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WO2025049742A1 - Systèmes et procédés de génération de nanoparticules hla mosaïcales multivalentes pour des applications d'immunothérapie personnalisée - Google Patents

Systèmes et procédés de génération de nanoparticules hla mosaïcales multivalentes pour des applications d'immunothérapie personnalisée Download PDF

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WO2025049742A1
WO2025049742A1 PCT/US2024/044431 US2024044431W WO2025049742A1 WO 2025049742 A1 WO2025049742 A1 WO 2025049742A1 US 2024044431 W US2024044431 W US 2024044431W WO 2025049742 A1 WO2025049742 A1 WO 2025049742A1
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vlp
cancer
hla
antibody
loadable
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Nikolas 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
    • 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
    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/001188—NY-ESO
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • 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
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16023—Virus like particles [VLP]
    • 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
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • VLP virus-like particle
  • TCR T cell receptor
  • TCRs have a low avidity and fast off-rates for MHC-I complexes as a result of thymic selection, as such monomeric MHC-I complexes are not sufficient to detect antigen- specific T cells for the development of therapeutics against various infectious diseases, allergies, or cancers 1,3 .
  • MHC tetramers More than two decades ago, multivalent MHC tetramers were introduced for the detection of antigen-specific T cells. These MHC tetramers show an increased avidity for their cognate TCRs and have been successfully used since their introduction to detect, isolate, and study antigen-specific T cells 4 .
  • MHC-I allotypes – which are the most polymorphic proteins in humans, encompassing 6 different allotypes in an individual and more than 35,000 alleles at the population levels 5 – individual tetramer reagents must be prepared for each MHC-I heavy chain.
  • tetramers cannot stimulate T cells due to the lack of size and co-stimulatory molecules, such as 4-1BBL, limiting applications to the detection of antigen specific T cells.
  • the present invention relates to a loadable, multivalent virus-like particle (VLP) system that presents a multitude of MHC-I molecules with different allotype and antigen specificities.
  • VLP virus-like particle
  • the system can comprise (a) one or more recombinant patient human leukocyte antigens (HLA), (b) one or more antigens capable of binding to the one or more recombinant 2 4889-2185-6222.1 PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019/6WO1 patient HLAs, (c) a VLP, wherein the one or more recombinant patient HLAs are conjugated to the VLP, and wherein the one or more recombinant patient HLAs conjugated to the VLP are loaded with the one or more antigens.
  • the one or more patient human leukocyte antigens are isolated from a subject.
  • the one or more antigens are selected from an infectious antigen or a tumor associated antigen (TAA).
  • TAA tumor associated antigen
  • the infectious antigen can, for example be selected from a bacterial antigen, a viral antigen, a parasitic antigen, or a fungal antigen.
  • the TAA can, for example, be an antigen from a solid or a liquid cancer.
  • the solid or liquid cancer can, for example, be selected from the group consisting of a lung cancer, a gastric cancer, a colon cancer, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, a metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, a glioma, a glioblastoma, a non-Hodgkin’s lymphoma (NHL), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a chronic myelogenous leukemia (CML), a multiple myeloma (MM), and an acute myeloid leukemia (AML).
  • NHL lymphoma
  • ALL acute lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • MM multiple my
  • the system further comprises a co-stimulatory molecule.
  • the co-stimulatory molecule can, for example, be conjugated to the VLP system.
  • the co- stimulatory molecule can, for example, be selected from a protein, an antibody, a small molecule, and/or a combination thereof.
  • the protein can, for example, be selected from an OX40 ligand (OX40L), a B7 ligand, a 4-IBB ligand (4-IBBL), or a CD40 ligand (CD40L).
  • the antibody can, for example be selected from an anti-CD28 antibody, an anti-CTLA4 antibody, or an anti-PD1 antibody.
  • the molar ratio of the VLP to the HLA is about 1 to 180.
  • cancer vaccines comprising the systems described herein.
  • the cancer vaccine can, for example comprise the system described herein and an agonist molecule capable of eliciting a polyclonal anti-tumor T cell response.
  • the agonist molecule can, for example, be a co-stimulatory molecule.
  • the co- stimulatory molecule can, for example, be selected from a protein, an antibody, a small molecule, and/or a combination thereof.
  • the protein can, for example, be selected from an OX40 ligand (OX40L), a B7 ligand, a 4-IBB ligand (4-IBBL), or a CD40 ligand (CD40L).
  • the antibody can, for example be selected from an anti-CD28 antibody, an anti-CTLA4 antibody, or an anti-PD1 antibody. 3 4889-2185-6222.1 PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019/6WO1 [0016] Also provided are methods of producing an expanded tumor infiltrating lymphocyte (TIL) population from a subject with cancer.
  • TIL tumor infiltrating lymphocyte
  • the methods comprise (a) administering to the subject the loadable, multivalent VLP system described herein; (b) expanding a tumor infiltrating lymphocyte (TIL) population in the subject; and (c) isolating one or more expanded TILs from the subject.
  • TIL tumor infiltrating lymphocyte
  • the cancer is a solid or a liquid cancer.
  • the solid or liquid cancer can, for example, be selected from the group consisting of a lung cancer, a gastric cancer, a colon cancer, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, a metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, a glioma, a glioblastoma, a non-Hodgkin’s lymphoma (NHL), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a chronic myelogenous leukemia (CML), a multiple myeloma (MM), and an acute myeloid leukemia (AML).
  • NHL lymphoma
  • ALL acute lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • MM multiple my
  • the subject is further administering a co-stimulatory molecule.
  • the co-stimulatory molecule can, for example, be selected from a protein, an antibody, a small molecule, and/or a combination thereof.
  • the protein can, for example, be selected from an OX40 ligand (OX40L), a B7 ligand, a 4-IBB ligand (4-IBBL) or a CD40 ligand (CD40L).
  • the antibody can, for example be selected from an anti-CD28 antibody, an anti-CTLA4 antibody, or an anti-PD1 antibody.
  • expanded TIL populations produced by the methods disclosed herein.
  • kits for screening for an agent that is capable of binding an antigen comprise (a) contacting the agent with the loadable, multivalent VLP system described herein; and (b) identifying an agent that binds the one or more antigens in the VLP system.
  • the agent is selected from an antibody or a synthetic immunoreceptor.
  • the synthetic immunoreceptor can, for example be a chimeric antigen receptor (CAR) and/or a bispecific T cell engager (BiTE).
  • CAR chimeric antigen receptor
  • BiTE bispecific T cell engager
  • antibodies, CARs or BiTEs identified by the methods disclosed herein.
