblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear. nose and throat (ENT) cancer, breast cancer, prostate cancer, cancer of the uterus, ovarian cancer and lung cancer and the metastases thereof.
  • ENT nose and throat
  • cancer according to the invention also comprises cancer metastases and relapse of cancer.
  • the term “autoimmunity'’ relates a system of immune responses of an organism against its own healthy cells, tissues and other normal body constituents. Any disease resulting from this type of immune response is termed an “autoimmune disease.”
  • Prominent examples include, but are not limited to, celiac disease, post- infectious IBS, diabetes mellitus type 1, Henoch-Schonlein purpura (HSP) sarcoidosis, systemic lupus erythematosus (SLE), Sjogren syndrome, eosinophilic granulomatosis with polyangiitis, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, Addison's disease, rheumatoid arthritis (RA), ankylosing spondylitis, polymyositis (PM), dermatomyositis (DM), Alopecia Areata and multiple sclerosis (MS).
  • HSP Henoch-Schonlein pur
  • infectious disease relates to a transmissible disease or communicable disease, or an illness resulting from an invasion of tissues by pathogens, their multiplication, and the reaction of host tissues to the infectious agent and the toxins they produce.
  • infectious agents pathogens
  • bacteria e.g. Mycobacterium tuberculosis, Staphylococcus aureus, Escherichia coli, Clostridium botulinum, and Salmonella spp.
  • viruses and related agents such as viroids.
  • HIV Rhinovirus
  • Lyssaviruses such as Rabies virus, Ebolavirus and Severe acute respiratory' syndrome coronavirus 2
  • fungi further subclassified into: Ascomycota, including yeasts such as Candida (the most common fungal infection); filamentous fungi such as Aspergillus; Pneumocystis species; and dermatophytes, a group of organisms causing infection of skin and other superficial structures in humans, basidiomycota, including the human-pathogenic genus Cryptococcus, parasites, which are usually divided into: unicellular organisms (e.g.
  • nematodes such as parasitic roundworms and pinworms, tapeworms (cestodes), and flukes (trematodes, such as schistosomes), arthropods such as ticks, mites, fleas, and lice
  • nematodes such as parasitic roundworms and pinworms
  • tapeworms such as tapeworms
  • flukes trematodes, such as schistosomes
  • arthropods such as ticks, mites, fleas, and lice
  • human disease which conceptually are similar to infections, but invasion of a human or animal body by these macroparasites is usually termed infestation, and prions (although they do not secrete toxins).
  • peptide disease target relates to compositions such as, but not limited to, allotypes, peptides, epitopes, cell populations, ligands, receptors, or binders that interact, or affect the expression of, a target of interest, which may be specific to a desired disease.
  • the protein of interest is an antigen, advantageously a tumor antigen.
  • an antigen may encompass a protein or a fragment, derivative or variant thereof as long as an immune response is capable of being elicited by the antigen or the fragment, derivative or variant thereof.
  • a tumor antigen may be a tumor-specific antigen or a tumor-associated antigen.
  • Tumor antigens include, but are not limited to, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), CA-I25, MUC-1, epithelial tumor antigen (ETA), tyrosinase, melanoma- associated antigen (MAGE), and abnormal products of ras, p53.
  • the invention also relates to an immune adjuvant produced by a T cell in a T cellbased immunotherapy comprising a T cell transduced with any of the herein disclosed CFPs.
  • the T cell-based immunotherapy is a chimeric antigen receptor (CAR)-T (see, e.g., international patent publication W02019210153A1), T-cell receptor (TCR)-T (see, e.g., international patent publication W02020038492A1) or tumor infiltrating lymphocyte (TIL) therapy (see, e.g., international patent publication WO2018182817A1).
  • CAR chimeric antigen receptor
  • TCR T-cell receptor
  • TIL tumor infiltrating lymphocyte
  • the invention also relates to a vaccine adjuvant comprising any of the herein disclosed CFPs.
  • the vaccine is an anti-viral vaccine or a cancer vaccine.
  • the invention also relates to a multivalent molecule comprising any of the herein disclosed CFPs conjugated to a protein or molecule that mediates targeting of the CFP to a target cell.
