Systems and methods for generating multivalent mosaic HLA nanoparticles for personalized immunotherapy applications

Abstract

The present invention relates to a loadable, multivalent virus-like particle (VLP) system which presents a multitude of MHC-I molecules comprising different allotype and antigen specificities, and methods for mating and using the same.

Description

PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 SYSTEMS AND METHODS FOR GENERATING MULTIVALENT MOSAIC HLA NANOPARTICLES FOR PERSONALIZED IMMUNOTHERAPY APPLICATIONS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 535,888, filed on August 31, 2023, the disclosure of which is incorporated herein by reference in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] This application contains a sequence listing, which is submitted electronically. The contents of the electronic sequence listing (074313.6WO1 Sequence Listing.xml; size: 8,559 bytes; and creation date of July 11, 2024) is herein incorporated by reference in its entirety. INCORPORATION BY REFERENCE

[0003] All documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. FEDERAL FUNDING LEGEND

[0004] This invention was made with government support under Grant Nos. AI143997, DK112217 and GM125034 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION

[0005] The present invention relates to a loadable, multivalent virus-like particle (VLP) system which presents a multitude of MHC-I molecules comprising different allotype and antigen specificities. 1 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 BACKGROUND OF THE INVENTION

[0006] A multitude of CD8+T cells expressed in the human body express a polyclonal and highly specific antigen receptor, called a T cell receptor (TCR) on the cell surface. These TCRs bind to the class I major histocompatibility complex (MHC-I), comprising of a polymorphic heavy chain, an invariant light chain (beta-2-microglobulin, ȕ2m) and displaying an antigenic peptide (termed antigen). Thus, TCRs can recognize the peptide in the context of a specific MHC-I molecule, a property known as MHC-I restriction of T cell antigen recognition1. This interaction is the first of many interactions necessary to kickstart the adaptive immune system2.

[0007] 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 cancers1,3. 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 cells4. However, one drawback of the current MHC tetramer technology is that in order to study multiple 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 levels5– individual tetramer reagents must be prepared for each MHC-I heavy chain. In addition, 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. Finally, in order to study different antigenic peptides, or other ligands (in the case of the non-polymorphic MR1 and CD1d MHC-like molecules), individual tetramers must be prepared using MHC-I molecules that have been exchanged or refolded with the different ligands.

[0008] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. SUMMARY OF THE INVENTION

[0009] 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. 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 4889-2185-6222.1PATENT 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.

[0010] In certain embodiments, the one or more patient human leukocyte antigens are isolated from a subject.

[0011] In certain embodiments, 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).

[0012] In certain embodiments, 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.

[0013] In certain embodiments, the molar ratio of the VLP to the HLA is about 1 to 180.

[0014] Also provided are 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.

[0015] 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 4889-2185-6222.1PATENT 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. 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.

[0017] In certain embodiments, 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).

[0018] In certain embodiments, 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.

[0019] Also provided are expanded TIL populations produced by the methods disclosed herein.

[0020] Also provided are methods of screening for an agent that is capable of binding an antigen, the methods 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. In certain embodiments, 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). Also provided are antibodies, CARs or BiTEs identified by the methods disclosed herein.

[0021] Also provided are methods of inducing a polyclonal T or CAR-T response in a subject in need thereof. 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 4889-2185-6222.1PATENT 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. 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.

[0023] Also provided are methods of inducing a polyclonal T or CAR-T response in a subject in need thereof. The methods comprise administeringto the subject any one of the above systems, vaccines, or TIL populations.

[0024] 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.

[0025] In certain embodiments, 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).

[0026] 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, 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.

[0027] Also provided are methods of using the loadable, multivalent VLP described herein to identify synthetic immunoreceptors. 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.

[0028] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product 5 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that 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. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

[0029] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0030] These and other embodiments are disclosed or are obvious from and encompassed by the following Detailed Description. BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES

[0031] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0032] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0033] Figure 1. 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 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 with tumor specific antigens. These 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. Subsequent identified binders are then advanced for further development into existing immunotherapy modalities such as CAR-T cells (3a) and / or BiTEs (3b), resulting in polyclonal populations of CARs or BiTEs.

[0034] Figures 2A-2D. Demonstration of proper formation of HLA, co-stimulatory, and virus-like particles. FIG. 2A. SEC traces of the Spytagged G120C / H31C “OPEN-A02” molecules refolded with NY-ESO-1CV9(blue) and NY-ESO-1W5A(green). The red triangle arrowhead indicates the complex peaks. FIG. 2B. SDS / PAGE analysis of OPEN-A02, 4- 1BBL, and SpyCatcher-mi3. FIG.2C. DSF shows thermal stability curves of the OPEN-A02 / NY-ESO-1CV9in blue (Tm= 59.2°C) and OPEN-A02 / NY-ESO-1W5A(Tm= 58.0°C). The average of three technical replicates (mean) is plotted. 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. Staining of 1G4-transduced and untransduced primary CD8+ T cells with FITC-labeled OPEN- A02-mi3 nanoparticles, with NY-ESO-1CV9(blue and orange) and NY-ESO-1W5A(red and green). FIG.3C. Staining of 1G4-transduced or untransduced primary CD8+ T cells with PE- tetramers of WT HLA-A*02:01 / NY-ESO-1CV9, 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.

[0036] Figure 4. 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.

[0037] 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 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 CD3 / antiCD28 beads for 3 days before flow cytometry analysis. 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. SDS-PAGE of the micro-conjugation reactions of different molar ratios between VLP-SpyCatcher003-mi3 with SpyTagged open HLA-A*02:01 / NY-ESO-1C9Vor open HLA-A*02:01 / NY-ESO-1W5A. 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.

[0039] Figures 7A-7B. Characterization of VLP-Open HLA-A*02:01 Particles. FIG. 7A. SDS-PAGE from the scaled-up conjugation reactions between VLP-SpyCatcher003-mi3 with SpyTagged open HLA-A*02:01 / NY-ESO-1C9Vor open HLA-A*02:01 / NY-ESO-1W5A. A 100-kDa spin column removed monomers or unreacted proteins. FIG.7B. DLS scans of VLP- SpyCatcher003-mi3 only, VLP-Open HLA-A*02:01 / NY-ESO-1C9V, and VLP-Open HLA- A*02:01 / NY-ESO-1W5A.

[0040] Figures 8A-8B. Exploring the Optimal Working Concentration Range of VLP- Open HLA-A*02:01 / NY-ESO-1C9Vfor 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-1C9V, VLP-Open HLA-A*02:01 / NY-ESO-1W5A, media only (buffer negative control), or anti-CD3 / anti-CD28 beads (positive control) for 3 days before flow cytometry analysis. 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. Average of 2 technical replicates is shown.

[0041] Figure 9. Loading and Binding ofTAMRATax9 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 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1

[0043] Figures 11A-11D. VLP-Open HLA-A*02:01 / gTax exchanged with NY-ESO-1C9Vcan 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-1C9Vor NY-ESO-1W5A, VLP-HLA-A*02:01 / gTax only (no exchange), VLP only, peptide only (NY-ESO-1C9Vor NY-ESO-1W5A), media only (buffer negative control), or anti- CD3 / anti-CD28 beads (positive control) for 3 days before flow cytometry analysis. 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. Frequency of 4-1BB+ cells in different conditions. FIG. 11D. Frequency of CD69+ cells in different conditions. Average of at least two technical replicates is shown.