  • the methods comprise administering the antibody, CAR, or BiTE identified by the methods disclosed herein to the subject and inducing a polyclonal T or CAR- T response in the subject. 4 4889-2185-6222.1 PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019/6WO1 [0022] Also provided are methods of producing a polyclonal chimeric antigen receptor (CAR) T-cell or bispecific T cell engager (BiTE) population comprising a library of synthetic immunoreceptors.
  • CAR chimeric antigen receptor
  • BiTE bispecific T cell engager
  • the methods comprise contacting the loadable, multivalent VLP systems disclosed herein with a library of synthetic immunoreceptors to produce a polyclonal chimeric antigen receptor (CAR) T-cell or a bispecific T cell engager (BiTE) populations, wherein the CAR T-cell or BiTE population are capable of binding a HLA/tumor antigen.
  • CAR polyclonal chimeric antigen receptor
  • BiTE bispecific T cell engager
  • methods of inducing a polyclonal T or CAR-T response in a subject in need thereof comprise administeringto the subject any one of the above systems, vaccines, or TIL populations.
  • the present invention also relates to the use of any one of the above systems, vaccines, or TIL populations to induce a polyclonal T or CAR-T response in a subject in need thereof.
  • the subject in need thereof has a solid or a liquid cancer.
  • the solid or liquid cancer can, for example, be selected from the group consisting of a lung cancer, a gastric cancer, a colon cancer, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, a metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, a glioma, a glioblastoma, a non- Hodgkin’s lymphoma (NHL), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a chronic myelogenous leukemia (CML), a multiple myeloma (MM), and an acute myeloid leukemia (AML).
  • NHL non- Hodgkin’s lymphoma
  • ALL acute lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous le
  • the methods comprise contacting the loadable, multivalent VLP with a population of T-cells, and determining the binding of the T-cell to the one or more antigens on the loadable, multivalent VLP, wherein binding of the T-cell to the one or more antigens on the VLP results in the identification and/or stimulation of the T cell.
  • the methods comprise contacting the loadable, multivalent VLP with a population of synthetic immunoreceptors, and determining the binding of the synthetic immunoreceptor with the one or more antigens on the loadable, multivalent VLP, wherein binding of the synthetic immunoreceptor to the one or more antigens on the VLP results in the identification of the synthetic immunoreceptor.
  • the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. ⁇ 112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art.53(c) EPC and Rule 28(b) and (c) EPC.
  • the VLP platform can be utilized to elicit a personalized (patient and tumor specific) polyclonal anti-tumor response.
  • top The patient is typed for their HLA genotype, and immunopeptidomics are performed on a tumor biopsy, resulting in a pool of tumor specific antigens.
  • Patient HLAs are synthesized and conjugated to the VLP, then loaded 6 4889-2185-6222.1 PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019/6WO1 with tumor specific antigens.
  • VLPs can then be used in the following applications: (1) Cancer vaccination: VLPs that also contain agonist molecules (e.g., costimulatory molecules such as 4-1BBL or CD40L) can be used to elicit polyclonal anti-tumor T cell response. (2) Ex vivo TIL expansion: Following tumor biopsy, TILs are isolated from the tumor and expanded with the VLPs from 1. The subsequent polyclonal population of TILs can then be used for in vivo immunotherapy. (3) Screening of patient HLA/tumor antigen specific synthetic immunoreceptors: VLPs are used to screen a library of synthetic immunoreceptors (e.g., scFvs) for the ability to bind HLA/tumor antigen.
  • synthetic immunoreceptors e.g., scFvs
  • FIG. 2A SEC traces of the Spytagged G120C/H31C “OPEN-A02” molecules refolded with NY-ESO-1 CV9 (blue) and NY-ESO-1 W5A (green). The red triangle arrowhead indicates the complex peaks.
  • FIG. 2B SEC traces of the Spytagged G120C/H31C “OPEN-A02” molecules refolded with NY-ESO-1 CV9 (blue) and NY-ESO-1 W5A (green). The red triangle arrowhead indicates the complex peaks.
  • FIG. 2B SEC traces of the Spytagged G120C/H31C “OPEN-A02” molecules refolded with NY-ESO-1 CV9 (blue) and NY-ESO-1 W5A (green). The red triangle arrowhead indicates the complex peaks.
  • FIG. 2B SEC traces of the Spytagged G120C/H31C “OPEN-A02” molecules refolded with NY-ESO-1 CV9
  • FIG.2D SEC traces of the SpyCatcher- mi3 particles. [0035]
  • Figures 3A-3D Open HLA-A*02:01 conjugated SpyCather-mi3 nanoparticles are an effective T cell detection system.
  • FIG. 3A Gating strategy for analysis in FIG. 3B.
  • FIG.3C Staining of 1G4-transduced or untransduced primary CD8+ T cells with PE- tetramers of WT HLA-A*02:01/NY-ESO-1 CV9 , blue and red, respectively.
  • FIG. 3D Comparison of the frequency of 1G4 positive T cell between OPEN-A02-mi3 nanoparticles and WT HLA-A*02:01 PE-tetramers.
  • FIG. 5A-5C Gating strategy for analysis shown in Figures 5A-5C.
  • CD8+ T cells were stimulated for 3 days before flow cytometry analysis. Analyzed cells in Figures 5A-5C were gated on Singlet+ LiveDead FarRed Low CD3+CD8+HLA-A2/C9V+ TCRVb13.1+ cells.
  • Figures 5A-5C pMHC loaded VLPs can stimulate T cell activation and proliferation in a cognate antigen dependent manner.
  • 1G4 Transduced or untransduced T cells were stimulated with different concentrations of HLA-A*02:01/C9V loaded VLPs or anti- 7 4889-2185-6222.1 PATENT Docket Nos.
  • FIG. 5A Cell proliferation was determined by dilution of CellTraceViolet dye. Frequency of proliferating 1G4+ cells (top) or untransduced cells (bottom) is shown.
  • FIG. 5B Frequency of 4-1BB+ cells.
  • FIG. 5C Frequency of CD69+ cells. Significance was determined by One-way ANOVA followed by multiple comparisons testing with PBS treatment as control. Average of 3 technical replicates is shown. [0038] Figure 6.
  • FIG.7B DLS scans of VLP- SpyCatcher003-mi3 only, VLP-Open HLA-A*02:01/NY-ESO-1 C9V , and VLP-Open HLA- A*02:01/NY-ESO-1 W5A .
  • Figures 8A-8B DLS scans of VLP- SpyCatcher003-mi3 only, VLP-Open HLA-A*02:01/NY-ESO-1 C9V , and VLP-Open HLA- A*02:01/NY-ESO-1 W5A .
  • FIG. 8A Representative flow cytometry data of 4-1BB+ CD8+ cell populations across different conditions.
  • FIG. 8B Frequency of 4-1BB+ cells in different conditions.