  • the protein that mediates targeting of the CFP to a target cell is a single chain fragment variable (scFV) antibody.
  • the target cell is a tumor cell or a viral infected cell.
  • the therapeutically active agents, vaccines and compositions described herein may be administered via any conventional route, including by injection or infusion.
  • the administration may be carried out, for example, orally, intravenously, intraperitoneally, intramuscularly, subcutaneously or transdermally.
  • administration is carried out intranodally such as by injection into a lymph node.
  • Other forms of administration envision the in vitro transfection of antigen presenting cells such as dendritic cells with nucleic acids described herein followed by administration of the antigen presenting cells.
  • an “effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses.
  • the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease.
  • the desired reaction in a treatment of a disease or of a condition may also be delay of the onset or a prevention of the onset of said disease or said condition.
  • an effective amount of an agent described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the agents described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient w ith an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.
  • compositions of the invention are preferably sterile and contain an effective amount of the therapeutically active substance to generate the desired reaction or the desired effect.
  • compositions of the invention are generally administered in pharmaceutically compatible amounts and in pharmaceutically compatible preparation.
  • pharmaceutically compatible refers to a nontoxic material which does not interact with the action of the active component of the pharmaceutical composition. Preparations of this kind may usually contain salts, buffer substances, preservatives, carriers, supplementing immunity- enhancing substances such as adjuvants, e.g. CpG oligonucleotides, cytokines, chemokines, saponin, GM-CSF and/or RNA and, where appropriate, other therapeutically active compounds.
  • the salts should be pharmaceutically compatible. However, salts which are not pharmaceutically compatible may be used for preparing pharmaceutically compatible salts and are included in the invention.
  • Pharmacologically and pharmaceutically compatible salts of this kind comprise in a non-limiting way those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic acids, and the like.
  • Pharmaceutically compatible salts may also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.
  • a pharmaceutical composition of the invention may comprise a pharmaceutically compatible carrier.
  • carrier refers to an organic or inorganic component, of a natural or synthetic nature, in which the active component is combined in order to facilitate application.
  • pharmaceutically compatible carrier includes one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a patient.
  • the components of the pharmaceutical composition of the invention are usually such that no interaction occurs which substantially impairs the desired pharmaceutical efficacy.
  • compositions of the invention may contain suitable buffer substances such as acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt.
  • compositions may. where appropriate, also contain suitable preservatives such as benzalkonium chloride, chlorobutanol, paraben and thimerosal.
  • compositions are usually provided in a uniform dosage form and may be prepared in a manner known per se.
  • Pharmaceutical compositions of the invention may be in the form of capsules, tablets, lozenges, solutions, suspensions, syrups, elixirs or in the form of an emulsion, for example.
  • compositions suitable for parenteral administration usually comprise a sterile aqueous or nonaqueous preparation of the active compound, w hich is preferably isotonic to the blood of the recipient.
  • compatible carriers and solvents are Ringer solution and isotonic sodium chloride solution.
  • sterile, fixed oils are used as solution or suspension medium.
  • the invention also encompasses a use of any one of the herein disclosed complexes, nucleic acids, vectors or pharmaceutical compositions to screen a panel of candidate epitopic peptides to identify relevant peptide disease targets.
  • the disease is cancer, autoimmunity or an infectious disease.
  • the invention also encompasses methods for enhancing peptide presentation and identification in tumor cells.
  • the methods comprise (a) contacting tumor cells with a nucleic acid encoding a chaperone fused to a nucleic acid encoding a protein of interest to form a fused nucleic acid encoding a chaperone fusion protein (CFP); (b) expressing the CFP in the tumor cells; (c) upregulating human leukocyte antigen (HLA) expression; (d) capturing and identify ing peptides that are bound on the upregulated HLAs.
  • the HLA is HLA-I.
  • Capturing and identifying peptides that are bound on the upregulated HLA peptides can, for example, be done using MHC I immunopeptidome isolation and analysis methods, such as those described in Purcell et al.. Nat. Protocol. 14(6): 1687-1707 (2019); Kuznetsov et al., Molecules 25(22):5409 (2020); tiling et al., Current Opinion Immunol. 77: 102216 (2022); and Koval chik et al., bioRxiv (2020); and those demonstrated in the Examples below.
