Vaccine to mobilize b cells for therapy

Abstract

An antigen (Ag) that elicits a polyclonal T cell receptor like antibody response from endogenous B cells. The Ag comprises a single chain peptide MHCI (pMHCI) complex, that directs B cell responses to the displayed peptide without reactivity to the rest of the pMHCI molecule. Also, methods of using the Ag to induce polyclonal T cell receptor like (TCRL) antibodies (Abs) and using the Ag to deliver the single chain peptide to a target cell.

Description

[0001] Vaccine to mobilize B cells for therapy

[0002] FIELD OF TECHNOLOGY

[0003] The present disclosure relates to vaccine to mobilize B cells for therapy.

[0004] BACKGROUND INFORMATION

[0005] Selective targeting of cancer cells is an essential component of therapeutic strategies in oncology. Antibodies (Abs) have long been used for this purpose although decades of research invested into Ab discovery have only yielded a handful of clinical options.1On average, therapeutic Abs require over 6 years to complete clinical trials with most candidates failing (5 - 10% success rate)2due to unexpected toxicity or immunogenicity such as the induction of anti-drug Abs.3The overall process for clinically translating an Ab is far from efficient, especially compared to our B cells. Over the span of only a few weeks, our immune system can identify B cell clones that recognize a foreign antigen (Ag), increase their binding affinities towards their targets, and continuously produce Abs that can neutralize pathogens. Hence, the mobilization of patients’ own B cells towards generating cancer targeting Abs could be a powerful strategy, altering the traditional concept of Ab therapeutics.

[0006] To achieve this, a vaccine antigen that induces B cell responses towards cancer would be required. However, there are limited types of tumor markers that can safely be targeted. Current monoclonal Abs (mAbs) recognize cancer associated Ags, which are surface markers upregulated on cancer cells but they also exist on healthy tissues.4mAbs such as Panitumumab that bind EGFR on colorectal cancer commonly generates adverse skin reactions5(that also expresses EGFR) while Rituximab that binds to CD20 on both malignant and healthy B cells causes severe infusion reactions in ~12% of patients.6Consequently, using B cells to target cancer associated Ags would ultimately result in unwanted autoimmune responses and side effects.

[0007] RECTIFIED SHEET (RULE 91.1 ) Alternatively, cancer cells express more specific types of markers that are in the form of cancer specific peptides displayed on the major histocompatibility complex I (MHCI) of cancer cells.4The peptide MHCI (pMHCI) complexes are mainly recognized by T cell receptors (TCR) on CD8+T cells although their cytotoxic functions are known to be heavily suppressed within solid tumors and within patients with hematological cancers.7 9Recently, TCR-like Abs (TCRL Abs) have been developed to precisely recognize pMHCI similar to CD8 T cells, but impart T cell independent mechanisms of cancer cell lysis through antibody and complement dependent cellular cytotoxicity. These Abs are effective within preclinical models and are primarily developed through synthetic approaches such as phage display.10However, none have entered clinical trials where the majority of TCRL Ab candidates will fail, rendering their use within the clinic uncertain.

[0008] SUMMARY OF DISCLOSURE

[0009] Presented herein is an Ag design that elicits a polyclonal TCRL Ab response from endogenous B cells. The Ag of the present disclosure comprises a single chain peptide MHCI (pMHCI) complex, that directs B cell responses to the displayed peptide without reactivity to the rest of the self-MHCI molecule. In one embodiment, the present disclosure relates to a series of multivalent pMHCI Ags containing T helper cell epitope. In aspects the pMHCI is a peptide self-major histocompatibility complex I (psMHCI).

[0010] In one embodiment, the present disclosure relates to a single chain polypeptide comprising a major histocompatibility complex I (MHCI) molecule and a T helper epitope linked to the C-terminus of the MHCI molecule.

[0011] In one embodiment of the single chain polypeptide of the present disclosure, the single chain polypeptide further comprises an Fc domain of an immunoglobulin linked to the C-terminus of the MHCI molecule.

[0012] RECTIFIED SHEET (RULE 91.1 ) In another embodiment of the single chain polypeptide of the present disclosure, the T helper epitope is linked to the C-terminus of the Fc domain (MHCI-Fc-Th).

[0013] In another embodiment of the single chain polypeptide of the present disclosure, the T helper epitope is placed between the C-terminus of the MHCI molecule and the Fc domain (MHCI-Th-Fc).

[0014] In another embodiment of the single chain polypeptide of the present disclosure, the polypeptide is a multivalent single chain polypeptide comprising two or more p-MHCI molecules, a Fc domain of an immunoglobulin linked to C-terminus of the two or more p-MHCI molecules and the T helper epitope linked to the C-terminus of the Fc domain.

[0015] In another embodiment of the single chain polypeptide of the present disclosure, the single chain polypeptide further comprises a single-chain variable fragment (scFv).

[0016] In another embodiment of the single chain polypeptide of the present disclosure, the MHCI molecule is a human leukocyte antigen (HLA) class I molecule.

[0017] In another embodiment of the single chain polypeptide of the present disclosure, the MHCI molecule is a peptide MHCI (p-MHCI) molecule.

[0018] In another embodiment of the single chain polypeptide of the present disclosure, the p-MHCI is a peptide self-major histocompatibility complex I (psMHCI).

[0019] In another embodiment of the single chain polypeptide of the present disclosure, the single chain polypeptide is presented in higher order multivalent displays on synthetic nano-particles and / or nanofibers.

[0020] In another embodiment of the single chain polypeptide of the present disclosure, the single chain polypeptide comprises (i) SEQ ID NO: 17 linked to SEQ ID NO: 21 , or (ii) SEQ ID NO: 25 linked to SEQ ID NO: 21.

[0021] RECTIFIED SHEET (RULE 91.1 ) In another embodiment of the single chain polypeptide of the present disclosure, the single chain polypeptide comprises SEQ ID NO: 17 linked to SEQ ID NOs: 19, 20 and 21 , or (ii) SEQ ID NO: 25 linked to SEQ ID NOs: 19, 20 and 21 .

[0022] In another embodiment, the present disclosure relates to an engineered nucleic acid polynucleotide encoding the single chain polypeptide according to the present disclosure.

[0023] In one embodiment of the engineered nucleic acid polynucleotide of the present disclosure, the nucleic acid polynucleotide is a deoxyribonucleic acid (DNA) polynucleotide or a messenger ribonucleic acid (mRNA) polynucleotide.

[0024] In another embodiment, the present disclosure provides for an immunogenic composition comprising antigens (Ags), wherein each Ag comprises a single chain polypeptide comprising a peptide major histocompatibility complex I (p-MHCI) molecule and a T helper epitope linked to the C-terminus of the major histocompatibility complex I (MHCI) molecule, wherein the peptide is known to be displayed on the MHCI molecule.

[0025] In one embodiment of the immunogenic composition of the present disclosure, the single chain polypeptide further comprises an Fc domain of an immunoglobulin (p- MHCI-Fc-Th) linked to the C-terminus of the MHCI molecule.

[0026] In another embodiment of the immunogenic composition of the present disclosure, the T helper epitope is linked to the C-terminus of the Fc domain.

[0027] In another embodiment of the immunogenic composition of the present disclosure, the T helper epitope is placed between the C-terminus of the MHCI molecule and the Fc domain.

[0028] In another embodiment of the immunogenic composition of the present disclosure, the single chain polypeptide is a multivalent fused polypeptide comprising two or more p- MHCI molecules, a Fc domain of an immunoglobulin linked to C-terminus of the two

[0029] RECTIFIED SHEET (RULE 91.1 ) or more p-MHCI molecules and the T helper epitope linked to the C-terminus of the Fc domain.

[0030] In another embodiment of the immunogenic composition of the present disclosure, the single chain polypeptide further comprises a single-chain variable fragment (scFv).

[0031] In another embodiment of the immunogenic composition of the present disclosure, the immunogenic composition is provided in the form of a messenger ribonucleic acid (mRNA) vaccine composition comprising: (a) an mRNA polynucleotide comprising an open reading frame encoding the Ags; and (b) an agent used to introduce the mRNA into cells.

[0032] In another embodiment of the immunogenic composition of the present disclosure, the antigens are presented in higher order multivalent displays on synthetic nanoparticles, nano-fibers, viral vectors, liposomes and / or nucleic acid origami.

[0033] In another embodiment of the immunogenic composition of the present disclosure, immunogenic composition further comprises an adjuvant.

[0034] In another embodiment of the immunogenic composition of the present disclosure, the MHCI molecule is a human leukocyte antigen (HLA) class I molecule.

[0035] In another embodiment of the immunogenic composition of the present disclosure, the p-MHCI is a peptide self-major histocompatibility complex I (psMHCI).

[0036] In another embodiment, the present disclosure relates to a method of inducing polyclonal T cell receptor like (TCRL) antibodies (Abs) in a subject, the method comprising, administering a subject the immunogenic composition according to an embodiment of the present disclosure, wherein the peptide is a target moiety recognized by the induced polyclonal Abs, and wherein the MHCI is a self-MHCI of the subject’s species.

[0037] RECTIFIED SHEET (RULE 91.1 ) I n one embodiment of the method of inducing polyclonal TCRL Abs in a subject of the present disclosure, the subject is a cancer patient, and the peptide is a cancer- associated antigen presented on cancer cells, and wherein the cancer is a hematological malignancy or a solid tumor cancer.

[0038] In another embodiment of the method of inducing polyclonal TCRL Abs in a subject of the present disclosure, the subject is infected by a pathogen and the peptide is derived from the pathogen.

[0039] In another embodiment of the method of inducing polyclonal TCRL Abs in a subject of the present disclosure, the immunogenic composition is administered in the form of a messenger ribonucleic acid (mRNA) vaccine composition comprising; (a) an mRNA polynucleotide comprising an open reading frame encoding the Ag; and (b) a transfection agent used to introduce the mRNA into cells.

[0040] In another embodiment of the method of inducing polyclonal TCRL Abs in a subject of the present disclosure, the subject is a human.

[0041] In another embodiment, the present disclosure relates to an immunogenic composition according to an embodiment of the present disclosure for use in inducing polyclonal T cell receptor like (TCRL) antibodies (Abs) in a subject, wherein the peptide is a target moiety recognized by the induced polyclonal Abs and wherein the MHCI is a self-MHCI of the subject’s species.

[0042] In one embodiment of the immunogenic composition for use of the present disclosure, the subject is a cancer patient, the peptide is a tumor-associated antigen presented on cancer cells, and wherein the cancer is a hematological malignancy or a solid tumor cancer.

[0043] In another embodiment of the immunogenic composition for use of the present disclosure, subject is infected by a pathogen and the peptide is derived from the pathogen.