  • FIG. 10 Loading and Binding of TAMRA Tax9 by VLP-Open HLA- A*02:01/gTax. Peptide exchange was measured by fluorescence polarization (mP) of 40nM TAMRATax9 as a function of the VLP-open HLA-A*02:01/TAX8 concentrations. Individual traces were fit to an exponential association model. The means from each condition’s replicates were plotted. [0042] Figure 10. SDS-PAGE from the conjugation reaction between VLP- SpyCatcher003-mi3 with SpyTagged open HLA-A*02:01/gTax. 8 4889-2185-6222.1 PATENT Docket Nos.
  • FIG.11A Representative flow cytometry data of 4-1BB+ CD8+ cell populations across different conditions.
  • FIG.11B Representative flow cytometry data of 4-1BB+ CD69+ cell populations across different conditions.
  • FIG.11C Representative flow cytometry data of 4-1BB+ CD69+ cell populations across different conditions.
  • FIG. 12A-12G The design, production, and characterization of VLP-Open HLA system.
  • FIG.12A Schematic of the design: a SpyTagged Open HLA molecule is conjugated to VLP-SpyCatcher to form VLP-Open HLA molecule.
  • FIG. 12B Computational modeling of the design of Open HLA-A*02:01 with an engineered disulfide bond as previously described by Sun et al.2023.
  • FIG.12C SEC traces of the SpyTagged G120C/H31C “Open A*02:01” molecules refolded with NY-ESO-1 CV9 (pink), NY-ESO-1 W5A (black), NY-ESO-1 gTAX (green). Representative SDS-PAGE analysis of SpyTagged Open A*02:01.
  • FIG. 12D Optimized conjugation reaction between SpyCatcher-mi3 VLP and Open A*02:01 molecules refolded with different peptides as validated with SDS PAGE gel. The gel showed pre- and post- conjugation products. In SDS-PAGE, the 60-mer complex broke apart and was observed as a monomer band at ⁇ 84 kDa.
  • FIG.13A Principle of peptide exchange on VLP-Open MHC system.
  • FIG.13B Loading and Binding of TAMRA Tax9 by VLP-Open A*02:01/gTax. Peptide exchange was measured by fluorescence polarization (mP) of 40nM TAMRATax9 as a function of the VLP- open HLA-A*02:01/TAX8 concentrations. Individual traces were fit to an exponential association model. The means from each condition’s replicates were plotted.
  • FIG. 13C Representative LC-MS Mass Spectrometry analysis of peptide bound to VLP-Open A*02:01; 9 4889-2185-6222.1 PATENT Docket Nos.
  • FIGs. 14A-14C (FIG. 14A) Labeling Scheme for VLP Open-HLA. (FIG. 14B) SDS-PAGE analysis of SpyCatcher-mi3 VLP pre- and post-labeling with fluorescein; without gel staining and under UV-light, the post-labeling sample showed a green band at the expected molecular weight of SpyCatcher-mi3, indicative of a fluorescently labeled VLP complex.
  • FIG.14C 1G4 CD8+ T cell staining with tetramers (controls) and with VLP-Open-HLA refolded with NY-ESO-1 C9V or NY-ESO-1 W5A .
  • FIGs.15A-15E. (FIG.15A) T-cell activation with Open HLA-VLP nanoparticles.
  • FIG.15B Exploring the Optimal Working Concentration Range of VLP-Open HLA-A*02:01/NY-ESO-1 C9V for Specific T cell Activation in a Cognate Antigen Dependent Manner.1G4 CD8+ T cells were stimulated with different concentrations of VLP-Open HLA- A*02:01/NY-ESO-1 C9V , VLP-Open HLA-A*02:01/NY-ESO-1 W5A , media only (buffer negative control), or anti-CD3/anti-CD28 beads (positive control) for 3 days before flow cytometry analysis.
  • FIG.15B Exploring the Optimal Working Concentration Range of VLP-Open HLA-A*02:01/NY-ESO-1 C9V for Specific T cell Activation in a Cognate Antigen Dependent Manner.1G4 CD8+ T cells were stimulated with different concentrations of VLP-Open HLA- A*02:01/NY-ESO-1 C9V , VLP-Open H
  • FIG. 15B Median fluorescence intensity (MFI) of Cell Trace Violet of CD8+ Live Cell population (lower intensity signals more population of proliferated cells).
  • FIG. 15C Frequency of 4-1BB+ cells in different conditions. Average of 2 technical replicates is shown. Representative flow cytometry data of 4-1BB+ CD8+ cell populations across different conditions.
  • FIG. 15D & (FIG. 15E) VLP-Open HLA-A*02:01/gTax exchanged with NY-ESO-1 C9V can trigger T cell activation in a cognate antigen dependent manner.
  • 1G4 CD8+ T cells were stimulated with VLP-Open HLA-A*02:01/gTax exchanged with different concentrations of NY-ESO-1 C9V or NY-ESO-1 W5A , VLP-HLA-A*02:01/gTax only (no exchange), VLP only, peptide only (NY-ESO-1 C9V or NY-ESO-1 W5A ), media only (buffer negative control), or anti-CD3/anti-CD28 beads (positive control) for 3 days before flow cytometry analysis.
  • FIG. 15D Frequency of 4-1BB+ cells in different conditions. Representative flow cytometry data of 4-1BB+ CD8+ cell populations across different conditions.
  • FIG.15E Frequency of CD69+ cells in different conditions. Representative flow cytometry data of CD69+ CD8+ cell populations across different conditions. Average of at least two technical replicates is shown. [0048] Table 1. Protein sequence of constructs used in this application. Protein sequences are given as single-letter codes. 10 4889-2185-6222.1 PATENT Docket Nos.
  • VLP loadable, multivalent virus-like particle
  • the technology builds on the existing SpyCatcher/SpyTag system 6 and previously described open MHC-I technology 7 . It enables a range of applications for the development of personalized T cell vaccines, autologous T cell-based therapies or synthetic immunoreceptors (Chimeric Antigen Receptors, CARs or Bispecific T cell engagers, BiTEs 8 ) aiming to stimulate and elicit a polyclonal T cell or a CAR-T cell that is specific to the HLA genotype and neoantigen composition of a patient’s tumor (Fig. 1).
  • VLP platform can be used in the following immunotherapy applications: [0050] As a cancer vaccination strategy: Patient tumor derived antigens are loaded on patient HLAs and conjugated to VLPs containing costimulatory molecules. After transfer to 11 4889-2185-6222.1 PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019/6WO1 patients, these particles recognize and activate tumor specific T cells, thereby boosting anti- tumor immunity (Fig.1-1).
  • TILs tumor infiltrating lymphocytes
  • the VLPs are also used to expand tumor-specific T cells that have been isolated directly from a tumor biopsy, resulting in expansion of a polyclonal population of tumor-antigen specific T cells (Figs.1 and 2A-2D).