  • MHC-I molecules can be immunoprecipitated using a pan allelic antibody, such as W6/32), and peptides will be eluted under mild acidic conditions and analyzed using liquid chromatography coupled with mass spectrometry (LC-MS).
  • Software such as MSFragger (Kong et al., Nat. Methods 14(5):513-520 (2017)) can be used to search the MS raw data for peptides using reference protein databases, further validated using MhcVizPipe (Kovalchik et al., Mol. Cell Proteomics 21(1): 100178 (2022)).
  • Embodiment 1 is a method of increasing surface human leukocyte antigen (HLA) expression comprising fusing a nucleic acid encoding a chaperone to a nucleic acid encoding a protein of interest, forming a fused nucleic acid encoding a chaperone fusion protein (CFP). administering the nucleic acid encoding the CFP to a cell, allowing the nucleic acid to express the CFP in the cell, wherein the CFP increases HLA expression in the cell.
  • HLA human leukocyte antigen
  • Embodiment 2 is the method of embodiment 1 , wherein the chaperone is a HLA class I chaperone.
  • Embodiment 6 is the method of embodiment 5, wherein the HLA is HLA-G.
  • Embodiment 9 is the method of any one of embodiments 1-8, wherein the protein of interest is an antigen.
  • Embodiment 13 is a vaccine adjuvant comprising the CFP of any one of embodiments 1-10.
  • Embodiment 14 is the vaccine adjuvant of embodiment 13, wherein the vaccine is an anti-viral vaccine or a cancer vaccine.
  • Embodiment 15 is a multivalent molecule comprising the CFP of any one of embodiments 1-10 conjugated to a protein or molecule that mediates targeting of the CFP to a target cell.
  • Embodiment 16 is the multivalent molecule of embodiment 1 , wherein the protein that mediates targeting of the CFP to a target cell is a single chain fragment vanable (scFV) antibody.
  • Embodiment 17 is the multivalent molecule of embodiment 15 or 16, wherein the target cell is a tumor cell or a viral infected cell.
  • Embodiment 18 is a pharmaceutical composition comprising the CFP of any one of embodiments 1-10, the immune adjuvant of embodiment 11 or embodiment 12, the vaccine adjuvant of embodiment 13 or embodiment 14, or the multivalent molecule of any one of embodiments 15-17 and a pharmaceutically acceptable carrier.
  • Embodiment 19 is a method of treating or preventing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the CFP of any one of embodiments 1-10, the immune adjuvant of embodiment 11 or embodiment 12, the vaccine adjuvant of embodiment 13 or embodiment 14, the multivalent molecule of any one of embodiments 15-17 or the pharmaceutical composition of embodiment 18.
  • Embodiment 20 is a use of the CFP of any one of embodiments 1-10, the immune adjuvant of embodiment 11 or embodiment 12, the vaccine adjuvant of embodiment 13 or embodiment 14, the multivalent molecule of any one of embodiments 15-17 or the pharmaceutical composition of embodiment 18 to increase surface human leukocyte antigen (HLA) expression.
  • HLA human leukocyte antigen
  • Embodiment 21 is a method for enhancing peptide presentation and identification in tumor cells, the method comprising:
  • Embodiment 21a is the method of embodiment 21, wherein the HLA is HLA-I.
  • Embodiment 22 is the method of embodiment 21, wherein the chaperone is a HLA class I chaperone.
  • Embodiment 23 is the method of embodiment 22, wherein the chaperone is tapasin or transporter associated with antigen processing (TAP)-binding protein related (TAPBR).
  • TAP antigen processing
  • TAPBR antigen processing-binding protein related
  • Embodiment 24 is the method of embodiment 23, wherein the tapasin or the TAPBR is fused to a transmembrane (TM) domain.
  • TM transmembrane
  • Embodiment 25 is the method of embodiment 24, wherein the TM domain is a human leukocyte antigen (HLA) TM domain.
  • Embodiment 26 is the method of embodiment 25, wherein the HLA is HLA-G.
  • Embodiment 27 is the method of embodiment 26, wherein the nucleic acid encoding the TM domain of HLA-G comprises SEQ ID NO:3.