[0044] FIGs. 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.2023.27(FIG.12C) SEC traces of the SpyTagged G120C / H31C “Open A*02:01” molecules refolded with NY-ESO-1CV9(pink), NY-ESO-1W5A(black), NY-ESO-1gTAX(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. 12E) Dynamic Light Scattering (DLS) scans of VLP- SpyCatcher003-mi3 only, VLP-Open A*02:01 / NY-ESO-1C9V, and VLP-Open A*02:01 / NY- ESO-1W5A. (FIG. 12F) Representative mass photometry data for VLP-Open A*02:01 / NY- ESO-1C9V, showing successful conjugation to most of the 60 sites of VLP. (FIG. 12G) Cryo- EM image of VLP-Open A*02:01 / NY-ESO-1C9V.

[0045] FIGs. 13A-13C. A loadable VLP-Open MHC System that allows rapid peptide exchange. (FIG.13A) Principle of peptide exchange on VLP-Open MHC system. (FIG.13B) Loading and Binding ofTAMRATax9 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 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 the peptide is from TCGA peptide library. VLP-Open A*02:01 / gTAX can load and capture different antigenic peptides.

[0046] 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. This was then successfully conjugated to Open A*02:01 / NY-ESO-1C9Vor Open A*02:01 / NY- ESO-1W5Afor T cell staining experiment. (FIG.14C) 1G4 CD8+ T cell staining with tetramers (controls) and with VLP-Open-HLA refolded with NY-ESO-1C9Vor NY-ESO-1W5A.

[0047] FIGs.15A-15E. (FIG.15A) T-cell activation with Open HLA-VLP nanoparticles. (FIG.15B) & (FIG.15C) Exploring the Optimal Working Concentration Range of VLP-Open HLA-A*02:01 / NY-ESO-1C9Vfor 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-1C9V, VLP-Open HLA-A*02:01 / NY-ESO-1W5A, media only (buffer negative control), or anti-CD3 / anti-CD28 beads (positive control) for 3 days before flow cytometry analysis. (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-1C9Vcan 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-1C9Vor NY-ESO-1W5A, VLP-HLA-A*02:01 / gTax only (no exchange), VLP only, peptide only (NY-ESO-1C9Vor NY-ESO-1W5A), 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 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 Construct Name Protein Sequence (SEQ ID NO) Addgene Plasmid # O A02 MGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDT F N / ADETAILED DESCRIPTION OF THE INVENTION

[0049] Described herein is a loadable, multivalent virus-like particle (VLP) system which can present a multitude of MHC-I molecules comprising different allotype and antigen specificities. Moreover, the 60 binding sites present on the VLP allow for further customization, including conjugation with co-stimulatory molecules, a multitude of scFvs that block checkpoint receptors, or indeed any other class of synthetic immunoreceptor. The technology builds on the existing SpyCatcher / SpyTag system6and previously described open MHC-I technology7. 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, BiTEs8) 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). Applicants envision that the 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 4889-2185-6222.1PATENT 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).

[0051] Ex vivo expansion of tumor infiltrating lymphocytes (TILs): 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).

[0052] 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).

[0053] Notably, common to all of the above is the induction of a polyclonal T or CAR-T response, which is facilitated by use of VLPs. This results in greater coverage of tumor- or neoantigens, leading to greater therapeutic efficacy for the patient.

[0054] The present invention is described with reference to particular embodiments having various features. It will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. One skilled in the art will recognize that these features may be used singularly or in any combination based on the requirements and specifications of a given application or design. One skilled in the art will recognize that the systems and devices of embodiments of the invention can be used with any of the methods of the invention and that any methods of the invention can be performed using any of the systems and devices of the invention. Embodiments comprising various features may also consist of or consist essentially of those various features. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. The description of the invention provided is merely exemplary in nature and, thus, variations that do not depart from the essence of the invention are intended to be within the scope of the invention.

[0055] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. 12 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1

[0056] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as would be commonly understood or used by one of ordinary skill in the art encompassed by this technology and methodologies.

[0058] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0059] Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, 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.

[0061] As used herein, 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. For example, 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. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A 13 4889-2185-6222.1PATENT 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).

[0062] As used herein, 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.

[0064] As used herein, the term “consists essentially of,” or variations such as “consist essentially of” or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition. See M.P.E.P. § 2111.03.

[0065] As used herein, “subject” means any animal, preferably a mammal, most preferably a human. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably a human.

[0066] The words “right,” “left,” “lower,” and “upper” designate directions in the drawings to which reference is made.

[0067] It should also be understood that the terms “about,” “approximately,” “generally,” “substantially,” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, 14 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

[0068] As used herein, the term “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. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms “peptide,” “polypeptide,” and “protein” can be used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0070] As used herein, the term “polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “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. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes 15 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “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.

[0071] 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.

[0072] 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. In one embodiment, 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.

[0073] 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. For instance, 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.

[0074] A used herein, 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 16 4889-2185-6222.1PATENT 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. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. 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)). 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. Moreover, 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.

[0075] 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. Within a recombinant expression vector, "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). With regards to recombination and cloning methods, mention is made of U.S. patent application Ser. No.10 / 815,730, published Sep.2, 2004 as US 2004-0171156 A1, the contents of which are herein incorporated by reference in their entirety.

[0076] The term “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 4889-2185-6222.1PATENT 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. Examples thereof are lung carcinomas, mamma carcinomas, prostate carcinomas, colon carcinomas, renal cell carcinomas, cervical carcinomas, or metastases of the cancer types or tumors described above. The term cancer according to the invention also comprises cancer metastases and relapse of cancer.

[0077] The term “autoimmunity” according to the invention 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).

[0078] The term “infectious disease” according to the invention 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 diseases are caused by infectious agents (pathogens) such as, but not limited to, bacteria (e.g., Mycobacterium tuberculosis, Staphylococcus aureus, Escherichia coli, Clostridium botulinum, and Salmonella spp.), viruses and related agents such as viroids. (e.g. 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. malaria, Toxoplasma, Babesia), macroparasites (worms or helminths) including nematodes such as parasitic roundworms and pinworms, tapeworms (cestodes), and flukes (trematodes, such as schistosomes), arthropods such as ticks, mites, fleas, and lice, can also cause human disease, which conceptually are similar to infections, but invasion of a human 18 4889-2185-6222.1PATENT 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).

[0079] The term “peptide disease target” according to the invention 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.

[0080] Virus-like particle (VLP) 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. Alternatively, cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question, and images recorded under appropriate exposure conditions.

[0081] 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), and 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.

[0082] Systems, Vaccines, and Tumor Infiltrating Lymphocyte (TIL) Populations

[0083] 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 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 specificities. 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.

[0084] In certain embodiments, the one or more patient human leukocyte antigens are isolated from a subject.

[0085] In certain embodiments, 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.

[0086] 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).

[0087] In certain embodiments, 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.

[0088] In certain embodiments, the molar ratio of the VLP to the HLA is about 1 to 180.

[0089] Also provided are 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.

[0090] 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 4889-2185-6222.1PATENT 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.

[0091] Also provided are methods of producing an expanded tumor infiltrating lymphocyte (TIL) population from a subject with cancer. 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.

[0092] In certain embodiments, 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).

[0093] In certain embodiments, 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.

[0094] Also provided are expanded TIL populations produced by the methods disclosed herein.

[0095] Methods of Screening and Methods of Use

[0096] Also provided are methods of screening for an agent that is capable of binding an antigen in the VLP system. The methods 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.

[0097] 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). 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 4889-2185-6222.1PATENT 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.

[0100] Also provided are methods of using the loadable, multivalent VLP described herein to identify synthetic immunoreceptors. The methods comprise contacting the loadable, multivalent VLP with a population of synthetic immunoreceptors, and determining the binding of the synthetic immunoreceptor to the one or more antigens on the loadable, multivalent VLP. Binding of the synthetic immunoreceptor to the one or more antigens on the VLP results in the identification of the synthetic immunoreceptor. The synthetic immunoreceptor can, for example, be a chimeric antigen receptor (CAR) and / or a bispecific T cell engager (BiTE).