  • Screening of patient tumor specific synthetic immunoreceptors VLPs are used to screen a library of synthetic immunoreceptors (for example but not limited to scFv antibodies) that are specific for patient tumor antigens. Identified binders can then be cloned into existing downstream therapeutic immunoreceptors such as CAR constructs or BiTEs (Figs. 1, 2A-2D, and 3A-3B).
  • a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).
  • the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise. [0060] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.
  • the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended.
  • a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus.
  • “or” refers to an inclusive or and not to an exclusive or.
  • a condition A or B is satisfied by any one of the following: A 13 4889-2185-6222.1 PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019/6WO1 is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
  • the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first.
  • a third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.” [0063] As used herein, the term “consists of,” or variations such as “consist of” or “consisting of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, but that no additional integer or group of integers can be added to the specified method, structure, or composition.
  • “subject” means any animal, preferably a mammal, most preferably a human.
  • isolated means a biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins.
  • nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. “Isolated” nucleic acids, peptides and proteins can be part of a composition and still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, or protein. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. [0069] As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule comprised of amino acids and can be recognized as a protein by those of skill in the art.
  • Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes 15 4889-2185-6222.1 PATENT Docket Nos.
  • “Modified” bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.
  • 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.
  • 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.
  • a vector is capable of replication when associated with the proper control elements.
  • 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 16 4889-2185-6222.1 PATENT Docket Nos.
  • CHOP 2024-012PCT and 074313.11019/6WO1 acid molecules that are single-chain, single-stranded, double-stranded, or partially double- stranded; 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 known 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 Another type of vector is a viral vector, wherein virally- derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)).
  • viruses e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)
  • 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).
  • vectors e.g., non-episomal mammalian vectors
  • Other 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.
  • certain 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.
  • 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 according to the invention comprises solid or liquid cancers.
  • Solid or liquid cancers can, for example, comprise 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 17 4889-2185-6222.1 PATENT Docket Nos.
  • CHOP 2024-012PCT and 074313.11019/6WO1 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.
  • the term 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–Schönlein purpura (HSP) sarcoidosis, systemic lupus erythematosus (SLE), Sjögren 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–Schönlein 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
  • human disease which conceptually are similar to infections, but invasion of a human 18 4889-2185-6222.1 PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019/6WO1 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.
  • Virus-like particle refers to a non-replicating, viral shell, derived from any of several viruses. VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins.
  • VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for producing particular VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. See, for example, Baker et al. (1991) Biophys. J. 60:1445-1456; and Hagensee et al. (1994) J. Virol. 68:4503- 4505. For example, VLPs can be isolated by density gradient centrifugation and/or identified by characteristic density banding.
  • a nanoparticle or ultrafine particle is usually defined as a particle of matter that is between 1 and 500 nanometres (nm) in diameter.
  • a self-assembling nanoparticle refers to a ball-shape protein shell with a diameter of tens of nanometers and well-defined surface gemoetry that is formed by identical copies of a non-viral protein capable of automatically assembling into a nanoparticle with a similar appearance to VLPs.
  • Known examples include ferritin (FR), which is conserved across species and forms a 24-mer, as well as B.
  • stearothermophilus dihydrolipoyl acyltransferase E2p
  • Aquifex aeolicus lumazine synthase LS
  • Thermotoga maritima encapsulin which all form 60-mers.
  • Self-assembling nanoparticles can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for nanoparticle production, detection, and characterization can be conducted using the same techniques developed for VLPs.
  • the present invention relates to a loadable, multivalent virus-like particle (VLP) system that presents a multitude of MHC-I molecules with different allotype and antigen 19 4889-2185-6222.1 PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019/6WO1 specificities.
  • VLP virus-like particle
  • the system can comprise (a) one or more recombinant patient human leukocyte antigens (HLA), (b) one or more antigens capable of binding to the one or more recombinant patient HLAs, (c) a VLP, wherein the one or more recombinant patient HLAs are conjugated to the VLP, and wherein the one or more recombinant patient HLAs conjugated to the VLP are loaded with the one or more antigens.
  • the one or more patient human leukocyte antigens are isolated from a subject.
  • the one or more antigens are selected from an infectious antigen or a tumor associated antigen (TAA).
  • the infectious antigen can, for example be selected from a bacterial antigen, a viral antigen, a parasitic antigen, or a fungal antigen.
  • the TAA can, for example, be an antigen from a solid or a liquid cancer.
  • the solid or liquid cancer can, for example, be selected from the group consisting of a lung cancer, a gastric cancer, a colon cancer, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, a metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, a glioma, a glioblastoma, a non-Hodgkin’s lymphoma (NHL), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a chronic myelogenous leukemia (CML), a multiple myeloma (MM), and an acute myeloid leukemia (AML).
  • NHL lymphoma
  • ALL acute lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • MM multiple my
  • the system further comprises a co-stimulatory molecule.
  • the co-stimulatory molecule can, for example, be conjugated to the VLP system.
  • the co- stimulatory molecule can, for example, be selected from a protein, an antibody, a small molecule, and/or a combination thereof.
  • the protein can, for example, be selected from an OX40 ligand (OX40L), a B7 ligand (composed of a CD80 and CD86), a 4-IBB ligand (4- IBBL), or a CD40 ligand (CD40L).
  • the antibody can, for example be selected from an anti- CD28 antibody, an anti-CTLA4 antibody, or an anti-PD1 antibody.
  • the molar ratio of the VLP to the HLA is about 1 to 180.
  • cancer vaccines comprising the systems described herein.
  • the cancer vaccine can, for example comprise the system described herein and an agonist molecule capable of eliciting a polyclonal anti-tumor T cell response.
  • the agonist molecule can, for example, be a co-stimulatory molecule.
  • the co- stimulatory molecule can, for example, be selected from a protein, an antibody, a small molecule, and/or a combination thereof.
  • the protein can, for example, be selected from an OX40L, a B7 ligand, a 4-IBB ligand (4-IBBL), or a CD40 ligand (CD40L).
  • the antibody can, 20 4889-2185-6222.1 PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019/6WO1 for example be selected from an anti-CD28 antibody, an anti-CTLA4 antibody, or an anti-PD1 antibody.
  • TIL tumor infiltrating lymphocyte
  • the methods comprise (a) administering to the subject the loadable, multivalent VLP system described herein; (b) expanding a tumor infiltrating lymphocyte (TIL) population in the subject; and (c) isolating one or more expanded TILs from the subject.
  • TIL tumor infiltrating lymphocyte
  • the cancer is a solid or a liquid cancer.
  • the solid or liquid cancer can, for example, be selected from the group consisting of a lung cancer, a gastric cancer, a colon cancer, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, a metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, a glioma, a glioblastoma, a non-Hodgkin’s lymphoma (NHL), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a chronic myelogenous leukemia (CML), a multiple myeloma (MM), and an acute myeloid leukemia (AML).