[0044] RECTIFIED SHEET (RULE 91.1 ) In another embodiment of the immunogenic composition for use of the present disclosure, the immunogenic composition is in the form of a messenger ribonucleic acid (mRNA) vaccine composition comprising; (a) an mRNA polynucleotide comprising an open reading frame encoding the Ag; and (b) an agent used to introduce the mRNA into cells.

[0045] In another embodiment of the immunogenic composition for use of the present disclosure, the subject is a human.

[0046] In another embodiment, the present disclosure relates to a use of a single chain polypeptide according to an embodiment of the present disclosure in the manufacture of a medicament or a drug for inducing polyclonal T cell receptor like (TCRL) antibodies (Abs) in a subject.

[0047] In another embodiment, the present disclosure relates to a use of a nucleic acid molecule encoding a single chain polypeptide according to an embodiment of the present disclosure in the manufacture of a medicament or a drug for inducing polyclonal T cell receptor like (TCRL) antibodies (Abs) in a subject.

[0048] In another embodiment, the present disclosure relates to an in vitro method of raising polyclonal T cell receptor like (TCRL) antibodies (Abs), the method comprising, contacting B cells with an immunogenic composition according to an embodiment of the present disclosure, wherein the peptide is a target moiety recognized by the induced polyclonal Abs, and wherein the MHCI is a self-MHCI of the B cell’s species.

[0049] In one embodiment of the in vitro method of raising polyclonal Abs of the present disclosure, the peptide is a cancer-associated antigen presented on cancer cells, and wherein the cancer is a hematological malignancy or a solid tumor cancer.

[0050] In another embodiment of the in vitro method of raising polyclonal Abs of the present disclosure, the peptide is derived from a pathogen.

[0051] RECTIFIED SHEET (RULE 91.1 ) In another embodiment of the in vitro method of raising polyclonal Abs of the present disclosure, the immunogenic composition is provided in the form of a messenger ribonucleic acid (mRNA) composition comprising: (a) an mRNA polynucleotide comprising an open reading frame encoding the Ag; and (b) an agent used to introduce the mRNA into cells.

[0052] In another embodiment of the in vitro method of raising polyclonal Abs of the present disclosure, the subject is a human.

[0053] In another embodiment, the present disclosure relates to a method of delivering a single chain polypeptide of the present disclosure to a target cell, the method comprising: (a) fusing to the single chain polypeptide a ligand that recognizes a marker on the surface of the target cell to form a fusion polypeptide, and (b) contacting the target cell with the fusion polypeptide.

[0054] In one embodiment of the method of delivering the single chain polypeptide of the present disclosure to a target cell, the ligand is a single chain variable fragment (scFv) that contains the antigen-binding domains of the heavy (VH) and light (VL) chains of an antibody that recognizes the marker on the surface of the target cell.

[0055] In another embodiment of the method of delivering the single chain polypeptide of the present disclosure to a target cell, (i) the target cell is a follicular dendritic cell (FDC) and the marker on the surface of the FDC is a complement receptor 1 (CD35), or (ii) the target cell is a dendritic cell and the marker on the surface of the dendritic cell is CD205, or (iii) the target cell is a B cell, and the marker on the surface of the B cell is a major histocompatibility complex (II).

[0056] In another embodiment, the present disclosure relates to a nanoparticle carrying multiple copies of a single chain polypeptide according to an embodiment of the present disclosure.

[0057] RECTIFIED SHEET (RULE 91.1 ) BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The following figures illustrate various aspects and preferred and alternative embodiments.

[0059] Figs. 1A to 1B - Technology overview. (1A) B cell vaccines with self-MHCI complexes displaying cancer peptides. (1 B) Secretion of Abs by plasma cells that target cancerous cells.

[0060] Fig. 2A-2D - Characterization of psMHCl-based Ags. (1A) Cartoon of pH2Kb antigens 20a and 20b. Reference number “24” indicates T helper epitope while reference number “21” indicates SIINFEKL (SEQ ID NO: 11 ) peptide. (2B) Nonreducing SDS-PAGE of pH2Kb-Th after recombinant expression and isolation. (2C) Non-reducing SDS-PAGE of pH2Kb-Fc-Th after recombinant expression and isolation. (2D) Analysis of pH2Kb folding via ELISA. High protein binding plates were coated with purified pH2Kb Ags followed by incubation with anti-SUNFEKL (SEQ ID NO: 11) H2-Kb monoclonal Ab (clone: 25-D1.16). SIINFEKL (SEQ ID NO: 11) H2-Kb complexes from the NIH Tetramer core were used as positive control.

[0061] Figs. 3A to 3M - Induction of TCRL Abs. (3A) Cartoon of Ags and dose delivered subcutaneously per mouse. All mice received 5 p.g of SMNP adjuvant. (3B) Experimental timeline. (3C) Analysis of serum IgGs that recognize SIINFEKL (SEQ ID NO: 11) H2-Kb after vaccinating mice with specified Ags for 21 days (n=4 mice). (3D) Specificity of TCRL Ab response within mice vaccinated with pH2Kb-Th and pH2Kb- Fc-Th for 21 days. Serially diluted sera were incubated in wells coated with H2-Kb complexes displaying either SIINFEKL (SEQ ID NO: 11 ) or Sendai Virus (SV) peptides to assess their specificity (n=4 mice). (3E) Longitudinal monitoring of TCRL Ab response within pH2Kb-Fc-Th vaccinated mice following a single priming dose. Area under the curve, AUC, of the serum IgG binding curves to SIINFEKL (SEQ ID NO: 11) or SV H2-Kb at different time points are shown (n=4 mice). (3F) Analysis of serum TCRL Ab isotypes within mice vaccinated with pH2Kb-Fc-Th for 21 days (n=4 mice).

[0062] RECTIFIED SHEET (RULE 91.1 ) (3G) ELISA-based specificity assessment of TCRL Abs after vaccinating with pH2Kb- Fc-Th and SNP for 21 days (n=3 mice). (3H) Prime-boost experimental timeline for pH2Kb-Fc-Th with LCMV T helper peptide or Trp2 peptide in place of SIINFEKL (SEQ ID NO: 11 ). (3I) ELISA readout of serum isolated from mice vaccinated with pH2Kb- Fc-Th containing LCMV helper peptide and SIINFEKL (SEQ ID NO: 11) (n=1 mice). (3J) ELISA readout of serum isolated from mice vaccinated with pH2Kb-Fc-Th containing PADRE helper peptide and Trp2 peptide (n=2 mice). (3K) Cartoon of targeted Ag constructs, tarH2Kb-Th and tarH2Kb-Fc-Th. Reference number “25” indicates the anti-CD35 single chain variable fragment. (3L) Non-reducing SDS-PAGE of targeted pH2Kb Ags after recombinant expression and isolation. (3M) ELISA-based specificity assessment of serum TCRL Abs after vaccinating with tarpH2Kb-Fc-Th or tarpH2Kb-Th and SMNP for 21 days (n=3 mice per group).

[0063] Figs. 4A to 4D - Cell-based assay to determine serum Ab specificity. (4A) Illustration of DC2.4 assay. Activated DC2.4 cells displaying SIINFEKL (SUN; SEQ ID NO: 11 ) or irrelevant peptides (Ir. Pep.) were incubated with serially diluted serum from vaccinated (Vac) or naive mice. Cells were then stained with anti-mouse IgG nanobody conjugated with Cy3. (4B) Histogram of Cy3 signal after incubation of DCs displaying SIINFEKL (SEQ ID NO: 11) (SUN) or irrelevant peptide (Ir. Pep) with 6% serum from pH2Kb-Fc-Th vaccinated (21 days) or naive mice. (4C) Cy3 MFI vs serially diluted serum for indicated groups.

[0064] Figs. 5A to 5C: GC response to follicle targeted pH2Kb-Fc-Th. (5A) Experimental timeline. (5B) Gating strategy for Ag specific GC B cells (B220+, CD38-, GL7+, SV peptide-, SIINFEKL (SEQ ID NO: 11) H2-Kb+ / +). (5C) Total Ag specific GC B cell number (n = 4 mice) within lymph nodes (LNs) of mice vaccinated with SMNP and Ova or pH2Kb-Fc-Th. Dashed boxes show cell populations of interest in the gating strategy.

[0065] Figs. 6A to 6C - pH2Kb-Fc-Th induces Ag specific T cell expansion. (6A) Experimental timeline. (6B) Representative flow plots of SIINFEKL (SEQ ID NO: 11)+ T cells amongst peripheral blood mononuclear cells (PBMCs) isolated from mice that

[0066] RECTIFIED SHEET (RULE 91.1 ) are naive, vaccinated with pH2Kb-Fc-Th for 7 days, and vaccinated with Ova for 7 days. (6C) Quantification of SIINFEKL (SEQ ID NO: 11)+ T cells amongst PBMCs of mice vaccinated with Ova or pH2Kb-Fc-Th (n = 3 mice).

[0067] Figs. 7A to 71 - Syngeneic cancer models. (7A) Representative flow plots showing CD8 and SIINFEKL (SEQ ID NO: 11) tetramer expression amongst CD3+ cells isolated from peripheral blood WT or CD8KO mice vaccinated with Ova for 7 days. (7B) ELISA readings showing the production of TCRL Abs that recognize SIINFEKL (SUN; SEQ ID NO: 11 ) and off-target Sendai virus (SV) at 14 days after vaccination of CD8 KO and WT mice with pH2Kb-Fc-Th. Naive sera is shown as a control. (7C-7D) Ex vivo cell-binding assay. Binding of serum IgG (6% v / v), isolated from pH2Kb-Fc-Th vaccinated (Vac) or naive CD8KO mice, to C1498-mC-SIIN and wildtype (WT) C1498 cells. Representative glow plots (C) and quantification of the mean fluorescence intensity (MFI) (7D) are shown (n=3mice). (7E) Experimental timeline for prophylactic model. (7F) Growth of subcutaneously implanted C1498-mC-SIIN tumors within pH2Kb-Fc-Th vaccinated and naive mice (n=3mice). (7G) Experimental timeline for active therapy model. (7H) Survival curves of mice vaccinated with pH2Kb-Fc-Th or control mice injected only with SMNP or SMNP and Ova (n = 4 mice for control, n = 2 mice for pH2Kb-Fc-Th vaccinated. (7I) Prophylactic model with wild type C57BI / 6 mice using B16-F10 melanoma cells that display SIINFEKL (SEQ ID NO: 11) peptide (B16- SIIN). Mice were vaccinated with SIINFEKL (SEQ ID NO: 11 ) containing pH2Kb-Fc- Th for 2 weeks prior to subcutaneous challenge with B16-SIIN. Tumor growth curves between naive control cohort and vaccinated mice (n = 3 mice per group).