[0101] Pharmaceutical Compositions and Methods of Administration

[0102] 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.

[0103] Also provided are methods of producting a polyclonal chimeric antigen receptor (CAR) T-cell or bispecific T cell engager (BiTE) population comprising a library of synthetic immunoreceptors. 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.

[0104] Also provided are methods of inducing a polyclonal T or CAR-T response in a subject in need thereof. The methods comprise administeringto the subject any one of the above systems, vaccines, TIL populations, antibodies, CARs, or BiTEs to the subject.

[0105] 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.

[0106] The therapeutically active agents, vaccines and compositions described herein may be administered via any conventional route, including by injection or infusion. The 22 4889-2185-6222.1PATENT 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.

[0107] 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. In the case of treatment of a particular disease or of a particular condition, 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.

[0108] 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 with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0109] 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.

[0110] The pharmaceutical compositions of the invention are generally administered in pharmaceutically compatible amounts and in pharmaceutically compatible preparation. The term “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. When used in medicine, 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 23 4889-2185-6222.1PATENT Docket Nos. 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.

[0111] A pharmaceutical composition of the invention may comprise a pharmaceutically compatible carrier. The term “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. According to the invention, 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.

[0112] 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.

[0113] The pharmaceutical compositions may, where appropriate, also contain suitable preservatives such as benzalkonium chloride, chlorobutanol, paraben and thimerosal.

[0114] The pharmaceutical 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.

[0115] 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. Examples of compatible carriers and solvents are Ringer solution and isotonic sodium chloride solution. In addition, usually sterile, fixed oils are used as solution or suspension medium.

[0116] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims. Examples

[0117] Study Design 24 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1

[0118] The most prevalent Human Leucocyte Antigen class I allele family (HLA, the MHC in humans), HLA-A*02:01 (A02), with a global population frequency of 20-40%9was used in the experiments described herein. As a model antigen, the NY-ESO-1 peptide (SLLMWITQV) (SEQ ID NO:4), a previously described tumor-associated antigen known to bind the engineered 1G4 T cell Receptor10was used. The previously developed open MHC-I platform7was 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 ȕ2m, which enables a loadable system. 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 CD2711), was used.4-1BB is upregulated after T-cell receptor recognition of pMHC antigens expressed on antigen presenting, or tumor cells12. Upon binding to 4-1BB ligand (4-1BBL or TNFSF9), 4-1BB signaling results in increased expression of pro-survival molecules via NF-^B signaling13. Initial studies demonstrated antitumor effects of agonistic 4-1BB antibodies with pronounced tumor regression in mastocytoma and sarcoma mouse models, and required both CD4+and CD8+T cells14. Agonistic antibodies against 4-1BB have been effective in reducing or eliminating multiple tumors in murine models of melanoma, glioblastoma, lymphoma, renal cell carcinoma, and colon cancer among others15–18. As a result, signaling domains for 4-1BB are commonly included in CAR construct designs19,20, exemplifying the importance of 4-1BB in amplifying T cell responses. In principle many other co-stimulatory molecules (such as the checkpoint inhibitors anti-CTLA4 antibodies (ipilimumab) and anti-PD1 antibodies (pembrolizumab)) are compatible with the platform. Demonstration of antigen specific recognition and stimulation of T cells was done using CD8+ T cells within peripheral blood mononuclear cells (PBMCs) that were lentivirally transfected with a construct expressing the 1G4 alpha and beta chains. It is demonstrated herein that the VLP platform can indeed both recognize the 1G4 TCR but also elicit activation and proliferation from 1G4 T cells. These data stand as a proof-of-principle that the platform can indeed function in the applications that are described in Figure 1, namely 1) as a cancer vaccine, 2) as a platform for ex vivo expansion of TILs and 3) as a method for screening synthetic immunoreceptors such as CARs or BiTEs.

[0119] Materials and Methods

[0120] Production of open-A*02:01 molecules. The ectodomain of open A*02:01 (G120C) heavy chain tagged at the C-terminus with SpyTag003 (RGVPHIVMVDAYKRYK) (SEQ ID NO:5) and ȕ2m (H31C) were refolded in the presence of excess peptide, NY-ESO-1 25 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 or NY-ESO-1W5Apeptide (SLLMAITQV (SEQ ID NO:6), used as a negative control). A 9 residue, Gly-rich linker was included between the isopeptide fusion tag, SpyTag003 and the Į3 domain of the heavy chain to prohibit unwanted interactions and interference between the functions of the tag and heavy chain. Plasmids encoding the luminal domain of the C-terminally linked SpyTag003 open A*02:01 (G120C) and open human ȕ2m (H31C) were transformed into E. coli cells. Cells were grown and harvested in the Luria-Broth medium, and inclusion bodies were pelleted and purified as previously described21. Ternary complexes were generated by in refolding, a 100 mg mixture of open A02-SpyTag003 and open ȕ2m at a 1:3 molar ratio was slowly diluted over 4 hours in 500 mL of refolding buffer containing 0.4 M L-Arginine HCl, 100 mM Tris, 2 mM EDTA, 5 mM reduced L-glutathione, 0.5 mM oxidized L- glutathione, pH 8.0 supplemented with 5 mg of the NY-ESO-1 or NY-ESO-1W5A. The refolding reaction proceeded for 4 days, and proteins were dialyzed into buffer containing 25 mM Tris, 150 mM NaCl, pH 8.0 (TBS) and purified by size exclusion chromatography. Purified proteins were further confirmed in reduced and non-reduced conditions using sodium dodecyl sulfate- polyacrylamide (SDS-PAGE) gel electrophoresis.

[0121] Production of Recombinant Human Trimeric 4-1BBL. As previously described22, SpyTag003-4-1BBL was produced as inclusion bodies in E. coli. The SpyTag003- 4-1BBL protein (10 mg) was allowed to refold in 500 ml of folding buffer (0.4 M L-Arginine, 100 mM Tris, pH 8, 2 mM EDTA, pH 8, 5 mM reduced L-glutathione, 0.5mM oxidized L- glutathione, 0.005% Tween 20, and a protease inhibitor mixture slowly stirred for 72 h at 4 °C, before concentration on a 10-kDa cut-off membrane. SpyTag003-4-1BBL was purified by SEC on Superdex 200 HR 16 / 60.

[0122] Expression and purification of SpyCatcher-mi3. The virus-like particle, His6- SpyCatcher-mi3 (including an N-terminal 6x-His tag) was produced as previously described23,24. Briefly, plasmids encoding the SpyCatcher-mi3 particles were transformed into E. coli cells The starter culture was diluted 1:100 into 1 L LB (50 ^g / mL) kanamycin and 0.8% (w / v) glucose and cultured at 37 °C at 200 rpm until an OD600 ~0.6. Protein expression was induced with isopropyl ȕ-D-1-thiogalactopyranoside (IPTG) (420 ^M) and incubated at 22°C at 200 rpm for an additional 16 hours. The culture was centrifuged, and the pellet was resuspended in 25 mL buffer containing 25mM Tris–HCl, 300mM NaCl, pH 8.5 with 0.1 mg / mL lysozyme, 1 mg / mL cOmplete mini EDTA-free protease inhibitor and 2 mM phenylmethanesulfonyl fluoride (PMSF). Cell suspension was incubated at 22°C for 30 min on a platform shaker and sonicated on ice four times for 60 sec at 40% amplitude. Cell lysate was 26 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 clarified at 21,000 × g for 30 min at 4°C. The supernatant was filtered through 0.45 and 0.22 ^m syringe filters, and mi3 particles were isolated by Ni-NTA chromatography using a pre- packed HisTrapTMHP column. Eluted particles were concentrated using a 30K concentrator and purified by SEC using a HiLoad® 16 / 600 Superdex® 200 column equilibrated with TBS supplemented with 0.02% NaN3. Endotoxin was removed from the SpyCatcher003-mi3 samples using Triton X-114 phase separation as previously described25. SpyCatcher-mi3 particles were stored at 4°C and used for conjugations for up to 1 month after filtering with a 0.2 ^m filter or spinning at 21,000 xg for 10 min.