  • NHL lymphoma
  • ALL acute lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • MM multiple my
  • the subject is further administered a co-stimulatory molecule.
  • the co-stimulatory molecule can, for example, be selected from a protein, an antibody, a small molecule, and/or a combination thereof.
  • the protein can, for example, be selected from an OX40L, a B7 ligand, a 4-IBB ligand (4-IBBL), or a CD40 ligand (CD40L).
  • the antibody can, for example be selected from an anti-CD28 antibody, an anti-CTLA4 antibody, or an anti-PD1 antibody.
  • expanded TIL populations produced by the methods disclosed herein.
  • Methods of Screening and Methods of Use are also provided.
  • methods of screening for an agent that is capable of binding an antigen in the VLP system comprise (a) contacting the agent with the loadable, multivalent VLP system described herein; and (b) identifying an agent that binds the one or more antigens in the VLP system.
  • the agent can, for example, be selected from an antibody or a synthetic immunoreceptor.
  • the synthetic immunoreceptor can, for example, be selected from a chimeric antigen receptor (CAR) and/or a bispecific T cell engager (BiTE).
  • CAR chimeric antigen receptor
  • BiTE bispecific T cell engager
  • the methods can comprise contacting the VLP system with a population of antibodies, CARs, and/or BiTEs and determining binding of the antibody, CAR, and/or BiTE with an antigen on the VLP system. 21 4889-2185-6222.1 PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019/6WO1 [0098] Also provided are antibodies, CARs, and/or BiTEs identified by the methods disclosed herein. [0099] Also provided are methods of using the loadable, multivalent VLP described herein to identify and/or stimulate T-cells.
  • the methods comprise contacting the loadable, multivalent VLP with a population of T-cells, and determining the binding of the T-cell to the one or more antigens on the loadable, multivalent VLP. Binding of the T-cell to the one or more antigens on the VLP results in the identification and/or stimulation of the T cell.
  • the synthetic immunoreceptor can, for example, be a chimeric antigen receptor (CAR) and/or a bispecific T cell engager (BiTE).
  • CAR chimeric antigen receptor
  • BiTE bispecific T cell engager
  • Pharmaceutical Compositions and Methods of Administration [00102] Antibodies, CARs, or BiTEs identified by the methods disclosed above can be used in methods of producing a polyclonal chimeric antigen receptor (CAR) T-cell or bispecific T cell engager (BiTE) population.
  • the CAR T-cell or BiTE populations are capable of binding a HLA/tumor antigen.
  • the methods comprise contacting the loadable, multivalent VLP systems disclosed herein with a library of synthetic immunoreceptors to produce a polyclonal chimeric antigen receptor (CAR) T-cell or a bispecific T cell engager (BiTE) population.
  • the CAR T- cell or BiTE populations are capable of bidning a HLA/tumor antigen.
  • the methods comprise administeringto the subject any one of the above systems, vaccines, TIL populations, antibodies, CARs, or BiTEs to the subject.
  • the present invention also relates to the use of any one of the above systems, vaccines, TIL populations, antibodies, CARs, or BiTEs to induce a polyclonal T or CAR-T response in a subject in need thereof.
  • the therapeutically active agents, vaccines and compositions described herein may be administered via any conventional route, including by injection or infusion. The 22 4889-2185-6222.1 PATENT Docket Nos.
  • CHOP 2024-012PCT and 074313.11019/6WO1 administration may be carried out, for example, orally, intravenously, intraperitoneally, intramuscularly, subcutaneously or transdermally. In one embodiment, 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. [00107] The agents described herein are administered in effective amounts.
  • 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.
  • the pharmaceutical 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.
  • the pharmaceutical 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.
  • salts which are not pharmaceutically compatible may be used for preparing pharmaceutically compatible salts and are included in the invention.
  • CHOP 2024-012PCT and 074313.11019/6WO1 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.
  • the term “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.
  • the pharmaceutical 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.
  • the pharmaceutical compositions may, where appropriate, also contain suitable preservatives such as benzalkonium chloride, chlorobutanol, paraben and thimerosal.
  • compositions suitable for parenteral administration usually comprise a sterile aqueous or nonaqueous preparation of the active compound, which is preferably isotonic to the blood of the recipient.
  • suitable carriers and solvents are Ringer solution and isotonic sodium chloride solution.
  • sterile, fixed oils are used as solution or suspension medium.
  • the NY-ESO-1 peptide SLLMWITQV (SEQ ID NO:4)
  • SLLMWITQV NY-ESO-1 peptide
  • the previously developed open MHC-I platform 7 was further adapted, as these molecules allow for efficient peptide exchange without the presence of any chaperoning proteins and are stable due to an engineered, interchain covalent linkage of the heavy chain to ⁇ 2 m, which enables a loadable system.
  • 4-1BBL As a representative co-stimulatory molecule, recombinant 4-1BBL, the ligand for 4-1BB, which is a member of the tumor necrosis factor receptor superfamily (TNFRSF, which also includes OX40, CD40, GITR, and CD27 11 ), was used.4-1BB is upregulated after T-cell receptor recognition of pMHC antigens expressed on antigen presenting, or tumor cells 12 . Upon binding to 4-1BB ligand (4-1BBL or TNFSF9), 4-1BB signaling results in increased expression of pro-survival molecules via NF- ⁇ B signaling 13 .
  • TNFRSF tumor necrosis factor receptor superfamily
  • Conjugated mi3 particles were separated from unconjugated HLA molecules by washing the protein mixture three times with PBS (20 mM sodium phosphate pH 7.5, 150 mM NaCl) using a 100-kDa cut-off membrane. Concentrations of conjugated mi3 particles were determined using a Bio-Rad Protein Assay. [00124] Conjugation of fluorophore to SpyCatcher-mi3. Spycatcher-mi3 particles at a concentration of 2 mg/mL were buffer exchanged using Micro Bio-Spin 6 Columns into Reaction Buffer (500 mM carbonate buffer, pH 9.5).
  • FITC NHS-fluorescein
  • 1 mg of NHS-fluorescein (FITC) was dissolved in 1 mL anhydrous DMSO and added at a ratio of 80 ⁇ g FITC per 1 mg of mi3 particles and incubated for 1 hour at room temperature at 100 rpm.
  • FITC-conjugated mi3 particles buffer exchanged and unreacted FITC was removed by gel filtration using a PD-10 column equilibrated with Storage Buffer (10 mM Tris, 150 mM NaCl, 0.1% NaN3, pH 8) and the concentration of FITC-conjugated mi3 particles were determined using a Bio-Rad Protein Assay. Conjugation of open A02 was performed as described above. [00125] In vitro stimulation and staining of human T cells.