[0068] Figs. 8A to 8B - Translating pH2Kb Ags into mRNA vaccines. (8A) Experimental schematic for detection of secreted pH2Kb-Fc-Th from mRNA transfected C2C12 cells. (8B) ELISA absorbance readout showing the presence of secreted Ags from transfected cells (n=4 replicates).

[0069] RECTIFIED SHEET (RULE 91.1 ) DETAILED DISCLOSURE

[0070] Definitions

[0071] As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0072] As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this invention.

[0073] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.

[0074] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments

[0075] RECTIFIED SHEET (RULE 91.1 ) of 0.1 or 1.0 as is appropriate. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about” which includes a standard deviation of about 15 %, or alternatively about 10% or alternatively about 5 %. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0076] “Effective amount” refers to an amount sufficient to induce a detectable therapeutic or preventive response in a subject.

[0077] In this document the terms “fused protein” and “single chain polypeptide” are used interchangeably to refer to a synthetic or artificial protein or polypeptide created by joining two or more genes of original proteins together, resulting in a single protein or polypeptide that exhibits characteristics of each original protein.

[0078] “Isolated” refers to a nucleic acid molecule or a polypeptide which is substantially separated from other cellular components.

[0079] The self-MHCI complex referred to in this disclosure is understood to be the specific MHCI molecule that is native to the organism, whether mouse, human or any other species of the animal kingdom, being vaccinated with an Ag of the present disclosure. For example, in the case of the findings that focuses on the C57BI / 6 mouse strain as a model system, the specific self-MHCI complex would be H2-Kb. Within this same strain, self-MHCI molecules could also include H2-Db while in the Balb / c mouse strain, self-MHCI molecules would include H2-Kd and H2-Dd. In humans, self-MHCI refers to Human Leukocyte Antigen (HLA), the human MHCI complex, that is native to the specific cancer patient that will receive a vaccine Ag of the present disclosure. As a non-limiting example, patients that express HLA-A:02:01 would receive a psMHCl-Fc- Th that comprises of a single chain HLA-A*02:0111displaying a cancer peptide of interest. This would further extend to other HLA class I molecules such as HLA-

[0080] RECTIFIED SHEET (RULE 91.1 ) A*24:02, HLA-A*11:01 , HLA-B*18:01 , or HLA-C*12:02, all of which are prevalent amongst humans of different ethnicities.12'13

[0081] The Fc domain included in the Ag construct of the present disclosure is understood to be the native Fc domain that is present within the organism that is to be vaccinated. In our preliminary findings, the Fc domain is that of murine lgG2cfound within C57BI / 6 mice and can be that of any other isotype found within this strain such as murine IgG 1 , lgG2b, lgG3, IgA, and IgM. Forthe use ofthe Ags of the present disclosure in humans, the Fc domain would refer to Ab isotypes found within humans such as lgG1 , lgG2, lgG3, lgG4, IgA, IgM, and IgE. The Fc domains included in the Ags of the present disclosure also refer to mutated versions of the different antibody isotypes that alter a number of Fc domain mediated functions such as the capacity to bind Fc receptors or induce complement deposition.

[0082] In the context of this document, the terms “linked” and “fused” are used interchangeably to refer to two peptides within a single chain polypeptide that are connected to one another either directly through a peptide bond, or indirectly through another molecule such a single amino acid, a peptide, a base, or a polynucleotide.

[0083] “Multivalent” in the context of pMHCI, including human leukocyte antigen (HLA), it refers to a pMHCI complex that can bind to multiple B cell receptors (BCRs) as well as TCRs simultaneously.

[0084] The term “subject” as used herein refers all members of the animal kingdom including mammals, preferably humans.

[0085] Overview

[0086] The present disclosure relates to a platform for vaccines that mobilize B cells for therapy.

[0087] In one embodiment, the present disclosure provides for a platform comprising a single chain polypeptide comprising, or consisting essentially of, or consisting of, a major

[0088] RECTIFIED SHEET (RULE 91.1 ) histocompatibility complex I (MHCI) molecule and a T helper epitope linked, directly or indirectly, to the C-terminus of the MHCI molecule. In one embodiment, the platform further comprises an Fc domain of an immunoglobulin linked to the C-terminus of the MHCI molecule. In one embodiment, the MHCI molecule is a peptide MHCI (p-MHCI) molecule, wherein the peptide is known to be displayed on the MHCI molecule.

[0089] With reference to Fig. 2A, in one embodiment, the present disclosure relates to a single chain or fused antigen 20a, b (also referred to as a single chain polypeptide) comprising, or consisting essentially of, or consisting of, a peptide 21 major histocompatibility complex I (pMHCI) 22, and a T helper cell epitope 24 at the C terminus (pMHCl-Th), the peptide 21 in the single chain is a specific peptide known to be displayed on the MHCI 22, such as a cancer specific peptide or a peptide specific of another condition or disorder. In one embodiment of the single chain antigen 20b, the MHCI 22 is linked or fused to, including directly or indirectly to, an IgG Fc domain 23. In one embodiment, the T helper epitope is placed between the C-terminus of the MHCI and the Fc domain (pMHCl-Th-Fc). In another embodiment, the Fc domain 23 is placed between the C-terminus of the MHCI 22 and the T helper epitope 24 (pMHCl- Fc-Th). In one aspect of the fused Ag of the present disclosure, the pMHCI is a peptide self-major histocompatibility complex I (psMHCI).

[0090] In one embodiment, the single chain polypeptide of the present disclosure includes a dimerization scheme using Fc domains, following the pMHCI domain, that maximizes TCRL Ab generation while also mounting T cell responses.

[0091] In another embodiment, the single chain polypeptide of the present disclosure is presented in higher order multivalent displays such as chemical conjugation to synthetic nano-particles and nano-fibers (e.g., polymeric or gold particles with about 10 nm diameter) or linked to protein subunits and peptides (e.g., lumazine synthase or ferritin) that self-assemble into nano-particles and nano-fibers or linked to other synthetic moieties (DNA or RNA nanostructures) that self-assemble into nanoparticles and -fibers. Given the unexpected finding that single chain polypeptides of

[0092] RECTIFIED SHEET (RULE 91.1 ) the present disclosure having higher valencies facilitate TCRL Ab response (see Fig. 3C), the single chain polypeptides of the present disclosure may be presented in different antigen formats to elicit polyclonal TCRL Abs. For example, in another embodiment, the present disclosure provides for nanoparticles carrying one or multiple (i.e. two or more) copies of the single chain polypeptides of the present disclosure.

[0093] The single chain polypeptides of the present disclosure can achieve targeted delivery of pMHCI to different target cell types. For example, the single chain polypeptides of the present disclosure achieve target delivery of pMHCI within a lymph node by fusing to the single chain polypeptide of the present disclosure a ligand such as a scFv (single-chain variable fragment) that contains the antigen-binding domains of the heavy (VH) and light (VL) chains of an antibody (pMHCl-ScFv), that recognizes a marker on the surface of the target cells, in this case within the lymph node. As such, in another embodiment, the single chain polypeptide of the present disclosure further comprises a scFv (single-chain variable fragment) that contains the antigen-binding domains of the heavy (VH) and light (VL) chains of an antibody (pMHCl-ScFv) that recognizes a marker on the surface of the cell type, such as, for example, the complement receptor 1 (CD35) on the surface of Follicular dendritic cells (FDCs) , CD205 (also known as DEC-205) on the surface of dendritic cells and major histocompatibility complex (II) on the surface of B cells.

[0094] In one embodiment, the MHCI molecule of the single chain polypeptide of the present disclosure is a MHCI of a particular species (i.e., H2-K for mice and HLA for humans). In another embodiment, the MHCI of the single chain polypeptide of the present disclosure is an artificial or synthetic MHCI molecule having one or different mutations such as disulfide traps,14linker accommodating mutations,11and so forth.

[0095] Nucleic Acid Molecules

[0096] In another embodiment, the present disclosure relates to an isolated nucleic acid molecule (DNA or RNA) that encodes for a single chain peptide major

[0097] RECTIFIED SHEET (RULE 91.1 ) histocompatibility complex I (pMHCI), linked, directly or indirectly, to a T helper cell epitope at the C terminus of the MHCI (pMHCl-Th), the peptide in the single chain known to be displayed on the MHCI. In one aspect of the isolated nucleic acid molecule encodes a psMHCl-Th.

[0098] In another embodiment the present disclosure relates to an isolated nucleic acid molecule (DNA or RNA) that encodes for a single chain peptide major histocompatibility complex I (pMHCI) linked at its C-terminus, directly or indirectly, to an IgG Fc domain and the T helper epitope, the peptide in the single chain known to be displayed on the MHCI. In one embodiment, the Fc is placed between the C- terminus of the MHCI and the T helper cell epitope (pMHCI -Fc-Th). In another embodiment, the T helper cell epitope is placed between the C terminus of the MHCI and the Fc domain (pMHCl-Th-Fc). In one aspect the isolated nucleic acid molecule encodes a psMHCl-Fc-Th or psMHCl-Th-Fc.

[0099] In another embodiment the present disclosure relates to a messenger ribonucleic acid (mRNA) vaccine composition comprising an mRNA polynucleotide comprising an open reading frame encoding an pMHCl-Th Ag or a pMHCl-Fc-Th Ag or pMHCl-Th- Fc Ag of the present disclosure. In one aspect, the mRNA vaccine further comprises an agent that facilitates the mRNA is introduced into cells, such as a cationic lipid- based transfection reagent used to introduce the mRNA into cells, or a viral vector used to introduce the mRNA into cells. In one aspect, the mRNA polynucleotide encodes a psMHCl-Th Ag, a psMHCl-Fc-Th Ag or psMHCl-Th-Fc Ag of the present disclosure.

[0100] Compositions

[0101] In another embodiment, the present disclosure relates to an immunogenic composition comprising, or consisting essentially of, or consisting of, a single chain polypeptide comprising a peptide major histocompatibility complex I (pMHCI), linked, directly or indirectly, to a T helper cell epitope at the C terminus of the MHCI (pMHCl-Th), the

[0102] RECTIFIED SHEET (RULE 91.1 ) peptide in the single chain known to be displayed on the MHCI molecule. This composition includes single chain MHCI molecules across different species (i.e. , H2- K for mice and HLA for humans) as well as artificial or synthetic single chain MHCI designs having different mutations such as disulfide traps,14linker accommodating mutations,11and so forth. In one aspect, the pMHCI is a psMHCI thereby forming psMHCl-Th.