[0123] Conjugation of HLAs and 4-1BBL to SpyCatcher-mi3. Purified SpyCatcher003- mi3 was incubated with a 8-fold molar excess (open A02 to mi3 subunit) of purified SpyTagged open A02 (either one HLA only for making homotypic open A02 particles or a 1:3 mixture of open A02 to 4-1BBL for making mosaic particles or an equimolar mixture of six distinct open- HLAs for making mosaic particles) overnight at 4°C in TBS. 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.

[0124] 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). 1 mg of NHS-fluorescein (FITC) was dissolved in 1 mL anhydrous DMSO and added at a ratio of 80 μg 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.

[0125] In vitro stimulation and staining of human T cells. To measure proliferation, cells were labeled with CellTrace Violet (Thermo) prior to stimulation using manufacturer recommendations. These cells were then cultivated in Advanced RPMI (Gibco), supplemented with 10% heat-inactivated FBS (Gibco), Glutamax (Gibco), penicillin / streptomycin (Gibco), 10mM HEPES (Gibco) and 300 U / mL of recombinant IL-2 (Peprotech). T cells were cultured at 1 million cells / mL and stimulated with VLPs or a 1:1 ratio of Dynabeads Human T-Activator CD3 / CD28 beads (Thermo) for a duration of 72 hours. Following this activation period, the beads were removed, and then stained for flow cytometry analysis and acquired on a BD 27 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 LSRFortessa (BD Biosciences). For VLP staining, cells were stained with a 100 Pg / mL solution of VLPs in PBS with 2.5% heat inactivated FBS and 0.02% sodium azide for 30 minutes prior to acquisition.

[0126] Lentiviral transduction of human T cells. Blood samples were obtained from healthy and consenting donors through the Human Immunology Core at the University of Pennsylvania. Subsequently, magnetic separation was employed to enrich total CD3+ or CD8+ T cells from the collected blood. 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. In parallel untransduced cells were also stimulated but not exposed to lentivirus.

[0127] Optimization of VLP-SpyCatcher003-mi3 Conjugation Reaction with SpyTagged open HLA. To saturate the binding sites (60 binding sites on VLP) and improve the efficiency of VLP conjugation to open HLA molecules, micro-reactions of 20 ^L / reaction were done to optimize the conjugation reaction. For each reaction, 50 nM of purified VLP- SpyCatcher003-mi3 was incubated with different molar ratio of SpyTagged open HLA- A*02:01 / NY-ESO-1C9Vor open HLA-A*02:01 / NY-ESO-1W5A(Table 2) in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN3at room temperature for 16-18 hours.

[0128] Table 2: Equivalent (VLP:HLA molar ration) and concentration for the conjugation reactions. Equivalent (VLP:HLA Molar Ratio) & Concentration

[0129] Scale-up Reaction and Characterization of VLP-Open HLA-A*02:01 Conjugates. Conjugation Reaction: 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 Pierce™ 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 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 open HLA-A*02:01 / NY-ESO-1C9Vor open HLA-A*02:01 / NY-ESO-1W5Ain 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN3at room temperature for 16-18 hours.

[0130] Dynamic Light Scattering (DLS): VLP-Open HLA protein complex and buffer (for any sample preparation) were centrifuged at 15,000 rpm for 10 minutes to remove any potential aggregates right before DLS experiment. In a cuvette, 60 ^L of each sample was loaded; the total protein concentration should be between 0.1-0.4 ^g / mL. Each sample was measured at 20oC on a Nanobrook 90Plus Submicron Particle Sizer (Brookhaven Instrument) for 2-4 runs. The intensity distribution was plotted and the particle size / radius average along with its standard deviation were calculated in Excel.

[0131] Exploration of working concentration of VLP-Open HLA-A*02:01 with in vitro stimulation of human T cells. CD8+ T cells 1G4 TCR, a human T cell engineered expressing the variants of a single TCR (1G4) specific for the cancer antigen NY-ESO-1, were used in these experiments. 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.

[0132] On Day 0 of the experiment, T cells were cultured at 1 x 106cells / mL at 37°C and 5% CO2 overnight. On Day 1, 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-1C9Vor VLP-Open HLA-A*02:01 / NY-ESO-1W5A) or at a 1:1 ratio of Dynabeads™ 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. For the conditions with VLPs and the negative control, 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. The cell supernatant was saved for later analysis with ELISA. For flow cytometry analysis, the cells were stained with 500 μL 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 μL FACS buffer. The cells were centrifuged again at 300g for 5 minutes then stained with 100 μL of an antibody cocktail (Brilliant Ultra Violet™ 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 minutes. 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 29 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 were ready to be analyzed with a flow cytometry. For this experiment, a CytoFLEX flow cytometer was used for analysis of the cells.

[0133] Development of a VLP-Open HLA Loadable System. Prior to the assays, the VLPs and all components of the assays were centrifuged for 15,000 rpm; VLP concentration was determined via Pierce™ BCA Protein Assay. In a 96-well plate, for each 100 ^L reaction, VLP-Open HLA-A*02:01 / gTax of different concentrations (2.5-100 nM) were incubated with 40 nM TAMRATax9 in FP buffer (150 mM NaCl, 20 mM sodium phosphate, 0.05% Tween-20, pH 7.4). The 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.

[0134] 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. In a 1000 ^L-reaction, 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 Pierce™ BCA Protein Assay.

[0135] On Day 2, the peptide exchange reactions were prepared according to Table 3. 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, 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. At the end of the reactions, the whole reaction was diluted 12.5X to create the 2X stocks for the T cell activation assays.

[0136] Table 3: Equivalent (VLP-HLA:peptide molar ratio) and concentrations for the conjugation reactions. 30 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 Equivalent (VLP-HLA:Peptide Molar Ratio) & Concentration 1600 1300 160 130ed with NY-ESO-1. 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. Since it was previously established that a concentration below 3.13 μg / mL of the VLP-HLA particle could minimize non-specific T cell activation, a VLP concentration of 0.8 μg / mL was used for all conditions. The VLP-Open HLA-A*02:01 / gTax was exchanged with NY-ESO-1C9Vor NY-ESO-1W5Apeptide as described above; the final peptide concentrations in the T cell activation assays were 25X the concentrations in Table 3 (96 nM, 48 nM, 9.6 nM, and 4.8 nM). A similar protocol for T cell activation as aforementioned was used for this assay. In addition to evaluate the VLP- Open HLA-A*02:01 / gTax exchanged with NY-ESO-1C9V, T cell activation with VLP-Open HLA-A*02:01 / gTax exchanged with NY-ESO-1W5Apeptide, VLP-HLA-A*02:01 / gTax only (no exchange), VLP only, peptide only (NY-ESO-1C9Vor NY-ESO-1W5A), media only (negative control), and a positive control with Dynabeads™ Human T-Activator CD3 / CD28 (Gibco) was also evaluated.