  • VSV-G pseudotyped lentivirus was generated by transfecting Lenti-X 293T cells (Takara) with a pSFFV-1G4 TCR expression vector, along with the viral packaging and envelope plasmids pcPAX2 and pMD2.G using Lipofectamine 3000 (Thermo). Following a 48-hour incubation, the viral supernatant containing the lentivirus was collected and concentrated. T cells were transduced with lentivirus 24 hours after in vitro stimulation with 1:1 ratio of Dynabeads Human T-Activator CD3/CD28 beads and cultured for an additional 48 hours. On the third day following T cell stimulation, Dynabeads were removed, and cells were expanded for an additional 5-7 days.
  • VLP- SpyCatcher003-mi3 was incubated with different molar ratio of SpyTagged open HLA- A*02:01/NY-ESO-1 C9V or open HLA-A*02:01/NY-ESO-1 W5A (Table 2) in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN 3 at room temperature for 16-18 hours.
  • Table 2 Equivalent (VLP:HLA molar ration) and concentration for the conjugation reactions. Equivalent (VLP:HLA Molar Ratio) & Concentration [00129] Scale-up Reaction and Characterization of VLP-Open HLA-A*02:01 Conjugates.
  • VLP-SpyCatcher003-mi3 Prior to the conjugation, the proteins were centrifuged at 15,000 rpm for 10 minutes to remove any potential aggregates. VLP-SpyCatcher003-mi3 was quantified using PierceTM BCA Protein Assay (Thermo Scientific, 23225). For a 500 ⁇ L- reaction, 50 nM of purified VLP-SpyCatcher003-mi3 was incubated with 9 ⁇ M SpyTagged 28 4889-2185-6222.1 PATENT Docket Nos.
  • the cells were cultivated in Advanced RPMI (Gibco), supplemented with 10% heat-inactivated FBS (Gibco), 1X Glutamax (Gibco), 1X Penicillin-Streptomycin (Gibco), 10 mM HEPES (Gibco), and 10 ng/mL IL-2 recombinant IL-2.
  • T cells were cultured at 1 x 10 6 cells/mL at 37°C and 5% CO2 overnight.
  • the cells were plated at 100,000 cells/well in a sterile U-bottomed 96-well plate; the cells were stained with CellTrace Violet proliferation dye (Thermo) prior to stimulation according to the manufacturer’s protocol.
  • the cells were stimulated with different concentrations of the VLP particles (VLP-Open HLA-A*02:01/NY-ESO-1 C9V or VLP-Open HLA-A*02:01/NY-ESO-1 W5A ) or at a 1:1 ratio of DynabeadsTM Human T-Activator CD3/CD28 for T Cell Expansion and Activation (Gibco) for 72 hours (3 days).
  • a negative control with media only was also included.
  • the media was also supplemented with 4 ⁇ g/mL CD28. Each condition was done in duplicate. On Day 3, the cells were harvested by centrifuging the plate at 300g for 5 minutes.
  • VLP- SpyCatcher003-mi3 50 nM of purified VLP- SpyCatcher003-mi3 was incubated with 9 ⁇ M SpyTagged open HLA-A*02:01/gTax in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN3 at room temperature for 16-18 hours.
  • Conjugated VLP-Open HLA complex was separated from unconjugated HLA and monomers by washing the reactions three times with 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN3 using a 100-kDa spin column.
  • the concentration of VLP-Open HLA complex was measured by PierceTM BCA Protein Assay.
  • VLP-SpyCatcher003-mi3 For a 500 ⁇ L-reaction, 50 nM of purified VLP-SpyCatcher003-mi3 was incubated with 9 ⁇ M SpyTagged Open HLA-A*02:01/NY-ESO-1 C9V , Open HLA- A*02:01/NY-ESO-1 W5A , or Open HLA-A*02:01/NY-ESO-1 gTAX in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN 3 at room temperature for 16-18 hours.
  • the VLP-SpyCatcher particle:SpyTag molar ratio explored was 1:180 as optimized. Coupling efficiency was visualized with SDS-PAGE with Coomassie staining.
  • VLP concentration was determined via PierceTM BCA Protein Assay.
  • VLP-Open HLA- A*02:01/gTax of different concentrations were incubated with 40 nM TAMRA Tax9 in FP buffer (150 mM NaCl, 20 mM sodium phosphate, 0.05% Tween-20, pH 7.4).
  • concentration of the TAMRA-labeled peptide was previously optimized so that the polarization baseline was between 0 and 50 mP.
  • the kinetic association of fluorescently labeled peptides loaded on MHC-I was monitored via FP for 4-6 hours.
  • the fluorescence of the TAMRA- labeled peptides were monitored at the excitation and emission wavelengths of 531 and 595 nm on a SpectraMax iD5 plate reader. All experiments were performed in triplicates at room temperature.
  • Raw parallel (III) and perpendicular emission intensities (I ⁇ ) were measured then converted to polarization (mP) values using the equation 1000*[(III-(G*I ⁇ ))/(III+(G*I ⁇ ))], in which the G-factor was 0.33 for TAMRA-labeled peptides.
  • the data was fitted in GraphPad Prism.
  • the cells were stimulated with different concentrations of the VLP particles (VLP-Open HLA-A*02:01/NY-ESO-1 C9V or VLP-Open HLA-A*02:01/NY-ESO-1 W5A ) or at a 1:1 ratio of DynabeadsTM Human T- Activator CD3/CD28 for T Cell Expansion and Activation (Gibco) for 72 hours (3 days).
  • a negative control with media only was also included.
  • the media was also supplemented with 4 ⁇ g/mL CD28. Each condition was done in duplicates. On Day 3, the cells were harvested by centrifuging the plate at 300g for 5 minutes. The cell supernatant was saved for later analysis with ELISA. For flow cytometry analysis, the cells were stained with 500 ⁇ L LIVE/DEAD IR 876 Fixable Dead Cell Stains (Invitrogen) for 10 minutes at room temperature being centrifuged at 300g for 5 minutes and washed with 500 ⁇ L FACS buffer.
  • the cells were centrifuged again at 300g for 5 minutes then stained with 100 ⁇ L of an antibody cocktail (Brilliant Ultra VioletTM 661 or 650 Dye* for CD69; FITC mouse anti-human for CD8, PE/Cyanine7 anti-human for CD137 or 4- 1BB) in 4oC fridge for 30 mins.
  • the cells were washed and centrifuged again, then fixed with 4% PFA for 30 minutes at room temperature. After being washed twice with FACS buffer, the cells were ready to be analyzed with a flow cytometry. For this experiment, CytoFLEX flow cytometer was used to analyze the cells.
  • CytoFLEX flow cytometer was used to analyze the cells.