[0103] In another embodiment the present disclosure relates to an immunogenic composition comprising, or consisting essentially of, or consisting of, a single chain polypeptide comprising a peptide major histocompatibility complex I (p-MHCI), the peptide in the single chain known to be displayed on the MHCI, fused, directly or indirectly, to an IgG Fc domain. In one embodiment, the Fc domain is linked (directly or indirectly) to the C-terminus of the MHCI and T helper epitope is linked (directly or indirectly) to the C- terminus of the Fc domain (pMHCl-Fc-Th). In another embodiment, the T helper cell epitope is placed in between the C-terminus of the MHCI and the Fc (pMHCl-Th-Fc). In one aspect of the immunogenic composition, the pMHCI is a psMHCI thereby forming psMHCl-Fc-Th or or psMHCl-Th-Fc.

[0104] In another embodiment, the present disclosure relates to an immunogenic composition comprising, or consisting essentially of, or consisting of, a nucleic acid molecule (DNA or RNA) that encodes for a single chain polypeptide comprising a peptide major histocompatibility complex I (pMHCI), linked, directly or indirectly, to a T helper cell epitope at the C terminus of the MHCI (pMHCl-Th), the peptide in the single chain known to be displayed on the MHCI. In one aspect, the pMHCI is a psMHCI thereby forming psMHCl-Th.

[0105] In another embodiment the present disclosure relates to an immunogenic composition comprising, or consisting essentially of, or consisting of, a nucleic acid molecule (DNA or RNA) that encodes for a single chain polypeptide comprising a peptide major histocompatibility complex I (pMHCI) linked, directly or indirectly, to an IgG Fc domain and a T helper cell epitope, the peptide in the single chain known to be displayed on

[0106] RECTIFIED SHEET (RULE 91.1 ) the MHCI. In one embodiment, the Fc is placed between the C-terminus of the MHCI and the T helper cell epitope (pMHCl-Fc-Th). In another embodiment, the T helper cell epitope is placed between the C terminus of the MHCI and the Fc domain (psMHCl-Th-Fc). In one aspect of the immunogenic composition, the pMHCI is a psMHCI thereby forming psMHCl-Fc-Th or psMHCl-Th-Fc.

[0107] The immunogenic compositions of the present disclosure comprise Ags of the present disclosure (or nucleic acid molecules encoding the Ags of the present disclosure) and a pharmaceutically acceptable carrier. The compositions can be used for administration of the Ags of the present disclosure to a subject. The compositions can further include an adjuvant. The adjuvant can enhance the biological activity of the Ag in the composition.

[0108] In embodiments, the Ags of the present disclosure, or nucleic acid molecules encoding the Ags of the present disclosure, can be provided in a transfer vector. These transfer vectors can be designed to target specific cells or tissues, enhance cellular uptake, and facilitate the release ofthe Ags of the present disclosure, or nucleic acid molecules encoding the Ags of the present disclosure. Examples of vectors include nucleic acid complexes such as nucleic acid origami, viral vectors, bacterial vectors, liposomes, lipids, peptides, nano particles and so forth.

[0109] In embodiments, the Ags ofthe present disclosure can be provided in a liposome (e.g., an immunoliposome) or lipid formulation.

[0110] Delivery of pMHCl-based vaccines of the present disclosure include either through depots, repeated injections, or replicon vaccines that achieve slow and extended release of antigens.

[0111] The compositions of the present disclosure can be formulated for injection (intramuscular, subcutaneous, intravenous, intraperitoneal, etc ), topical administration, inhalation, oral administration, or intranasal administration.

[0112] RECTIFIED SHEET (RULE 91.1 ) Examples of pharmaceutically acceptable carriers are well known to those skilled in the art. Non-limiting examples include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, aga, pectin, peanut oil, olive oils, sesame oil, and deionized water.

[0113] In embodiments, the compositions of the present disclosure further comprise one or more stabilizers. For example, the stabilizer can comprise a carbohydrate (e.g., sorbitol, mannitol, starch, sucrose, dextrin, glucose, or a combination thereof), a protein such as albumin or casein, and / or a buffer (e.g., an alkaline phosphate).

[0114] Compositions for injection may include one or more pharmaceutically acceptable vehicles or diluents. Compositions for injection can comprise buffered solutions that have a suitable pH and are iso-osmotic with physiological fluids. Any pharmaceutically suitable diluent may be used in the composition for injections (e.g., distilled water, a salt solution, and / or a buffer solution). Compositions for injection may be prepared by conventional volume-weight procedures. A certain amount of the peptide may be diluted to the necessary volume with a diluent or solvent. The solution may then filtered through sterilized filters and then bottled or ampouled. The resultant solution is suitably a stable transparent liquid and preferably does not contain any chemical or other impurities.

[0115] Applications

[0116] In another embodiment, the present disclosure relates to a method of inducing polyclonal TCRL antibodies in a subject, the method comprising, or consisting essentially of, or consisting of, administering a subject the immunogenic composition of the present disclosure wherein the peptide is a target moiety recognized by the induced polyclonal Abs and wherein the MHCI is a self-MHCI of the subject’s species.

[0117] In another embodiment, the present disclosure relates to a method of treating cancer in a subject, the method comprising, or consisting essentially of, or consisting of, administering to the subject the immunogenic composition comprising a psMHCI of

[0118] RECTIFIED SHEET (RULE 91.1 ) the present disclosure, the peptide of the psMHCI being a cancer-associated antigen presented on transformed cells such as cancer cells. In one embodiment, the cancer is a hematological malignance (blood cancer) or a solid tumor cancer.

[0119] In another embodiment, the present disclosure relates to a method of treating a disease or condition, the method comprising, or consisting essentially of, or consisting of, administering to the subject the immunogenic composition comprising a psMHCI of the present disclosure, wherein the peptide of the psMHCI is an antigen presented on cells or pathogens associated with the disease or condition being treated. Examples of pathogens include bacteria, virus, protozoan, fungus. In embodiment, the peptide of the psMHCI can be presented on transformed cells (i.e. cancer cells) or plant cells.

[0120] In another embodiment, the present disclosure relates to a use of the immunogenic composition of the present disclosure in (a) inducing polyclonal TCRL antibodies, and / or (b) treating cancer. In one embodiment the cancer is a hematological malignancy or blood cancer (i.e., leukemia, lymphoma, and myeloma. In another embodiment, the cancer is a solid tumor cancer. The peptide of the psMHCI is a cancer-associated antigen presented on cancer cells (solid or blood cancers).

[0121] In another embodiment, the present disclosure relates to a use of the immunogenic composition of the present disclosure for treating a disease or condition. The peptide of the psMHCI is an antigen presented on cells or pathogens associated with the disease or condition.

[0122] In another embodiment, the present disclosure relates to a use of a single chain polypeptide according to an embodiment of the present disclosure in the manufacture of a medicament or a drug for inducing polyclonal T cell receptor like (TCRL) antibodies (Abs) in a subject.

[0123] In another embodiment, the present disclosure provides for an in vitro method of raising polyclonal T cell receptor like (TCRL) antibodies (Abs). In one embodiment,

[0124] RECTIFIED SHEET (RULE 91.1 ) the method comprises, contacting in vitro B cells with the immunogenic composition of the present disclosure, wherein the peptide in the p-MHCI is a target moiety recognized by the induced polyclonal Abs, and wherein the MHCI is a self-MHCI of the B cell’s species. In aspects, the peptide is a cancer-associated antigen presented on cancer cells or derived from a pathogen, or associated with another disease or condition. In aspects, the immunogenic composition is provided in the form of a messenger ribonucleic acid (mRNA) composition comprising: (a) an mRNA polynucleotide comprising an open reading frame encoding the Ag; and (b) a cationic lipid-based transfection reagent used to introduce the mRNA into the B cells. In one embodiment, the B cells are human B cells.

[0125] In order to aid in the understanding and preparation of the present disclosure, the following illustrative, non-limiting examples are provided.

[0126] EXAMPLES

[0127] Example 1 - Inducing TCRL Ab responses through psMHCI Antigens

[0128] \Ne sought to generate T cell receptor-like (TCRL) antibodies (Abs) against a model foreign peptide SIINFEKL (derived from ovalbumin; SEQ ID NO: 11) known to be displayed on the H2-Kb complex, which is the self-MHCI complex native to C57BI6 / J mice. We developed two distinct types of Ags, monomeric peptide H2-Kb (pH2kB-Th) and dimerized peptide H2-Kb (pH2kB-Fc-Th) with T helper cell epitopes, whose amino acid sequences are listed below.

[0129] These Ags 20a, b consist of a single chain SIINFEKL (SEQ ID NO: 11 ) H2-Kb complex with a disulfide trap14to stabilize the peptide within the H2-Kb groove alone or fused to a murine lgG2c Fc domain (Fig. 2A). In addition, both Ags 20a, b in this Example possess a universal CD4 T cell epitope 24, known as PADRE,15to enable CD4 T cell

[0130] RECTIFIED SHEET (RULE 91.1 ) help during the Ab response. It should be understood that T helper cell epitopes other than PADRE may be used in the Ags of the present disclosure.

[0131] Following their expression and isolation, these Ags were validated by using nondenaturing SDS PAGE where the molecular weights (Mw) for pH2Kb-Th and pH2Kb- Fc-Th were ~50 and ~180 kDa (~90 kDa for monomer), respectively (Fig. 2B-C). These values are well within the range of the Mw based on the amino acid sequences of these Ags. For further in vitro characterization, we performed enzyme-linked immunosorbent assay (ELISAs) to verify the proper folding of the SIINFEKL (SEQ ID NO: 11) H2-Kb single chain MHCI by using a preexisting monoclonal TCRL Ab (clone: 25-D1.16) that recognizes this construct (Fig. 2D).

[0132] 2. Analyzing TCRL Abs within serum after vaccination.

[0133] \Ne examined whether Ags comprising our psMHCl-based constructs 20a, b and other traditional Ags such as Ovalbumin (Ova) 30 and SIINFEKL (SEQ ID NO: 11 ) peptide 21 with or without T helper epitope 24, PADRE, can induce a TCRL Ab response (Fig. 3A). To this end, C57BL / 6 mice were vaccinated with these Ags along with a saponinmonophosphoryl lipid A nanoparticle (SMNP)16for 21 days prior to isolation of serum for ELISA (Fig. 3B). In this assay, we coated high protein binding 96-well plates with SIINFEKL (SEQ ID NO: 11) H2-Kb tetramers (NIH Tetramer Core), blocked the wells with bovine serum albumin (BSA), and exposed them with serially diluted sera. We failed to detect any Ag specific serum IgGs across all constructs that lacked the T helper epitope. However, for Ag constructs with the PADRE peptide, we detected high levels of SIINFEKL (SEQ ID NO: 11 ) H2-Kb specific serum IgGs within mice vaccinated with pH2Kb-Fc-Th while pH2Kb-Th exhibited low levels of ag specific IgGs (Fig. 3C). These findings collectively suggest CD4 T cell help is required to induce Abs against peptide self-MHCI while the multivalent display of psMHCI 20b (e.g., pH2Kb-Fc-Th) enhances such responses.