[0138] Conjugation of SpyTag Open HLA to SpyCatcher003: 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 Pierce™ BCA Protein Assay (Thermo Scientific, 23225). 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-1C9V, Open HLA- A*02:01 / NY-ESO-1W5A, or Open HLA-A*02:01 / NY-ESO-1gTAXin 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN3at 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. 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 Pierce™ BCA Protein Assay. 31 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1

[0139] Dynamic Light Scattering (DLS): VLP-Open HLA protein complex and buffer (for any sample preparation) were centrifuged at 15,000 rpm for 10 minutes to remove any potential aggregates right before DLS experiment. In a cuvette, 60 ^L of each sample was loaded; the total protein concentration should be between 0.1-0.4 ^g / mL. Each sample was measured at 20oC on a Nanobrook 90Plus Submicron Particle Sizer (Brookhaven Instrument) for 2-4 runs. The intensity distribution was plotted and the particle size / radius average along with its standard deviation were calculated in Excel.

[0140] Fluorescence Polarization: VLP-Open HLA-A*02:01 / gTax conjugation was conducted in the same way as described in the previous section. Open A*02:01 was refolded with the placeholder peptide, gTAX, was exchanged and loaded with labeleled peptide TAMRATAX9 (TAMRA-KLFGYPVYV). Prior to the assays, the VLPs and all components of the assays were centrifuged for 15,000 rpm; VLP concentration was determined via Pierce™ BCA Protein Assay. In a 96-well plate, for each 100 ^L reaction, VLP-Open HLA- A*02:01 / gTax of different concentrations (2.5-100 nM) were incubated with 40 nMTAMRATax9 in FP buffer (150 mM NaCl, 20 mM sodium phosphate, 0.05% Tween-20, pH 7.4). The 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 (I٣) were measured then converted to polarization (mP) values using the equation 1000*[(III-(G*I٣)) / (III+(G*I٣))], in which the G-factor was 0.33 for TAMRA-labeled peptides. The data was fitted in GraphPad Prism.

[0141] T Cell Activation Assay: A T cell activation assay was conducted with VLP-Open HLA-A*02:01 / NY-ESO-1C9V(active construct) and VLP-Open HLA-A*02:01 / NY-ESO-1W5A(inactive construct as a negative control). 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. 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.

[0142] On Day 0 of the experiment, T cells were cultured at 1 x 106cells / mL at 37°C and 5% CO2 overnight. On Day 1, the cells were plated at 100,000 cells / well in a sterile U-bottomed 32 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 96-well plate; the cells could be 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-1C9Vor VLP-Open HLA-A*02:01 / NY-ESO-1W5A) or at a 1:1 ratio of Dynabeads™ 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. For the conditions with VLPs and the negative control, 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 μL 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 μL FACS buffer. The cells were centrifuged again at 300g for 5 minutes then stained with 100 μL of an antibody cocktail (Brilliant Ultra Violet™ 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.

[0143] For gating strategy, singlets from FSC-A vs. 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.

[0144] Results

[0145] The system design was sought to be experimentally validated by expressing the Spytagged G120C variant of HLA-A*02:01 in E. coli, isolating denatured proteins from inclusion bodies, and refolding it in vitro with the H31C variant of the ȕ2m in the presence of either, NY-ESO-1C9V(CV9) or NY-ESO-1W5A(W5A) peptide. Size-exclusion chromatography (SEC) and SDS / PAGE confirmed the formation of a Spytagged G120C / H31C HLA- A*02:01 / ȕ2m complex (hereafter referred to as OPEN A02) (Fig.2A-B). Differential scanning fluorimetry (DSF) was then performed to experimentally validate complex formation of OPEN-A02, and the thermal stability of the OPEN-A02-Spytag003 refolded with CV9 and W5A was observed with melting temperatures (Tm) of 59.2°C and 58.0°C, respectively, showing properly conformed peptide:MHC protein complexes (Fig. 2C). In addition, for 33 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 conjugation to the virus-like particle, Spycatcher-mi3 particles (mi3-VLP), the VLP particles were produced and purified (Fig. 2B, D). The co-stimulatory molecule, 4-1BBL was also produced and purified for conjugation to the mi3-VLPs (Fig.2B). To produce HLA-conjugated (homotypic) nanoparticles, 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. 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 trimer22. SDS / PAGE analysis confirmed the formation of both homotypic and heterotypic nanoparticles (Fig.2B).

[0146] 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. 3B), showing that the 1G4 was not able to recognize OPEN-A02 when either W5A peptide is present, or the cells were untransduced. Applicants also, utilized wild-type (WT) tetramers refolded with C9V peptide as a positive control (Fig.3C). Comparing staining levels between the OPEN-A02-mi3 particles and A02-tetramers, similar levels of staining were observed (Fig.3D). These data demonstrated that the OPEN-A02-mi3 particles were capable of TCR recognition and provided a rational for their use in stimulating T cells.

[0147] OPEN-A02 / CV9 conjugated mi3-VLPS were then utilized to test their ability to stimulate proliferation from 1G4 T cells. When treated with VLPs, 1G4 T cells were observed to both proliferate, and upregulate T cell activation markers 4-1BB and CD69 (Fig. 4, 5) in a dose-dependent manner. Importantly, T cell proliferation and activation was dependent on interaction between HLA-A*02:01 / CV9 and 1G4 TCR, as untransduced cells treated with VLPs did not show dose-dependent responses. These data demonstrated that the VLP platform has capacity to elicit TCR-dependent responses when loaded with cognate pMHC complexes.

[0148] The optimal ratio of components for theVLP-SpyCatchter003-mi3 conjugation reaction with SpyTagged Open HLA was then determined. The VLP-SpyCatcher 34 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 particle:SpyTag molar ratio explored was 1:60, 1:120, and 1:180. Coupling efficiency was visualized with SDS-PAGE with Coomassie staining (Fig. 6). The concentration of VLP- SpyCatcher003-mi3 pre-conjugation and post-conjugation was measured by Pierce™ BCA Protein Assay (Thermo Scientific, 23225). The results suggested that a ratio of 1:180, with final concentrations such as 50 nM VLP-SpyCatcher003-mi3 and 9 ^M SpyTag Open HLA resulted in the best conjugation reaction.

[0149] The VLP-Open HLA-A*02:01 conjugates were then characterized. The VLP- SpyCatcher particle:SpyTag molar ratio explored was 1:180 as previously optimized. Coupling efficiency was visualized with SDS-PAGE with Coomassie staining (Fig. 7A). 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 Pierce™ BCA Protein Assay.

[0150] The size of the VLP-Open HLA-A*02:01 conjugates was then determined via dynamic light scattering. The size of the VLP only particle (RH= 16.02 r 0.12 nm) was consistent with the literature (RH= 18.6 r 4.8 nm from Tan et al., Nat. Comm., 2021). The increase in particle size upon conjugation with open HLA-A*02:01 / NY-ESO-1C9Vor open HLA-A*02:01 / NY-ESO-1W5Awas consistent with the increase in size when conjugated of the size HLA molecule (~47 kDa and 411 amino acids) (Figure 7B).

[0151] The working concentration of VLP complex that would not result in unspecific activation was then investigated. To this end, a T cell activation assay with VLP-Open HLA- A*02:01 / NY-ESO-1C9V(active construct) and VLP-Open HLA-A*02:01 / NY-ESO-1W5A(inactive construct as a negative control) was conducted. For gating strategy, 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). It was observed that at a concentration of 3.13 μg / mL VLP particle or above, there was unspecific activation as was observed with similar 4-1BB expression by VLP-Open HLA-A*02:01 / NY-ESO-1W5A(inactive construct as a negative control) as the VLP-Open HLA-A*02:01 / NY-ESO-1C9V(active construct). However, below 3.13 μg / mL of the VLP particle, little to none of the non-specific T cell activation using 4-1BB marker was observed. This helped to identify the ideal working concentration of the VLPs. 35 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1

[0152] Through fluorescence polarization (FP) it was demonstrated that Open A*02:01 refolded with the placeholder peptide, gTax, could be exchanged and loaded with other peptides (labelled or unlabeled peptides) such as TAMRATax9 (TAMRA-KLFGYPVYV) (SEQ ID NO:7). Herein, it was demonstrated that similar peptide exchange occurred on open MHC- I conjugated on VLP. Raw parallel (III) and perpendicular emission intensities (I٣) were measured and then converted to polarization (mP) values using the equation 1000*[(III- (G*I٣)) / (III+(G*I٣))], in which the G-factor was 0.33 for TAMRA-labeled peptides. The data was fitted in GraphPad Prism (Figure 9).