  • Embodiment 28 is the method of embodiment 27, wherein the nucleic acid encoding the CFP comprises SEQ ID NO:4 or SEQ ID NO:5.
  • Embodiment 29 is the method of any one of embodiments 21-28, wherein the protein of interest is an antigen.
  • Lentiviral Production Lentivirus was produced by co-transfection of Lenti-X 293T cells (Takara Bio) with pSFFV transfer vector containing gene of interest along with psPAX2 packaging vector and pMD2.G envelope vector. Transfections were performed using lipofectamine 3000 according to manufacturer instructions. Virus containing supernatant was then collected each day for up to 3 days and concentrated using Lenti-X concentrator (Takara Bio) according to manufacturer instructions.
  • T cell Activation and Transduction and Purification were obtained from the Human Immunology Core of the University of Pennsylvania. T cells were then stimulated with anti-CD3/anti-CD28 Dynabeads at a 1: 1 ratio in complete RPMI medium supplemented with 10 ng/mL IL-2 for 24 hours prior to transduction. T cells were then replated on tissue culture treated plates coated with 50 ug/mL retronectin (Takara Bio) along with lentivirus. After an additional 48 hours of culture, dynabeads were removed and cells were expanded by adding fresh media supplemented with IL-2. Cells were expanded for several days before purification.
  • 1G4 purification was performed by labeling transduced cells with NYESO with PE-conjugated, HLA-A01*01 tetramer refolded with NYESO157-165 followed by magnetically activated cell sorting (MACS) using anti-PE microbeads. CFP expressing cells were then purified by a second round of MACS using anti-FLAG-PE antibody conjugate. Tumor cells were also transduced using the same method as above.
  • MCS magnetically activated cell sorting
  • T cell Killing Assays 1G4 T cells were co-cultured at different E:T ratios with tumor cells overnight. Killing was assayed by flow cytometry. Cell number was determined using CountBright counting beads (Thermo Fisher).
  • Example 2 CFPs Tapasin-TM and TAPBPR-TM increase cell surface pHLAs [00111]
  • increased cell surface pHLA may be achieved by leveraging the chaperoning functions of tapasin and TAPBPR.
  • Tapasin and TAPBPR variants were engineered that are targeted to the plasma membrane (called Tapasin- TM & TAPBPR- TM), allowing them to associate with HLAs from their point of synthesis in the ER to the cell surface [7], This has been accomplished by replacing the endogenous transmembrane (TM) domain, which normally retains tapasin and TAPBPR within the ER/Golgi compartments, with the HLA-G TM domain, allowing for trafficking along the secretory pathway (Table 1).
  • TM endogenous transmembrane
  • HLA-G TM domain allowing for trafficking along the secretory pathway
  • CFPs can, at least in principle, be delivered to tumor cells via T cells.
  • huCD8 T cells were co-transduced with 1G4 TCR and either tapasin-TM or TAPBPR- TM CFPs. These T cells were then purified to near 100% purity by PE-conjugated HLA- A2/NY-ESO-1157-165 tetramer staining followed by magnetically activated cell sorting (MACS) with anti-PE microbeads (Fig 3A). CFP expressing cells were then purified by a second round of MACS using the same principle (Fig. 3A, right). Given that this is the most likely application of CFPs, Applicants then sought to directly test whether these cells had enhanced capacity to kill target tumor cells.
  • CFP expressing 1G4 T cells have superior ability to recognize and kill EBcl cells pulsed with NYESOI 157-165 (Figs. 3B, 3C).
  • EBcl cells were washed thoroughly to remove all excess peptide prior to incubation with 1G4 T cells.
  • Improved killing correlated with increased expression activation markers CD69 and 4-1BB (Figs. 3E, 3F).
  • FLAG-TAG could be detected on the surface of EBcl cells (Figs. 3D, 3G). The likely mode of this transfer is via EVs.
  • CD8 T cell membrane markers were detected on the surface of EBcl cells, including CD8 (data not shown).
  • Peptide/MHC and CD58 are both required for efficient tumor killing, as shown by transduction of NY-ESO-1 peptide and CD58 in SUDHL4 cells which are NY-ESO-17CD58" (Fig. 3H).