[0134] RECTIFIED SHEET (RULE 91.1 ) TCRL Abs are expected to recognize a small region formed by union of the peptide and MHCL Despite observing positive serum IgG binding in our ELISA, these Abs could potentially bind to regions outside of the peptide cleft, thereby requiring additional analysis for epitope specificity. Hence, we repeated our ELISA using H2-Kb tetramers that displayed an irrelevant peptide (FAPGNYJAL) (SEQ ID NO: 51) from the Sendai Virus (NIH Tetramer Core) where positive binding to this control would suggest that serum IgGs are recognizing unwanted off-target epitopes on the self- MHCI protein. Strikingly, sera from mice vaccinated with pH2Kb-Fc-Th and pH2Kb-Fc exhibited negligible binding to the irrelevant control, suggesting that they are TCRL Abs that specifically recognized the displayed SIINFEKL (SEQ ID NO: 11 ) peptide (Fig. 3D). Longitudinal analysis of serum within pH2Kb-Fc-Th vaccinated mice indicates that these TCRL Abs persisted for at least 5 months after a single priming dose and are composed of a range of murine Ab isotypes (e.g., lgG1 , lgG2b, and lgG2c), suggesting the capacity of the TCRL Abs to induce Ab and complement dependent cellular cytotoxicity (Fig. 3E-F).

[0135] We also evaluated alternative approaches that could be used to induce TCRL Abs. Firstly, we tested adjuvants other than SMNP (an exploratory pre-clinical adjuvant) can elicit TCRL Ab response. To this end, we vaccinated C57BL / 6 mice with pH2Kb- Fc-Th displaying SIINFEKL (SEQ ID NO: 11) peptide along with saponin nanoparticle (SNP) that resembles Novavax’s Matrix-M, a clinically approved adjuvant for 21 days and evaluated the TCRL Ab responses. Similar to our previous findings, we found that these adjuvants can also induce TCRL Abs without off-target responses against the H2-Kb molecule (Fig. 3G). Secondly, we determined whether the PADRE sequence was specifically required by generating psMHCl-Fc-Th constructs that displayed SIINFEKL (SEQ ID NO: 11) but contain Lymphocytic Choriomeningitis Virus (LCMV) glycoprotein6i-80 (GLKGPDIYKGVYQFKSVEFD; SEQ ID NO: 50), a well-known CD4 T cell specific peptide displayed on the MHCII of antigen presenting cells within C57BL / 6 mice, at the C-terminus in place of the PADRE sequence. Following priming and boosting doses administered 14 days apart, sera from vaccinated mice were

[0136] RECTIFIED SHEET (RULE 91.1 ) isolated at 14 days after the boost and observed serum TCRL Abs that predominantly recognized SIINFEKL (SEQ ID NO: 11) H2-Kb with minimal binding to SV peptide H2-

[0137] Kb (Fig. 3H-I).

[0138] We then sought to explore the type of peptides that can be targeted with the vaccine platform of the present disclosure. In addition to SIINFEKL (a foreign peptide; SEQ ID NO: 11 ), we examined whether self-peptides that are found to be upregulated on cancer cells, such as Trp2 peptide, found in both murine and human melanoma cells, can also be targeted with the Ags of the present disclosure. Using the prime-boost scheme shown in Fig. 3H, we vaccinated C57BI / 6 mice with pH2Kb-Fc-Th containingTrp2i8o-188 peptjde, (Sequence: SVYDFFVWL (SEQ ID NO: 12) and assessed the TCRL Abs within serum at 2 weeks post boost. Our findings showed that serum Abs can also be raised against Trp2 while again exhibiting minimal reactivity to H2-Kb (Fig. 3J). Collectively, these findings suggests that the vaccine Ag of the present disclosure can target a range of peptides including foreign peptides caused by frame-shift mutations in cancer (e.g., Type A Nucleophosmin 1 mutations found in leukemia)17and upregulated self-peptides (e.g., Trp2 peptide,18Wilms Tumor 1 peptide19). This targeting capacity can be extended towards neoepitopes which are cancer specific peptides bearing missense mutations such as those found in RAS20(e.g., KRASG12D) and p5321 22(TP53R175H) proteins.

[0139] Lastly, we investigated whether targeted delivery of psMHCI Ags to specific cell types2324within the lymph node can yield TCRL Ab responses. Focusing on the delivery of Ags to the follicular dendritic cells (FDCs) as a proof-of-principle (WO 2024 / 107477 ,23we developed two new Ags that incorporate a single chain variable fragment (ScFv) that recognizes complement receptor 1 (CD35) on the surface of FDCs (25) in between the H2Kb and Th in H2Kb-Th and between the Fc and Th in H2Kb-Fc-Th to generate tarH2Kb-Th and tarH2Kb-Fc-Th, respectively (Fig. 3K). These targeted Ags exhibited molecular weights of ~75 and 205 kDa for tarH2Kb-Th and tarH2Kb-Fc-Th, respectively, after isolation and non-reducing SDS-PAGE analysis (Fig. 3L). Following a single priming dose, we performed ELISA on sera

[0140] RECTIFIED SHEET (RULE 91.1 ) isolated from mice and observed the higher levels TCRL Abs after vaccination with tarH2Kb-Fc-Th that bound to the SIINFEKL (SEQ ID NO: 11) peptide with minimal off- target binding to H2-Kb complexes displaying the irrelevant SV peptide (Fig. 3M).

[0141] Adding targeting moieties to the Ags of the present disclosure (such as ScFv fragment that recognizes CD35, will advantageously elicit similar, if not more, TCRL Ab responses (relative to delivering the Ags without a targeting moiety) at lower doses given the increased efficiency of delivering Ags to target cells that are critical in propagating immune responses.

[0142] 3. Vaccine-induced TCRL Abs can recognize cell surface psMHCL

[0143] To confirm that TCRL Abs from pH2Kb-Fc-Th vaccinated mice can recognize cells displaying the target peptide, we developed a cell-based assay using a dendritic cell (DC) line, DC2.4, that expresses H2-Kb. These cells were pulsed with SIINFEKL (SEQ ID NO: 11 ) or left unpulsed to display irrelevant peptides, exposed to Fc Block, and incubated in the sera from vaccinated (n=4) or naive (n=3) mice. After removing the serum, DCs were stained with Cy3-labeled anti-mouse IgG nanobodies to determine the extent of surface bound serum Abs (Fig. 4A). Using flow cytometry, we detected significantly higher surface IgG binding with vaccinated sera compared to naive, irrespective of serum concentration (Fig. 4B-C). In addition, no binding differences were observed on DCs with irrelevant peptides (Fig. 4C-D), confirming that vaccine- induced TCRL Abs specifically targeted the peptide displayed on the MHCI without cross-reacting with the MHCI molecule itself.

[0144] 4. TCRL Abs undergo affinity maturation following vaccination.

[0145] Potent Ab responses require B cells to undergo affinity maturation within germinal centers (GCs). This process enables B cells to increase the binding affinities of their Abs towards an Ag during the course of an Ab response. We examined whether pH2Kb-Fc-Th (with PADRE sequence) could potentially induce affinity maturation amongst vaccine activated B cells. To this end, we vaccinated C57BL / 6 mice with this

[0146] RECTIFIED SHEET (RULE 91.1 ) construct along with Ova, as a control Ag, and SMNP adjuvant for 13 days (Fig. 5A) and processed the cells from the draining LNs for flow cytometric analysis. To identify GC B cells and their Ag specificity, the gating strategy shown in Fig. 5B was used. Firstly, we designated GC B cells as viable cells that are B220+, GL7+, and CD38-. To identify Ag specific GC B cells, we removed the off-target GC B cell population by using a dye labeled H2-Kb tetramer with the Sendai peptide (irrelevant peptide). Amongst the non-binders to the irrelevant peptide probe, we set Ag specific GC B cells as the double positive population that captured SIINFEKL (SEQ ID NO: 11) H2-Kb tetramers labeled with two distinct dyes (PE and APC) to ensure that these cells are specific to SIINFEKL (SEQ ID NO: 11 ) H2-Kb and not to the individual dyes. Using these gating strategies, we discovered approximately ~160-fold higher number of SIINFEKL (SEQ ID NO: 11 ) H2-Kb specific GC B cells after vaccination with pH2Kb- Fc-Th compared to Ova (Fig. 5D). Taken together, our findings indicate that the psMHCI Ags of the present disclosure can induce GC reactions and enable TCRL Ab- expressing B cells to potentially undergo affinity maturation.

[0147] 5. T cell responses following vaccination with pH2Kb-Fc-Th.

[0148] In addition to Ab responses, we assessed Ag specific CD8 T cell responses following vaccination with pH2Kb-Fc-Tn. At 7 days after priming (Fig. 6A), we isolated peripheral blood mononuclear cells (PBMCs) from sera of C57BI / 6 mice and performed flow cytometry to quantify the proportion of SIINFEKL (SEQ ID NO: 11 ) H2- Kb specific T cells using dye labeled tetramers (NIH Tetramer core) (Fig. 6B). Relative to mice primed with Ova for the same duration, we observed similar extent of Ag specific T cell expansion after vaccinating with pH2Kb-Fc-Th (Fig. 6C), indicating that our Ag can simultaneously activate both CD8 T cells and TCRL Ab producing B cells.

[0149] 6. Treatment of syngeneic cancer models

[0150] We evaluated the efficacy of the vaccine of the present disclosure in protecting against and treating C57BL / 6 mice inoculated with a syngeneic AML cell line, C1498, that

[0151] RECTIFIED SHEET (RULE 91.1 ) expresses H2-Kb.25To isolate the effect of vaccine-induced TCRL Abs, we used CD8 knock out (CD8KO) mice (B6.129S2-Cd8ato?Ma / ( / J)26that express H2-Kb but do not possess any CD8 T cells. Consequently, at 7 days after vaccination with Ova, we failed to observe any SIINFEKL (SEQ ID NO: 11 )+T cells amongst the PBMCs isolated from CD8KO mice in contrast to the wild type (WT) counterpart (Fig. 7A). Coupled with the absence of C1498 specific CD4 T cell epitopes within pH2Kb-Fc-Th, the use of CD8KO mice eliminated any contributions from cell-mediated immunity towards anti-cancer responses.

[0152] Similar to the wild type (WT) strain, CD8K0 mice developed TCRL Abs at 2 weeks after vaccination SMNP and pH2Kb-Fc-Th containing the SIINFEKL (SEQ ID NO: 11 ) peptide (Fig. 7B). To evaluate whether the vaccine induced TCRL Abs are effective against cancer, we engineered C1498 cells that express mCherry and SIINFEKL (SEQ ID NO: 11 ) (C1498-mC-SIIN) (Fig. 7C). Using the approach similar to our DC assay (Fig. 4), we found that serum TCRL Abs isolated from CD8KO mice vaccinated with pH2Kb-Fc-Th can successfully bind to the C1498-mC-SIIN cells (Fig. 7C-D).