[0153] It was observed that loading of the TAMRATax9 as the probe exchanged out the gTax peptide on the VLP- Open HLA-A*02:01 complex. This exchange 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 free Open HLA-A*02:01 / gTax (Sun et al., PNAS, 2023). Thus, it was demonstrated and confirmed that the VLP-Open HLA-A*02:01 / gTax design could accommodate antigen peptide exchange and loading.

[0154] Then it was demonstrated that antigen loadable VLP-Open HLA particles could specifically stimulate human T cells in vitro. The VLP-SpyCatcher particle:SpyTag molar ratio explored was 1:180 as previously optimized. Coupling efficiency was visualized with SDS-PAGE with Coomassie staining (Figure 10).

[0155] For gating strategy, singlets from FSC-A vs. FSC H were identified, then live cells from FSC-A vs. Live-Dead IR 876-A were identified. From this subpopulation of cells, CD8+ expressing CD69 or 4-1BB cells 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 (Figure 11).

[0156] For both markers, a higher frequency of CD8+ expressing CD69 and 4-1BB (i.e., more T cell activations) was observed only for VLP-Open HLA-A*02:01 / gTax exchanged with HLA-A*02:01 / gTax and the positive control with CD3 / CD28 beads. For all other conditions, including the HLA-A*02:01 / gTax exchanged with NY-ESO-1W5Apeptide, VLP-HLA- A*02:01 / gTax only (no exchange), VLP only, peptide only (NY-ESO-1C9Vor NY-ESO-1W5A), 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).

[0157] Design and Characterization of VLP-Open HLA Conjugates: To generate VLP- Open HLA nanoparticles, Spy-tagged open MHC on VLP-SpyCatcher003-mi3 was displayed. For the SpyTag open MHC, the heavy chain ectodomain of the open HLA-A*02:01 was 36 4889-2185-6222.1PATENT 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 ȕ2m (H31C) (FIGs.12A-12B). The design of the open HLA construct was developed in the laboratory as previously described in Sun et al. (2023).7Compared to native HLA molecule, 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.7In this work, SpyTag HLA-A*02:01 was successfully refolded with high- affinity antigenic peptide NY-ESO-1C9V(SLLMWITQV) (SEQ ID NO:4), the mutated decoy peptide NY-ESO-1W5A(SLLMAITQV), (SEQ ID NO:6) and moderate-affinity placeholder peptide gTAX (KLFGYPVYV) (SEQ ID NO:9) (FIG. 12C). These spy-tagged open MHC also had enhanced thermal stability, comparable with what previously observed in the non- tagged open MHC construct. In Sun et al, using differential scanning fluorometry (DSF), 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.7In this work, it was demonstrated that adding 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.

[0158] Regarding the choice for the nanoparticle platform, the protein-based nanoparticle platform virus-like particle (VLP) was chosen due to its versatility, ease of assembly, stability, and geometry ideal for antigen display.26,27Specifically, 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.26,23For the conjugation reaction, 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. This molar ratio and concentrations were used to conjugate SpyTag HLA-A*02:01 refolded with either NY-ESO-1C9V, NY-ESO-1W5A, or gTAX peptide (FIG.12D).

[0159] The robust conjugation reaction via SpyCatcher-SpyTag chemistry resulted in the formation of VLP-Open HLA protein nanoparticle, as confirmed by dynamic light scattering (DLS). The size of the VLP only particle (RH= 16.02 r 0.12 nm) was consistent with the literature (RH= 18.6 r 4.8 nm from Tan et al., 2021) (FIG. 12E).23The increase in particle 37 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 size upon conjugation with open HLA-A*02:01 / NY-ESO-1C9Vor open HLA-A*02:01 / NY- ESO-1W5Awas consistent with the increase in size when conjugated of the size HLA molecule (~47 kDa and 411 amino acids). The particles were further characterized with mass photometry, and the masses observed were consistent with the expected mass for the unconjugated and conjugated particles (FIG. 12F). Cryo-electron microscopic (cryo-EM) image was also taken to visualize the particle and confirmed its cage-like structural integrity of the VLP-Open HLA nanoparticle (FIG.12G).

[0160] Development of a VLP-Open HLA Loadable System: Open MHC-VLP 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.7In this work, it was shown that 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. Via 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. In the presence of large molar excess of the labeled peptide TAMRATAX9, the moderate-affinity gTAX peptide was rapidly displaced from the open HLA-A*02:01 (FIG. 13B). The kinetic association of fluorescently labeled peptides loaded on MHC-I was monitored via FP for 4-6 hours, and a second biological replicate was conducted with a different batch of VLP and similar FP data was obtained. 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).7Thus, it was demonstrated and confirmed that the VLP-Open HLA-A*02:01 / gTAX design could accommodate efficient antigen peptide exchange and loading.

[0161] Furthermore, a mass spectrometry experiment was conducted to demonstrate that the platform can be used as a tool for identification of potential antigenic bound peptides. VLP- Open HLA-A*02:01 / gTAX was exchanged with either individual or mixture peptides from the Cancer Genome Atlas (TCGA) epitope library. The particle was washed on spin columns to identify bound peptides. The bound peptide then could be identified using mass spectrometry 38 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 (MS). As a proof of concept, it was shown that the predicted antigenic peptide from the TCGA could bind and be retained on the VLP-Open HLA nanoparticle, and that the bound peptide identified from VLP-Open HLA-A*02:01 was of correct identity (FIG.13C).

[0162] Open HLA-VLP nanoparticles can be fluorescently labeled and conjugated to Open HLA molecules for antigen-specific T cell staining: SpyCatcher-VLP (SpyCatcher003-mi3) can be fluorescently labeled by reaction with NHS-fluoresceine prior to conjugation with molar excess amount of Open HLA molecule without compromising much of its conjugation sites (FIG. 14A). From SDS-PAGE analysis, under UV radiation and prior to Coomassie staining, it was observed that post-labeled sample showed a green band corresponding to SpyCatcher003-mi3 (VLP is shown as monomer in reducing condition on SDS-PAGE gel) (FIG. 14B). SpyCatcher-VLP was then successfully conjugated to Open HLA-A*02:01 refolded with NY-ESO-1C9Vor NY-ESO-1W5Ausing the same conjugation protocol as described in the previous section (FIG. 14B). Significantly, the fluorescently labeled VLP-Open HLA-A*02:01 / NY-ESO-1C9Vnanoparticle specifically stained antigen- specific NY-ESO-1 TCR (1G4) CD8+ T cells, a human T cells engineered expressing the variants of a single TCR (1G4) specific for the cancer antigen NY-ESO-1 (FIG. 14C).28As controls, tetramer of Open HLA-A*02:01 / NY-ESO-1C9Vwas used to show that these cells can be stained specifically (positive control), while tetramer of Open HLA-A*02:01 / NY-ESO- 1W5Ashowed no staining compared to the positive control and the buffer only control (FIG. 14C; left panel). VLP-Open HLA-A*02:01 / NY-ESO-1C9Vshowed a shift indicative of specific staining of NY-ESO-1 TCR (1G4) CD8+ T cells (FIG.14C; right panel).