  • FSC H were identified, then live cells from FSC-A vs. Live-Dead IR 876-A were identified. From this subpopulation of cells, CD8+ was identified by gating FSC-A vs. CD8+. The CD8+ cell subpopulation was gated against 4- 1BB to identify the population expressing 4-1BB, a marker of T cell activation. [00144] Results [00145] The system design was sought to be experimentally validated by expressing the Spytagged G120C variant of HLA-A*02:01 in E.
  • the co-stimulatory molecule, 4-1BBL was also produced and purified for conjugation to the mi3-VLPs (Fig.2B).
  • OPEN A02 molecules refolded with either C9V and W5A were incubated with mi3-VLPs in a 1:8 molar ratio and excess OPEN A02 was removed.
  • HLA-4-1BBL-conjugated (heterotypyic) nanoparticles To produce HLA-4-1BBL-conjugated (heterotypyic) nanoparticles, an 8-fold molar excess mixture of OPEN A02 molecules refolded (either C9V or W5A) were incubated with and conjugated to 4-1BBL in a 1:3 molar ratio (HLA:4-1BBL), as previous data suggest that 4-1BBL is functional as a trimer 22 . SDS/PAGE analysis confirmed the formation of both homotypic and heterotypic nanoparticles (Fig.2B). [00146] The use of OPEN A02-conjugated mi3-VLPs as modular, ready-to-load reagent in T cell detection strategies, was evaluated next.
  • FITC-labeled mi-VLPs were incubated with OPEN-A02 molecules refolded with either CV9 or W5A peptide. Applicants then stained primary CD8+ T cells transduced with the TCR 1G4 that recognizes the NY-ESO-1 peptide restricted by HLA-A*02:01. Compared to untransduced 1G4 T cells, OPEN-A02/CV9 conjugated mi3-VLPS exhibited substantial staining levels (Fig. 3B). OPEN-A02/W5A conjugated mi3-VLPS molecules were used as negative controls. Analysis by flow cytometry showed minimal levels of background staining using the OPEN-A02-mi3 particles (Fig.
  • Applicants identified singlets from FSC-A vs. FSC H, then identified live cells from FSC-A vs. Live-Dead IR 876-A. From this subpopulation of cells, CD8+ cells were identified by gating FSC-A vs. CD8+. The CD8+ cell subpopulation was gated against 4-1BB to identify the population expressing 4-1BB, a marker of T cell activation (Figure 8A-8B).
  • VLP-HLA- A*02:01/gTax exchanged with NY-ESO-1 W5A peptide VLP-HLA- A*02:01/gTax only (no exchange), VLP only, peptide only (NY-ESO-1 C9V or NY-ESO-1 W5A ), the frequencies of CD69+ ( Figures 11A and 11C) and 4-1BB ( Figures 11B and 11D) were approximately those of the negative control (media/buffer control).
  • VLP-Open HLA Conjugates To generate VLP- Open HLA nanoparticles, Spy-tagged open MHC on VLP-SpyCatcher003-mi3 was displayed.
  • the heavy chain ectodomain of the open HLA-A*02:01 was 36 4889-2185-6222.1 PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019/6WO1 genetically encoded to have its C-terminal domain fused to SpyTag003 (RGVPHIVMVDAYKRYK (SEQ ID NO:5)) via a Gly-rich linker (GGSGGGSGG) (SEQ ID NO:8).
  • the engineered open HLA-A*02:01 features an engineered interchain disulfide bond between its heavy chain (G120C) and the light chain ⁇ 2 m (H31C) (FIGs.12A-12B).
  • the engineered open HLA-A*02:01 has enhanced stability and peptide-receptive conformation, allowing rapid ligand exchange and enabling ready-to-load MHC I system suitable for probing T cell activity and low-affinity ligand screening.
  • SpyTag HLA-A*02:01 was successfully refolded with high- affinity antigenic peptide NY-ESO-1 C9V (SLLMWITQV) (SEQ ID NO:4), the mutated decoy peptide NY-ESO-1 W5A (SLLMAITQV), (SEQ ID NO:6) and moderate-affinity placeholder peptide gTAX (KLFGYPVYV) (SEQ ID NO:9) (FIG. 12C).
  • SLLMWITQV high- affinity antigenic peptide NY-ESO-1 C9V
  • SLLMAITQV the mutated decoy peptide NY-ESO-1 W5A
  • SEQ ID NO:6 the mutated decoy peptide NY-ESO-1 W5A
  • SEQ ID NO:6 moderate-affinity placeholder peptide gTAX
  • KLFGYPVYV moderate-affinity placeholder peptide gTAX
  • open HLA-A*02:01 had a melting temperature (Tm) of 48.8 oC, which was higher than that of the wild-type of 41.6oC. 7
  • Tm melting temperature
  • the Spytag did not compromise the thermal stability of the protein, evident by the high Tm values of the Spy- tagged open HLA-A*02:01 refolded with different peptides between 51.8oC and 58.9oC.
  • VLP protein-based nanoparticle platform virus-like particle
  • the SpyCatcher003-mi3 scaffold (with 60 binding sites on VLP) that enabled efficient VLP conjugation and enhanced stability was chosen, and the SpyCatcher003-mi3 was expressed in E.coli based on previously published works by other groups. 26,23
  • the molar ratio of SpyCatcher003-mi3 and SpyTagged open HLA-A*02:01 were optimized. A ratio of 1:180 was suggested, as final concentrations such as 50 nM SpyCatcher003-mi3 and 9 ⁇ M SpyTag Open HLA resulted in the best conjugation reaction.
  • VLP-Open HLA nanoparticles allow efficient peptide exchange and can be used to screen for antigenic peptides: It was previously established that the engineered open MHC-I design allows conformational stability that enables enhanced peptide kinetics due to acceleration between the open and closed states of the MHC molecule.
  • open HLA Open HLA-A*02:01 conjugated to VLP still retain enhanced peptide exchange kinetic, thereby showing its potential as an effective screening tool for antigenic peptides (FIG. 13A).
  • Open A*02:01 refolded with the placeholder peptide, gTAX, prior to be conjugated on the VLP scaffold SpyCatcher003-mi3.
  • FP it was observed that rapid peptide exchange occurred between gTAX and labelled peptide TAMRATAX9 (TAMRA-KLFGYPVYV) (SEQ ID NO:7) on VLP-Open HLA.
  • the loading of the TAMRATAX9 and exchange out of gTAX peptide on the VLP- Open HLA-A*02:01 complex was dependent on the concentration of the VLP- Open HLA-A*02:01, which was similar to the effect observed in previous study with the Open HLA-A*02:01/gTAX (efficient peptide exchange within 4 hours with open HLA-A*02:01 and little to no exchange with wild-type HLA-A*02:01). 7 Thus, it was demonstrated and confirmed that the VLP-Open HLA-A*02:01/gTAX design could accommodate efficient antigen peptide exchange and loading.