[0153] To determine whether the ex vivo binding translates to in vivo efficacy, we first utilized a prophylactic model wherein CD8KO mice were vaccinated with SMNP and pH2Kb- Fc-Th containing SIINFEKL (SEQ ID NO: 11) for 14 days prior to subcutaneous inoculation with 0.5 million C1498-mC-SIIN cells (Fig. 7E). While significant tumor growth was observed within the naive control group of CDKO mice, we encouragingly failed to observe any tumor growth within the same time frame (Fig. 7F). We then determined whether our vaccine could be effective in treating AML caused by C1498 cells. To this end, we intravenously inoculated CD8KO mice with 1 million C1498-mC- SIIN cells and subcutaneously vaccinated these mice with SMNP and pH2Kb-Fc-Th at 2 days after cancer inoculation (Fig. 7G). For our control group, mice inoculated with 1 million C1498-mC-SIIN cells were administered with either SMNP and Ova or SMNP alone. The survival of our treated and control mice were then monitored. Notably, we chose to administer the cancer and our vaccine within a short time frame since ~ 2 weeks are required to observe any detectable serum TCRL Abs. At ~ 23

[0154] RECTIFIED SHEET (RULE 91.1 ) days after inoculation, 50% of within the control group succumbed to the cancer while no deaths have been observed within our vaccine treated group for up to 34 days (Fig. 7H). Taken together, these findings show that our vaccine-induced TCRL Abs can effectively recognize cancer cells, based on appropriate peptide selection, and eliminate the cancer cells in vivo.

[0155] In addition to the C1498 model, we also tested the vaccine of the present disclosure within wild type C57BI / 6 mice inoculated with SIINFEKL (SEQ ID NO: 11) displaying B16-F10 melanoma cells (B16-SIIN). Using our prophylactic challenge model (Fig. 7E), we first vaccinated C57BI / 6 mice with pH2KB-Fc-Th containing the SIINFEKL (SEQ ID NO: 11 ) peptide for 2 weeks prior to subcutaneous injection of 1 million B16- SIIN cells. Compared to the naive control cohort that was also inoculated with melanoma cells, we failed to detect the growth of tumors within the same time timepoint (Fig. 7I).

[0156] 7. Translation of protein based psMHCI Ags into mRNA vaccines.

[0157] To aid the clinical translation of our vaccine (i.e. , the vaccine of the present disclosure), we sought to determine whether our psMHCl-based protein Ags could similarly elicit TCRL Abs as a mRNA vaccine. To this end, we encoded our psMHCl-Fc-Th construct displaying SIINFEKL (SEQ ID NO: 11) and PADRE sequences into a T7 plasmid flanked by a poly T tail that is 100 base pairs in length. Following in vitro transcription and purification, we first characterized our mRNA construct’s capacity to express functional Ags in vitro by encapsulating the purified mRNA with commercially available Lipofectamine MessengerMax and transfecting murine muscle cell line, C2C12, for 3 days. To assess for the secreted psMHCl-Fc-Th in the soluble milieu, we collected the supernatant and performed ELISA by using an anti-His Tag capture antibody to pull-down our Ags and biotinylated anti-SUNFEKL (SEQ ID NO: 11) H2-Kb antibody to verify the proper folding of the psMHCI domain (Fig. 8A). Our findings showed significantly higher signal within wells incubated with C2C12 transfected with our mRNA compared to non-transfected controls (Fig. 8B). Thus, we illustrate that the

[0158] RECTIFIED SHEET (RULE 91.1 ) mRNA encoded psMHCl-Fc-Th Ag of the present disclosure can still be produced and secreted, implicating its potential to elicit TCRL Abs irrespective of whether our Ag is protein or nucleic acid based.

[0159] Summary

[0160] We describe Ags, psMHCl-Th and psMHCl-Fc-Th, that induce TCRL Abs that can recognize a model SIINFEKL(SEQ ID NO: 11 ) peptide displayed in the MHCI complex of C57BL / 6 mice. The Abs induced by the Ags of the present disclosure were found to be highly specific with minimal off-target binding, illustrating the novel discovery that peptide self-MHCI molecules exhibit a unique immunogenicity profile that drives Ab responses towards the presented peptide. Moreover, the Ags of the present disclosure also enabled affinity maturation to occur within the GC and follicle targeted psMHCI Ags of the present disclosure generally showed effectiveness in significantly delaying tumor growth in vivo.

[0161] Advantages of the Ags of the present disclosure inclue: (i) the use of peptide self- MHCI complexes in a vaccine to induce polyclonal TCRL Abs, (ii) psMHCl-Th or psMHCl-Fc-Th or psMHCl-Th-Fc structure that facilitates the induction of TCRL Abs, (iii) application of this technology as a cancer immunotherapy, (iv) the use of the psMHCI complexes for targeted delivery to different cell types .

[0162] There are Human Leukocyte Ags (HLA) types that occur in high frequencies, such as HLA-A*0201 found in ~20% of individuals of African, European, Latino, and Native American descent.18By targeting these common HLA types, the vaccine of the present disclosure can still significantly impact the patient community. Moreover, given the single chain nature of the entire Ag of the present disclosure, we anticipate the capacity to use nucleic acid vaccines in place of manufactured proteins as the means of inducing TCRL Abs. In this manner, the technology of the present disclosure can have significant far-reaching impacts and could also be applicable in personalized

[0163] RECTIFIED SHEET (RULE 91.1 ) medicine where unique cancer peptides can be replaced in our modular Ag design to treat different patients.

[0164] Example 2 - Higher order multivalent displays on synthetic nanoparticles

[0165] Based on the findings that pHMCI-Fc-Th elicits a more potent TCRL Ab response than pHMCI-Th (see Fig. 3C, 3D), one can extrapolate that the display of pMHCI at higher valencies could further improve the induction of TCRL Abs. This is achieved through a number of different approaches starting with the genetic fusion of pMHCI (including psMHCI) to protein domains such as lumazine synthase, ferritin, or 153, all of which self-assemble into virus-like particles (VLPs). These VLPs will display on the order of 10s of pMHCI molecules, pMHCl-Fc-Th and pMHCl-Th and can be recombinantly expressed, isolated, and administered as a TCRL Ab inducing vaccine Ag of the present disclosure.

[0166] The pMHCI molecules of the present disclosure can also be multivalently displayed on synthetic nanoparticles to elicit TCRL Ab responses. This is achieved by first conjugating pMHCl-Th with heterobifunctional chemical linker that can then covalently react with surface functionalized nanoparticles, such as gold nanoparticles with surface amines. In addition, pMHCI of the present disclosure can be displayed on nucleic acid-based nanostructures such as DNA origami-based nanostructures. This is achieved by conjugating DNA strands onto the pMHCI of the present disclosure that can anneal to the origami structures. In these cases, the conjugation of DNA oligos or chemical linkers to pMHCI can occur in either a site specific or random manner. Following conjugation to the synthetic nanoparticles and removal of excess moieties, these particles can be administered for use as vaccine Ags.

[0167] Example 3 - Targeted delivery to cells

[0168] In addition to targeted delivery of pMHCl-Fc-Th to FDCs that successfully elicited TCRL Ab response, antibody mediated delivery of pMHCls to other cell types could also achieve similar responses. Other possible cell types include dendritic cells and

[0169] RECTIFIED SHEET (RULE 91.1 ) B cells that can be targeted through surface markers such as CD205 and major histocompatibility complex (II), respectively. To enable the delivery of pMHCI of the present disclosure to these cells, pMHCI is genetically fused to single chain variable fragments derived from monoclonal antibodies such as DEC205 to achieve a single chain construct similar to tarpH2Kb-Th or tarpH2Kb-Fc-Th (Fig. 3K). In this embodiment, reference number “25” would indicate the anti-CD205 single chain variable fragment. Alternatively, pMHCI can also be genetically fused to the C terminus of the antibody heavy chain and combined with the wild type light chain of the antibody or C terminus of the antibody light chain and combined with the wild type heavy chain of the antibody. These constructs will yield antibody-pMHCI fusion proteins that require two polypeptide chains to properly fold. Lastly, pMHCI of the present disclosure can also be chemically fused to antibodies to achieve targeted delivery. Upon generating these targeted Ag constructs, they can be administered to elicitTCRL Ab responses.

[0170] SEQUENCES pH2Kb-Th: (Mw: 50 kPa; SEQ ID NO: 1)

[0171] SIINFEKLGCGASGGGGSGGGGSIQKTPQIQVYSRHPPENGKPNILNCYVTQFHPP HIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILAHTEFTPTETDTYACRVKHASMAEP KTVYWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYM EVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRV DLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKT WTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDSPKA HVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVETRPAGDGTFQK WASVWPLGKEQYYTCHVYHQGLPEPLTLRWESTVSN pH2Kb-Fc-Th: (Mw: 180 kPa; SEQ ID NO: 2)

[0172] SIINFEKLGCGASGGGGSGGGGSIQKTPQIQVYSRHPPENGKPNILNCYVTQFHPP

[0173] HIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILAHTEFTPTETDTYACRVKHASMAEP

[0174] RECTIFIED SHEET (RULE 91.1 ) KTVYWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYM

[0175] EVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRV

[0176] DLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKT

[0177] WTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDSPKA

[0178] HVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVETRPAGDGTFQK

[0179] WASVWPLGKEQYYTCHVYHQGLPEPLTLRWEPPPSTVSNGGGSEPRVPITQNPC

[0180] PPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISW

[0181] FVNNVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMSGKEFKCKVNNRALPSPI

[0182] EKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQ

[0183] NYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSWHEGLHNHLTTKTISRSL

[0184] GKGGGGSGGGGSDIVMTQTPSSLAVSAGEKVTMSCKSSQSLLYSKNKKNYLAWY

[0185] QQKPGQSPKLLISWASSRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCEQYY

[0186] N I PYTFGGGTKLELKRGGHH H HH HGSAKFVAAWTLKAAA tarpH2Kb-Th: (Mw: 77 kDa; SEQ ID NO: 3)

[0187] SIINFEKLGCGASGGGGSGGGGSIQKTPQIQVYSRHPPENGKPNILNCYVTQFHPP

[0188] HIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILAHTEFTPTETDTYACRVKHASMAEP

[0189] KTVYWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYM

[0190] EVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRV

[0191] DLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKT

[0192] WTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDSPKA

[0193] HVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVETRPAGDGTFQK

[0194] WASWVPLGKEQYYTCHVYHQGLPEPLTLRWEPPPSTVSNGGHHHHHHGSAKFV

[0195] AAWTLKAAA tarpH2Kb-Th: (Mw: 207 kDa; SEQ ID NO: 4)