[0163] Open HLA-VLP nanoparticles with refolded or exchanged antigenic peptide can trigger antigen-specific T cell activation in vitro

[0164] T Cell Activation Assays with the VLP-Open HLA-A*02:01 / gTax refolded with NY-ESO-1: It was demonstrated that VLP-Open HLA-A*02:01 can activate T cells (FIG. 15A). The working concentration of VLP complex that would not result in unspecific activation was investigated (FIG. 15B & 15C). To this end, a T cell activation assay with VLP-Open HLA-A*02:01 / NY-ESO-1C9V(active construct) and VLP-Open HLA-A*02:01 / NY-ESO-1W5A(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.

[0165] The cells were stimulated with different concentrations of the VLP particles (VLP- Open HLA-A*02:01 / NY-ESO-1C9Vor VLP-Open HLA-A*02:01 / NY-ESO-1W5A) or at a 1:1 39 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 ratio of Dynabeads™ 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. The cell supernatant was saved for later analysis with ELISA.

[0166] For gating strategy, singlets from FSC-A vs. FSC H were identified, and 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 (FIG. 15C). It was observed that at concentration of 3.13 μg / mL VLP particle or above, there was unspecific activation, as similar 4-1BB expression by VLP-Open HLA-A*02:01 / NY-ESO- 1W5A(inactive construct as a negative control) as the VLP-Open HLA-A*02:01 / NY-ESO-1C9V(active construct) was observed. However, below 3.13 μg / mL of the VLP particle, little to none of the non-specific T cell activation using 4-1BB marker was observed. This helped identify the ideal working concentration of our VLPs.

[0167] T Cell Activation Assays with the VLP-Open HLA-A*02:01 / gTax exchanged with NY-ESO-1

[0168] 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. In a 1000 ^L-reaction, 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% NaN3at 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. 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% NaN3using a 100-kDa spin column. The concentration of VLP-Open HLA complex was measured by Pierce™ BCA Protein Assay.

[0169] 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. At the end of 40 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 the reactions, the whole reaction was diluted 12.5X to create the 2X stocks for the T cell activation assays.

[0170] 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. Since it was previously established that a concentration below 3.13 μg / mL of the VLP-HLA particle could minimize non-specific T cell activation, a VLP concentration of 0.8 μg / mL was used for all conditions. The VLP-Open HLA-A*02:01 / gTax was exchanged with NY-ESO-1C9Vor NY-ESO-1W5Apeptide 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. In addition to evaluate the VLP-Open HLA-A*02:01 / gTax exchanged with NY-ESO-1C9V, T cell activation with VLP-Open HLA- A*02:01 / gTax exchanged with NY-ESO-1W5Apeptide, VLP-HLA-A*02:01 / gTax only (no exchange), VLP only, peptide only (NY-ESO-1C9Vor NY-ESO-1W5A), media only (negative control), and a positive control with Dynabeads™ Human T-Activator CD3 / CD28 (Gibco) was also evaluated.

[0171] For gating strategy, singlets from FSC-A vs. FSC H were identified, and then live cells from FSC-A vs. Live-Dead IR 876-A were identified. From this subpopulation of cells, 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).

[0172] For both markers, a higher frequency of CD8+ expressing CD69 and 4-1BB (i.e. more T cell activations) was observed only for VLP-Open HLA-A*02:01 / gTax exchanged with HLA-A*02:01 / gTax and the positive control with CD3 / CD28 beads. For all other conditions, including the HLA-A*02:01 / gTax exchanged with NY-ESO-1W5Apeptide, VLP-HLA- A*02:01 / gTax only (no exchange), VLP only, peptide only (NY-ESO-1C9Vor NY-ESO-1W5A), the frequencies of 4-1BB+ and CD69+ were approximately those of the negative control (media / buffer control). References 1. Szeto, C., Lobos, C. A., Nguyen, A. T. & Gras, S. TCR Recognition of Peptide–MHC- I: Rule Makers and Breakers. Int. J. Mol. Sci.22, 68 (2020). 2. Smith-Garvin, J. E., Koretzky, G. A. & Jordan, M. S. T Cell Activation. Annu. Rev. 41 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 Immunol.27, 591–619 (2009). 3. Arstila, T. P. et al. A Direct Estimate of the Human Įȕ T Cell Receptor Diversity. Science 286, 958–961 (1999). 4. Altman, J. D. et al. Phenotypic Analysis of Antigen-Specific T Lymphocytes. Science 274, 94–96 (1996). 5. Barker, D. J. et al. The IPD-IMGT / HLA Database. Nucleic Acids Res. 51, D1053– D1060 (2023). 6. Reddington, S. C. & Howarth, M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr. Opin. Chem. Biol.29, 94–99 (2015). 7. Sun, Y. et al. Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes. Proc. Natl. Acad. Sci.120, e2304055120 (2023). 8. Goebeler, M.-E. & Bargou, R. C. T cell-engaging therapies — BiTEs and beyond. Nat. Rev. Clin. Oncol.17, 418–434 (2020). 9. Song, S., Han, M., Zhang, H., Wang, Y. & Jiang, H. Full screening and accurate subtyping of HLA-A*02 alleles through group-specific amplification and mono-allelic sequencing. Cell. Mol. Immunol.10, 490–496 (2013). 10. Chen, J.-L. et al. Structural and kinetic basis for heightened immunogenicity of T cell vaccines. J. Exp. Med.201, 1243–1255 (2005). 11. Mayes, P. A., Hance, K. W. & Hoos, A. The promise and challenges of immune agonist antibody development in cancer. Nat. Rev. Drug Discov.17, 509–527 (2018). 12. Pollok, K. E., Kim, S. H. & Kwon, B. S. Regulation of 4-1BB expression by cell-cell interactions and the cytokines, interleukin-2 and interleukin-4. Eur. J. Immunol. 25, 488–494 (1995). 13. Wang, C., Lin, G. H. Y., McPherson, A. J. & Watts, T. H. Immune regulation by 4- 1BB and 4-1BBL: complexities and challenges. Immunol. Rev.229, 192–215 (2009). 14. Melero, I. et al. Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat. Med.3, 682–685 (1997). 15. Houot, R. et al. Therapeutic effect of CD137 immunomodulation in lymphoma and its enhancement by Treg depletion. Blood 114, 3431–3438 (2009). 16. Curran, M. A., Kim, M., Montalvo, W., Al-Shamkhani, A. & Allison, J. P. Combination CTLA-4 Blockade and 4-1BB Activation Enhances Tumor Rejection by Increasing T-Cell Infiltration, Proliferation, and Cytokine Production. PLoS ONE 6, e19499 (2011). 17. Westwood, J. A. et al. Routes of Delivery for CpG and Anti-CD137 for the Treatment 42 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 of Orthotopic Kidney Tumors in Mice. PLoS ONE 9, e95847 (2014). 18. Belcaid, Z. et al. Focal Radiation Therapy Combined with 4-1BB Activation and CTLA-4 Blockade Yields Long-Term Survival and a Protective Antigen-Specific Memory Response in a Murine Glioma Model. PLoS ONE 9, e101764 (2014). 19. Dai, Q. et al. 4-1BB Signaling Boosts the Anti-Tumor Activity of CD28-Incorporated 2nd Generation Chimeric Antigen Receptor-Modified T Cells. Front. Immunol. 11, 539654 (2020). 20. Zhao, X. et al. Efficacy and Safety of CD28- or 4-1BB-Based CD19 CAR-T Cells in B Cell Acute Lymphoblastic Leukemia. Mol. Ther. - Oncolytics 18, 272–281 (2020). 21. Li, H., Natarajan, K., Malchiodi, E. L., Margulies, D. H. & Mariuzza, R. A. Three- dimensional structure of H-2Dd complexed with an immunodominant peptide from human immunodeficiency virus envelope glycoprotein 120. J. Mol. Biol.283, 179–191 (1998). 22. Rabu, C. et al. Production of Recombinant Human Trimeric CD137L (4-1BBL). J. Biol. Chem.280, 41472–41481 (2005). 23. Tan, T. K. et al. A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses. Nat. Commun.12, 542 (2021). 24. Cohen, A. A. et al. Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice. (2021). 25. Aida, Y. & Pabst, M. J. Removal of endotoxin from protein solutions by phase separation using triton X-114. J. Immunol. Methods 132, 191–195 (1990). 26. Rahikainen, R.; Rijal, P.; Tan, T. K.; Wu, H.-J.; Andersson, A.-M. C.; Barrett, J. R.; Bowden, T. A.; Draper, S. J.; Townsend, A. R.; Howarth, M. Overcoming Symmetry Mismatch in Vaccine Nanoassembly through Spontaneous Amidation. Angewandte Chemie International Edition 2021, 60 (1), 321–330. https: / / doi.org / 10.1002 / anie.202009663. 27. Bruun, T. U. J.; Andersson, A.-M. C.; Draper, S. J.; Howarth, M. Engineering a Rugged Nanoscaffold To Enhance Plug-and-Display Vaccination. ACS Nano 2018, 12 (9), 8855–8866. https: / / doi.org / 10.1021 / acsnano.8b02805.Z.; Wang, Q. J.; Robbins, P. F.; Yu, L. Y. L.; Li, Y.; Molloy, P. E.; Dunn, S. M.; Jakobsen, B. K.; Rosenberg, S. A.; Morgan, R. A. High-Affinity TCRs Generated by Phage Display Provide CD4+ T Cells with the Ability to Recognize and Kill Tumor Cell Lines1. The Journal of Immunology 2007, 179 (9), 5845–5854. https: / / doi.org / 10.4049 / jimmunol.179.9.5845. 43 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 * * *