  • Example 4 CFPs enhance T-cell mediated tumor killing
  • SUDHL4 cells are a diffuse B cell lymphoma cell line which are a liquid cancer. SUDHL4 cells that were either transduced with NYESO-1 or empty 7 vector were utilized. In contrast to 1G4 T cells which had only 20% NYESO-1 specific killing, 1G4 T cells transduced with TAPBPR-TM had enhanced ability to kill target SUDHL4 cells, with -40% NYESO-1 specific killing (Fig. 4A). Number of surviving NYESO- 1 SUDHL4 cells and Empty vector SUDHL4 cells with 1G4 T cells and CFPs Tapasin-TM or TAPBPR-TM shown in Figs.
  • Example 5 CFPs enhance the immunogenicity’ of neuroblastoma cells.
  • EBc- 1 cells were pulsed with bJTE/SO— 1157-165 and co— cultured with 1G4 TCR— T cells, and it was found that expression of CFPs enhanced T cell-mediated killing (Fig. 5A,B). Similarly, cells presenting NYESO-1 antigen via the endogenous pathway also showed increased sensitivity 7 to 1G4-TCR-T cell killing (Fig. 5C). To further confirm these results, the kinetics of killing in overnight cultures of cells presenting NYESO-1 antigen via the endogenous pathway was also measured through xCELLigence-based impedance measurements. Similarly, enhanced killing of EBcl cells expressing the Tapasin-TM CFP was observed. These data suggest that CFPs can increase the immunogenicity' of neuroblastoma, a bona fide cold tumor model.
  • Example 7 CFP expressing T cells kill B cell lymphoma cells more efficiently.
  • SUDHL4 cells were transduced with either NYESO-1 or the empty vector and co-cultured with 1G4 T cells that express CFPs.
  • CFP expressing 1G4 T cells had superior ability’ to recognize and kill SUDHL4 cells expressing NYESO-1 (Fig. 7A).
  • Enhanced killing correlated with increased expression of T cell activation marker 4-1BB, suggesting superior target recognition (Fig. 7B).
  • CFPs could be detected on the surface of surviving target cells, showing they have been transferred to the target cells (Fig. 7C).
  • Example 8 CFPs enhance immunoprecipitation of folded HLA-I molecules for immunopeptidomics .
  • B2M beta-2-microglobulin

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Abstract

La présente invention concerne des variants modifiés de protéines chaperon pour médier la production et la conception complexes d'antigène leucocytaire humain peptidique mature (pHLA) et leur procédé de fabrication et d'utilisation.
PCT/US2024/055092 2023-11-08 2024-11-08 Plateforme pour augmenter la présentation d'antigène hla de classe i à l'aide de protéines de fusion chaperon modifiées Pending WO2025101877A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170252417A1 (en) * 2016-03-07 2017-09-07 Massachusetts Institute Of Technology Protein-chaperoned t-cell vaccines
US20200148743A1 (en) * 2017-07-24 2020-05-14 Commissariat A L'energie Atomique Et Aux Energies Alternatives Hla-g transcripts and isoforms and their uses
US20210054046A1 (en) * 2010-07-21 2021-02-25 Sangamo Therapeutics, Inc. Methods and compositions for modification of a hla locus
WO2023163980A1 (fr) * 2022-02-22 2023-08-31 The Children's Hospital Of Philadelphia Systèmes et procédés d'échange de ligand à médiation par chaperon sur des molécules associées au cmh-i et au cmh à l'aide d'un tapbpr de poulet

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210054046A1 (en) * 2010-07-21 2021-02-25 Sangamo Therapeutics, Inc. Methods and compositions for modification of a hla locus
US20170252417A1 (en) * 2016-03-07 2017-09-07 Massachusetts Institute Of Technology Protein-chaperoned t-cell vaccines
US20200148743A1 (en) * 2017-07-24 2020-05-14 Commissariat A L'energie Atomique Et Aux Energies Alternatives Hla-g transcripts and isoforms and their uses
WO2023163980A1 (fr) * 2022-02-22 2023-08-31 The Children's Hospital Of Philadelphia Systèmes et procédés d'échange de ligand à médiation par chaperon sur des molécules associées au cmh-i et au cmh à l'aide d'un tapbpr de poulet

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