[0196] SIINFEKLGCGASGGGGSGGGGSIQKTPQIQVYSRHPPENGKPNILNCYVTQFHPP

[0197] HIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILAHTEFTPTETDTYACRVKHASMAEP

[0198] KTVYWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYM

[0199] EVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRV

[0200] RECTIFIED SHEET (RULE 91.1 ) DLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKT

[0201] WTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDSPKA HVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVETRPAGDGTFQK WASVWPLGKEQYYTCHVYHQGLPEPLTLRWEPPPSTVSNGGGSEPRVPITQNPC

[0202] PPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVWDVSEDDPDVQISW FVNNVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMSGKEFKCKVNNRALPSPI EKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQ

[0203] NYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSWHEGLHNHLTTKTISRSL

[0204] GKGGGGSGGGGSDIVMTQTPSSLAVSAGEKVTMSCKSSQSLLYSKNKKNYLAWY

[0205] QQKPGQSPKLLISWASSRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCEQYY

[0206] NIPYTFGGGTKLELKRGGGGSGGGGSGGGGSQVKLQESGGGLVQPGRSLKLSCA

[0207] ASGFTFSNYDMAVWRQAPTKGLEWVASINYDGSSTYYRDSVKGRFTISRDNAKST LYLQMDSLRSEDTATYYCTTLYNWYVMDAWGQGTTVTVSSGGHHHHHHGSAKFV AAWTLKAAA

[0208] Beta-2M and H2-Kb (SEQ ID NO: 5)

[0209] IQKTPQIQVYSRHPPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFS

[0210] KDWSFYILAHTEFTPTETDTYACRVKHASMAEPKTVYWDRDMGGGGSGGGGSGG

[0211] GGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYE

[0212] PRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGCYNQSKGGSHTIQVISG

[0213] CEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAER

[0214] LRAYLEGTCVEWLRRYLKNGNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYA

[0215] DITLTWQLNGEELIQDMELVETRPAGDGTFQKWASVWPLGKEQYYTCHVYHQGL PEPLTLRWESTVSN post pH2Kb linker in SEQ ID NO:1 (SEQ ID NO: 6)

[0216] GGGGSGGGGSGGGGS post pH2Kb linker in SEQ ID NO:2 (SEQ ID NO: 7)

[0217] RECTIFIED SHEET (RULE 91.1 ) GGGS murine lqG2c Fc domain in SEQ ID NO: 2 (SEQ ID NO: 8)

[0218] EPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCWVDVS EDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMSGKEFKCK VNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVD WTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHN

[0219] HLTTKTISRSLGK

[0220] Anti-murine CD35 single chain variable fragment (ScFv) in SEQ ID NO: 9

[0221] DIVMTQTPSSLAVSAGEKVTMSCKSSQSLLYSKNKKNYLAWYQQKPGQSPKLLIS WASSRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCEQYYNIPYTFGGGTKLE LKRGGGGSGGGGSGGGGSQVKLQESGGGLVQPGRSLKLSCAASGFTFSNYDMA WVRQAPTKGLEWVASINYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSED

[0222] TATYYCTTLYNWYVMDAWGQGTTVTVSS

[0223] PADRE Sequence (SEQ ID NO: 10)

[0224] AKFVAAWTLKAAA

[0225] Peptide from Ovalbumin (SEQ ID NO: 11)

[0226] SIINFEKL

[0227] Trp2180-188peptide (SEQ ID NO: 12)

[0228] SVYDFFVWL

[0229] Below are non-limiting example sequences of human version of the psMHCl-Fc-Th molecules. These constructs also show different single chain HLA designs with traps and stabilizing mutations.

[0230] RECTIFIED SHEET (RULE 91.1 ) pHLA-Fc-Th, version 1

[0231] Single chain mutant HLA-A*02:01 displaying NPM1 peptide fused to human lgG1 Fc domain and a T helper peptide (SEQ ID NO:13)

[0232] CLAVEEVSLGGGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGF HPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTL SQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEP RFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTL RVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKE DLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRT DAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAG DGTFQKWAAVWPSGQEQRYTCHVQHEGLPKPLTLRWEGGGSEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPELGS AKFVAAWTLKAAA

[0233] Peptide from NPM1 Type A Mutant (AML) (SEQ ID NO: 14)

[0234] CLAVEEVSL

[0235] Normal Linker (SEQ ID NO: 15)

[0236] GGGGSGGGGSGGGG

[0237] Human beta 2 macroglobulin (SEQ ID NO: 16)

[0238] IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFS

[0239] KDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

[0240] Human HLA-A*02:01 alpha chain; H74L and Y84A stabilizing mutations (SEQ ID

[0241] NO: 17)

[0242] GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQE

[0243] GPEYWDGETRKVKAHSQTLRVDLGTLRGAYNQSEAGSHTVQRMYGCDVGSDWR

[0244] FLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGT

[0245] CVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQ

[0246] RDGEDQTQDTELVETRPAGDGTFQKWAAVWPSGQEQRYTCHVQHEGLPKPLTL

[0247] RWE

[0248] Human lgG1 hinge (SEQ ID NO: 18)

[0249] EPKSCDKTHTCPPC

[0250] Human lgG1 CH2 domain (SEQ ID NO: 19)

[0251] RECTIFIED SHEET (RULE 91.1 ) PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH

[0252] NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0253] Human lgG1 CH3 domain (SEQ ID NO: 20)

[0254] GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP

[0255] PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPEL

[0256] T helper peptide (SEQ ID NO: 21)

[0257] AKFVAAWTLKAAA pHLA-Fc-Th, version 2

[0258] Single chain mutant HLA-A*02:01 (version 2) displaying Wilm’s Tumor 1 peptide fused to human I gG1 Fc domain and to a T helper peptide (SEQ ID NO: 22)

[0259] RMFPNAPYLGCGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGF HPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTL SQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEP RFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTL RVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKE DLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRT DAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAG DGTFQKWAAVWPSGQEQRYTCHVQHEGLPKPLTLRWEGGGSEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPELGS AKFVAAWTLKAAA

[0260] Peptide from Wilm’s Tumor 1 protein (SEQ ID NO: 23)

[0261] RMFPNAPYL

[0262] Disulfide trap linker (SEQ ID NO: 24)

[0263] GCGGSGGGGSGGGGS

[0264] Human beta 2 macroglobulin (SEQ ID NO: 16)

[0265] Human HLA-A*02:01 alpha chain; H74L stabilizing mutation and Y84C Disulfide Trap (SEQ ID NO: 25)

[0266] GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQE GPEYWDGETRKVKAHSQTLRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWR FLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGT CVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQ

[0267] RECTIFIED SHEET (RULE 91.1 ) RDGEDQTQDTELVETRPAGDGTFQKWAAVWPSGQEQRYTCHVQHEGLPKPLTL RWE

[0268] Human IgG 1 hinge (SEQ ID NO: 18)

[0269] Human IgG 1 CH2 domain (SEQ ID NO: 19)

[0270] Human IgG 1 CH3 domain (SEQ ID NO: 20)

[0271] T helper peptide (SEQ ID NO: 21) mRNA sequences

[0272] H2Kb-Fc-Th displaying SIINFEKL peptide with 100 base pair length polyA tail

[0273] (RNA sequence) (SEQ ID NO: 26)