[0173] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention. 44 4889-2185-6222.1

Claims

PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 WHAT IS CLAIMED IS:

1. A loadable, multivalent virus-like particle (VLP) system that presents a multitude of MHC-I molecules comprising different allotype and antigen specificities, said system comprising: (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.

2. The loadable, multivalent VLP system of claim 1, wherein the one or more recombinant patient human leukocyte antigens are isolated from a subject.

3. The loadable, multivalent VLP system of claim 1, wherein the one or more antigens are selected from an infectious antigen or a tumor associated antigen (TAA).

4. The loadable, multivalent VLP system of claim 3, wherein the infectious antigen is selected from a bacterial antigen, a viral antigen, a parasitic antigen, or a fungal antigen.

5. The loadable, multivalent VLP system of claim 3, wherein the TAA is an antigen from a cancer selected from a solid or liquid cancer.

6. The loadable, multivalent VLP system of claim 5, wherein the solid or liquid cancer is 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).

7. The loadable, multivalent VLP system of claim 1, wherein the system further comprises a co-stimulatory molecule.

8. The loadable, multivalent VLP system of claim 7, wherein the co-stimulatory molecule is conjugated to the VLP. 45 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 9. The loadable, multivalent VLP system of claim 7, wherein the co-stimulatory molecule is selected from a protein, an antibody, a small molecule, and / or a combination thereof.

10. The loadable, multivalent VLP system of claim 9, wherein the co-stimulatory molecule is a protein, and wherein the protein is an OX40 ligand (OX40L), a B7 ligand, a 4-1BB ligand (4- IBBL), or a CD40 ligand (CD40L).

11. The loadable, multivalent VLP system of claim 9, wherein the co-stimulatory molecule is an antibody, and wherein the antibody is selected from an anti-CD28 antibody, an anti-CTLA4 antibody, or an anti-PD1 antibody.

12. The loadable, multivalent VLP system of claim 1, wherein the molar ration of the VLP to the HLA is about 1 to 180.

13. A cancer vaccine comprising the system of claim 1 and an agonist molecule capable of eliciting a polyclonal anti-tumor T cell response.

14. The cancer vaccine of claim 13, wherein the agonist molecule is a co-stimulatory molecule.

15. The cancer vaccine of claim 14, wherein the co-stimulatory molecule is selected from a protein, an antibody, a small molecule, and / or a combination thereof.

16. The cancer vaccine of claim 15, wherein the co-stimulatory molecule is a protein, and wherein the protein is an OX40 ligand (OX40L), a B7 ligand, a 4-1BB ligand (4-IBBL), or a CD40 ligand (CD40L).

17. The cancer vaccine of claim 15, wherein the co-stimulatory molecule is an antibody, and wherein the antibody is selected from an anti-CD28 antibody, an anti-CTLA4 antibody, or an anti-PD1 antibody.

18. A method of producing an expanded tumor infiltrating lymphocyte (TIL) population from a subject with a cancer, the method comprising: (a) administrating to the subject the loadable, multivalent VLP system of claim 1; (b) expanding a tumor infiltrarting lymphocyte (TIL) population in the subject; and (c) isolating one or more expanded TILs from the subject.

19. An isolated TIL population produced by the method of claim 18.

20. The method of claim 18, wherein the cancer is a solid or a liquid cancer.

21. The method of claim 20, wherein the solid or liquid cancer is selected from the group consisting of a lung cancer, a gastric cancer, a colon cancer, a hepatocellular carcinoma, a renal 46 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 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).

22. The method of claim 18, wherein the subject is further administered a co-stimulatory molecule.

23. The method of claim 22, wherein the co-stimulatory molecule is is selected from a protein, an antibody, a small molecule, and / or a combination thereof.

24. The method of claim 23, wherein the co-stimulatory molecule is a protein, and wherein the protein is an OX40 ligand (OX40L), a B7 ligand, a 4-1BB ligand (4-IBBL), or a CD40 ligand (CD40L).

25. The method of claim 23, wherein the co-stimulatory molecule is an antibody, and wherein the antibody is selected from an anti-CD28 antibody, an anti-CTLA4 antibody, or an anti-PD1 antibody.

26. A method of screening for an agent that is capable of binding an antigen, the method comprising: (a) contacting the agent with the loadable, multivalent VLP system of claim 1; and (b) identifying an agent that binds the antigen in the VLP system.

27. The method of claim 26, wherein the agent is selected from an antibody or a synthetic immunoreceptor, wherein the synthetic immunoreceptor is a selected from a chimeric antigen receptor (CAR) and / or a bispecific T cell engager (BiTE).

28. An antibody, CAR, or BiTE identified by the method of claim 27.

29. A method of inducing a polyclonal T or CAR-T response in a subject in need thereof, the method comprising administering the antibody, CAR, or BiTE of claim 28to the subject.

30. The method of claim 29, wherein the subject in need thereof has a solid or a liquid cancer.

31. The method of claim 30, wherein the solid or liquid cancer is 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 47 4889-2185-6222.1PATENT Docket Nos. CHOP 2024-012PCT and 074313.11019 / 6WO1 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).

32. A method of using the loadable, multivalent VLP of claim 1 to identify and / or stimulate T-cells, the method comprising 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..

33. A method of using the loadable, multivalent VLP of claim 1 to identify synthetic immunoreceptors, the method comprising contacting the loadable, multivalent VLP with a population of synthetic immunoreceptors, and determining the binding of the synthetic immunoreceptor to 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. 48 4889-2185-6222.1

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