  • T cell activation assay with VLP-Open HLA-A*02:01/NY-ESO-1 C9V (active construct) and VLP-Open HLA-A*02:01/NY-ESO-1 W5A (inactive construct as a negative control) ws conducted.
  • CD8+ T cells 1G4 TCR a human T cells engineered expressing the variants of a single TCR (1G4) specific for the cancer antigen NY-ESO-1, were used in these experiments.
  • VLP- Open HLA-A*02:01/NY-ESO-1 C9V or VLP-Open HLA-A*02:01/NY-ESO-1 W5A were stimulated with different concentrations of the VLP particles (VLP- Open HLA-A*02:01/NY-ESO-1 C9V or VLP-Open HLA-A*02:01/NY-ESO-1 W5A ) or at a 1:1 39 4889-2185-6222.1 PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019/6WO1 ratio of DynabeadsTM Human T-Activator CD3/CD28 for T Cell Expansion and Activation (Gibco) for 72 hours (3 days). A negative control with media only (no VLP and no beads) was also included. On Day 3, the cells were harvested by centrifuging the plate at 300g for 5 minutes.
  • T Cell Activation Assays with the VLP-Open HLA-A*02:01/gTax exchanged with NY-ESO-1 [00168] Loading the VLP-Open HLA-A*02:01/gTax Complex with Antigenic Peptides: On Day 1, prior to the conjugation, the proteins were centrifuged at 15,000 rpm for 10 minutes to remove any potential aggregates.
  • VLP- SpyCatcher003-mi3 50 nM of purified VLP- SpyCatcher003-mi3 was incubated with 9 ⁇ M SpyTagged open HLA-A*02:01/gTax in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN 3 at room temperature for 16-18 hours.
  • the VLP- SpyCatcher particle:SpyTag molar ratio explored was 1:180 as previously optimized. Coupling efficiency was visualized with SDS-PAGE with Coomassie staining.
  • VLP-Open HLA complex was separated from unconjugated HLA and monomers by washing the reactions three times with 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN 3 using a 100-kDa spin column. The concentration of VLP-Open HLA complex was measured by PierceTM BCA Protein Assay. [00169] On Day 2, the peptide exchange reactions were prepared. Keeping in the mind that there are 60 potential binding sites for these peptides on the HLA molecules conjugated to the 60 binding sites of the VLP, the concentrations, a peptide concentration of more than 0.24 ⁇ M would theoretically saturate all binding sites (1:60 VLP-HLA:Peptide molar ratio).
  • Peptides of the same concentrations (without VLP) were also prepared as peptide controls for the T cell activation assays.
  • the peptide exchange reactions were done at 4oC overnight.
  • the whole reaction was diluted 12.5X to create the 2X stocks for the T cell activation assays.
  • the resulted reactions from the peptide exchange step were diluted directly with the cell medium for the T cell activation assay without a wash step.
  • VLP-Open HLA-A*02:01/gTax was exchanged with NY-ESO-1 C9V or NY-ESO-1 W5A peptide as described above; the final peptide concentrations in the T cell activation assays were 25X the concentrations (96 nM, 48 nM, 9.6 nM, and 4.8 nM).
  • a similar protocol for T cell activation as aforementioned were used for this assay.
  • VLP-Open HLA-A*02:01/gTax exchanged with NY-ESO-1 C9V T cell activation with VLP-Open HLA- A*02:01/gTax exchanged with NY-ESO-1 W5A peptide, VLP-HLA-A*02:01/gTax only (no exchange), VLP only, peptide only (NY-ESO-1 C9V or NY-ESO-1 W5A ), media only (negative control), and a positive control with DynabeadsTM Human T-Activator CD3/CD28 (Gibco) was also evaluated. [00171] For gating strategy, singlets from FSC-A vs.
  • FSC H live cells from FSC-A vs. Live-Dead IR 876-A were identified.
  • CD8+ expressing CD69 or 4-1BB were identified by gating CD69 vs. CD8 and 4-1BB vs. CD8.
  • the CD8+ cell subpopulation was gated against CD69 or 4-1BB to identify the population expressing CD69 and 4-1BB, two markers of T cell activation (FIGs. 15D and 15E).
  • FSC H live cells from FSC-A vs. Live-Dead IR 876-A were identified.
  • CD8+ expressing CD69 or 4-1BB were identified by gating CD69 vs. CD8 and 4-1BB vs. CD8.
  • the CD8+ cell subpopulation was gated against CD69 or 4-1BB to identify the population expressing CD69 and 4-1BB, two markers of T cell activation (FIGs. 15D and 15E).
  • a higher frequency of CD8+ expressing CD69 and 4-1BB
  • 4-1BB Signaling Boosts the Anti-Tumor Activity of CD28-Incorporated 2nd Generation Chimeric Antigen Receptor-Modified T Cells. Front. Immunol. 11, 539654 (2020).
  • Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice. (2021).

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Abstract

La présente invention concerne un système de particules pseudovirales (PPV) multivalentes pouvant être chargées qui présente une multitude de molécules CMH I comprenant différentes spécificités d'allotype et d'antigène, et des procédés de correspondance et d'utilisation de ceux-ci.
PCT/US2024/044431 2023-08-31 2024-08-29 Systèmes et procédés de génération de nanoparticules hla mosaïcales multivalentes pour des applications d'immunothérapie personnalisée Pending WO2025049742A1 (fr)

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CN120098143A (zh) * 2025-03-10 2025-06-06 蚌埠医科大学 一种自组装铁蛋白纳米颗粒重组蛋白sft及其制备方法和应用

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US20190085048A1 (en) * 2001-05-22 2019-03-21 Dako Denmark A/S MHC Multimers, Methods for Their Generation, Labeling and Use
US20210155670A1 (en) * 2019-09-13 2021-05-27 The Regents Of The University Of California Systems and methods for the preparation of peptide-mhc-i complexes with native glycan modifications

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190085048A1 (en) * 2001-05-22 2019-03-21 Dako Denmark A/S MHC Multimers, Methods for Their Generation, Labeling and Use
US20210155670A1 (en) * 2019-09-13 2021-05-27 The Regents Of The University Of California Systems and methods for the preparation of peptide-mhc-i complexes with native glycan modifications

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120098143A (zh) * 2025-03-10 2025-06-06 蚌埠医科大学 一种自组装铁蛋白纳米颗粒重组蛋白sft及其制备方法和应用
CN120098143B (zh) * 2025-03-10 2025-09-09 蚌埠医科大学 一种自组装铁蛋白纳米颗粒重组蛋白sft及其制备方法和应用

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