[0274] AUGGAGACAGAUACCCUGCUCCUGUGGGUCUUGCUCUUGUGGGUACCUGGC

[0275] UCAACUGGATCTATTATTAATTTTGAGAAACTGGGUUGUGGGGCCUCAGGCGG

[0276] CGGGGGUUCUGGCGGUGGGGGCAGUAUACAAAAAACGCCCCAGAUACAAGU

[0277] AUACUCUAGACACCCGCCAGAAAAUGGGAAGCCCAAUAUCCUGAACUGCUAU

[0278] GUGACACAGUUUCAUCCCCCACAUAUAGAGAUACAGAUGUUGAAAAACGGUA

[0279] AAAAGAUCCCAAAAGUGGAAAUGUCCGACAUGAGCUUUAGUAAAGAUUGGUC

[0280] CUUCUACAUACUUGCUCAUACGGAGUUCACGCCCACGGAAACCGAUACUUAC

[0281] GCGUGCCGAGUGAAGCAUGCUUCCAUGGCAGAGCCCAAGACUGUUUACUGG

[0282] GACAGAGAUAUGGGUGGGGGUGGGUCAGGUGGCGGAGGAUCAGGAGGCGG

[0283] GGGCUCCGGAGGCGGCGGAUCUGGUCCUCAUUCUCUUAGAUACUUUGUUAC

[0284] AGCGGUCUCUCGGCCCGGGCUGGGGGAGCCGAGAUAUAUGGAAGUAGGCUA

[0285] UGUUGACGAUACCGAGUUUGUCCGAUUCGAUUCCGAUGCAGAAAAUCCGCGA

[0286] UAUGAGCCUCGAGCGCGGUGGAUGGAACAAGAGGGUCCGGAAUAUUGGGAG

[0287] CGGGAGACUCAGAAAGCAAAGGGGAAUGAGCAAUCAUUUAGAGUUGACUUGA

[0288] GAACGUUGUUGGGGUGUUAUAAUCAGAGCAAGGGCGGGUCACAUACGAUCC

[0289] AGGUUAUCUCUGGAUGCGAGGUCGGCUCUGACGGUCGAUUGUUGCGAGGCU

[0290] AUCAACAGUAUGCGUACGACGGUUGCGAUUAUAUUGCUCUCAACGAAGAUCU

[0291] UAAAACGUGGACGGCAGCUGACAUGGCUGCCUUGAUUACUAAACACAAGUGG

[0292] GAGCAAGCAGGAGAGGCCGAAAGAUUGCGGGCGUAUCUGGAGGGCACAUGU

[0293] GUAGAAUGGCUUAGACGCUACCUCAAAAAUGGCAAUGCCACACUCCUUCGGA

[0294] CCGACAGCCCAAAAGCGCACGUUACGCAUCACUCUAGGCCGGAAGACAAGGU

[0295] UACCCUCCGAUGCUGGGCCCUUGGAUUCUAUCCAGCAGACAUAACCCUCACC

[0296] UGGCAGCUUAACGGCGAAGAGCUGAUACAGGAUAUGGAACUCGUGGAGACLIA

[0297] GACCAGCAGGCGAUGGUACGUUCCAAAAAUGGGCUUCAGUUGUCGUACCGU

[0298] UGGGCAAGGAACAGUAUUAUACGUGCCACGUAUAUCACCAGGGAUUGCCUGA

[0299] ACCUUUGACCCUGAGGUGGGAACCUCCACCUAGUACCGUCUCUAAUGGGGG

[0300] UGGCUCAGAACCUCGGGUCCCUAUUACCCAAAACCCCUGCCCGCCAUUGAAG

[0301] GAAUGCCCUCCUUGUGCUGCACCGGACUUGUUGGGCGGUCCAAGUGUUUUU

[0302] AUAUUCCCGCCGAAAAUAAAAGACGUCUUGAUGAUCUCACUCAGUCCAAUGG

[0303] UCACCUGCGUCGUUGUCGAUGUUAGCGAAGAUGACCCUGACGUUCAGAUUA

[0304] RECTIFIED SHEET (RULE 91.1 ) GCUGGUUUGUUAACAACGUCGAGGUCCAUACGGCACAAACACAAACUCACAG

[0305] GGAAGACUACAACAGUACGCUCCGGGUGGUGUCAGCGCUCCCCAUACAGCAC

[0306] CAAGAUUGGAUGUCAGGUAAGGAGUUUAAGUGUAAGGUCAAUAAUAGAGCCU

[0307] UGCCGUCCCCGAUUGAAAAGACUAUCAGCAAACCUCGGGGGCCCGUUAGAGC

[0308] CCCACAAGUCUACGUUCUGCCACCCCCGGCUGAGGAAAUGACGAAAAAGGAA

[0309] UUUAGUCUCACAUGUAUGAUUACAGGGUUCCUUCCUGCUGAAAUAGCUGUUG

[0310] ACUGGACGUCUAACGGCAGGACCGAGCAAAAUUAUAAAAAUACUGCCACCGU

[0311] CUUGGAUAGUGAUGGCAGUUAUUUUAUGUAUAGUAAGCUCAGGGUGCAAAAA

[0312] UCUACAUGGGAGCGCGGCAGCUUGUUCGCGUGUAGUGUUGUCCACGAGGGG

[0313] CUGCAUAAUCACCUGACGACUAAAACUAUUUCCCGCUCUCUGGGAAAAGGAA

[0314] GUGCGAAAUUUGUGGCCGCUUGGACAUUGAAGGCAGCAGCGTAGTGAUGUA

[0315] CAAGUAGUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUU

[0316] CUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG

[0317] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0318] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0319] I g -Kappa Signal Peptide (RNA sequence) (SEQ ID NO: 27)

[0320] AUGGAGACAGAUACCCUGCUCCUGUGGGUCUUGCUCUUGUGGGUACCUGGC UCAACUGGA

[0321] SIINFEKL (SEQ ID NO: 11) peptide (RNA sequence) (SEQ ID NO: 28)

[0322] TCTATTATTAATTTTGAGAAACTG

[0323] Disulfide trap Linker (RNA sequence) (SEQ ID NO: 29)

[0324] GGUUGUGGGGCCUCAGGCGGCGGGGGUUCUGGCGGUGGGGGCAGU

[0325] Mouse beta-2m (RNA sequence) (SEQ ID NO: 30)

[0326] AUACAAAAAACGCCCCAGAUACAAGUAUACUCUAGACACCCGCCAGAAAAUGG

[0327] GAAGCCCAAUAUCCUGAACUGCUAUGUGACACAGUUUCAUCCCCCACAUAUA

[0328] GAGAUACAGAUGUUGAAAAACGGUAAAAAGAUCCCAAAAGUGGAAAUGUCCG

[0329] ACAUGAGCUUUAGUAAAGAUUGGUCCUUCUACAUACUUGCUCAUACGGAGUU

[0330] CACGCCCACGGAAACCGAUACUUACGCGUGCCGAGUGAAGCAUGCUUCCAUG

[0331] GCAGAGCCCAAGACUGUUUACUGGGACAGAGAUAUG

[0332] Long flexible linker (RNA sequence) (SEQ ID NO: 31)

[0333] GGUGGGGGUGGGUCAGGUGGCGGAGGAUCAGGAGGCGGGGGCUCCGGAGG CGGCGGAUCU

[0334] Mouse H2-Kb, Y84C mutation (RNA sequence) (SEQ ID NO: 32)

[0335] GGUCCUCAUUCUCUUAGAUACUUUGUUACAGCGGUCUCUCGGCCCGGGCUG

[0336] GGGGAGCCGAGAUAUAUGGAAGUAGGCUAUGUUGACGAUACCGAGUUUGUC

[0337] CGAUUCGAUUCCGAUGCAGAAAAUCCGCGAUAUGAGCCUCGAGCGCGGUGG

[0338] AUGGAACAAGAGGGUCCGGAAUAUUGGGAGCGGGAGACUCAGAAAGCAAAGG

[0339] GGAAUGAGCAAUCAUUUAGAGUUGACUUGAGAACGUUGUUGGGGUGUUAUAA

[0340] RECTIFIED SHEET (RULE 91.1 ) UCAGAGCAAGGGCGGGUCACAUACGAUCCAGGUUAUCUCUGGAUGCGAGGU CGGCUCUGACGGUCGAUUGUUGCGAGGCUAUCAACAGUAUGCGUACGACGG UUGCGAUUAUAUUGCUCUCAACGAAGAUCUUAAAACGUGGACGGCAGCUGAC AUGGCUGCCUUGAUUACUAAACACAAGUGGGAGCAAGCAGGAGAGGCCGAAA

[0341] GAUUGCGGGCGUAUCUGGAGGGCACAUGUGUAGAAUGGCUUAGACGCUACC UCAAAAAUGGCAAUGCCACACUCCUUCGGACCGACAGCCCAAAAGCGCACGU UACGCAUCACUCUAGGCCGGAAGACAAGGUUACCCUCCGAUGCUGGGCCCU UGGAUUCUAUCCAGCAGACAUAACCCUCACCUGGCAGCUUAACGGCGAAGAG

[0342] CUGAUACAGGAUAUGGAACUCGUGGAGACUAGACCAGCAGGCGAUGGUACG UUCCAAAAAUGGGCUUCAGUUGUCGUACCGUUGGGCAAGGAACAGUAUUAUA

[0343] CGUGCCACGUAUAUCACCAGGGAUUGCCUGAACCUUUGACCCUGAGGUGGG AACCUCCACCUAGUACCGUCUCUAAU

[0344] Short flexible linker (RNA sequence) (SEQ ID NO: 33)

[0345] GGGGGUGGCUCA

[0346] Murine lgG2c Fc Domain (RNA sequence) (SEQ ID NO: 34)

[0347] GAACCUCGGGUCCCUAUUACCCAAAACCCCUGCCCGCCAUUGAAGGAAUGCC

[0348] CUCCUUGUGCUGCACCGGACUUGUUGGGCGGUCCAAGUGUUUUUAUAUUCC

[0349] CGCCGAAAAUAAAAGACGUCUUGAUGAUCUCACUCAGUCCAAUGGUCACCUG

[0350] CGUCGUUGUCGAUGUUAGCGAAGAUGACCCUGACGUUCAGAUUAGCUGGUU UGUUAACAACGUCGAGGUCCAUACGGCACAAACACAAACUCACAGGGAAGAC UACAACAGUACGCUCCGGGUGGUGUCAGCGCUCCCCAUACAGCACCAAGAUU

[0351] GGAUGUCAGGUAAGGAGUUUAAGUGUAAGGUCAAUAAUAGAGCCUUGCCGUC CCCGAUUGAAAAGACUAUCAGCAAACCUCGGGGGCCCGUUAGAGCCCCACAA GUCUACGUUCUGCCACCCCCGGCUGAGGAAAUGACGAAAAAGGAAUUUAGUC UCACAUGUAUGAUUACAGGGUUCCUUCCUGCUGAAAUAGCUGUUGACUGGAC

[0352] GUCUAACGGCAGGACCGAGCAAAAUUAUAAAAAUACUGCCACCGUCUUGGAU AGUGAUGGCAGU U AU U U UAU GUAU AG UAAGCUCAGGGU GCAAAAAUCUACAU GGGAGCGCGGCAGCUUGUUCGCGUGUAGUGUUGUCCACGAGGGGCUGCAUA AUCACCUGACGACUAAAACUAUUUCCCGCUCUCUGGGAAAA

[0353] Amino acid ‘GS’ (RNA sequence) (SEQ ID NO: 35) GGAAGU

[0354] PADRE Sequence (RNA sequence) (SEQ ID NO: 36)

[0355] GCGAAAUUUGUGGCCGCUUGGACAUUGAAGGCAGCAGCG

[0356] Stop Codons (RNA) (SEQ ID NO: 37)

[0357] UAGUGA

[0358] 3’UTRs (RNA) (SEQ ID NO: 38)

[0359] RECTIFIED SHEET (RULE 91.1 ) UGUACAAGUAGUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCU UCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAG GAAG

[0360] PolyA Tail (RNA) (SEQ ID NO: 39)

[0361] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0362] Protein sequence (SEQ ID NO: 40)

[0363] METDTLLLWVLLLWVPGSTGSIINFEKLGCGASGGGGSGGGGSIQKTPQIQVYSRH PPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILAHTEF TPTETDTYACRVKHASMAEPKTVYWDRDMGGGGSGGGGSGGGGSGGGGSIQKT PQIQVYSRHPPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDW SFYILAHTEFTPTETDTYACRVKHASMAEPKTVYWDRDMGGGSGPHSLRYFVTAV SRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQ KAKGNEQSFRVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDG CDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNG NATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELV ETRPAGDGTFQKWASWVPLGKEQYYTCHVYHQGLPEPLTLRWEPPPSTVSNGS AKFVAAWTLKAAA

[0364] Ig-Kappa Signal Peptide (SEQ ID NO: 41)

[0365] METDTLLLWVLLLWVPGSTG

[0366] Disulfide trap Linker (SEQ ID NO: 42)

[0367] GCGASGGGGSGGGGS

[0368] Mouse beta-2m (SEQ ID NO: 43)

[0369] IQKTPQIQVYSRHPPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFS

[0370] KDWSFYILAHTEFTPTETDTYACRVKHASMAEPKTVYWDRDM

[0371] Long flexible linker (SEQ ID NO: 44)

[0372] GGGGSGGGGSGGGGSGGGGS

[0373] Mouse H2-Kb, Y84C mutation (SEQ ID NO: 45)

[0374] IQKTPQIQVYSRHPPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFS

[0375] KDWSFYILAHTEFTPTETDTYACRVKHASMAEPKTVYWDRDM

[0376] Short Flexible linker (SEQ ID NO: 46)

[0377] GGGS

[0378] Murine lgG2c Fc Domain (SEQ ID NO: 47)

[0379] RECTIFIED SHEET (RULE 91.1 ) GPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQ

[0380] EGPEYWERETQKAKGNEQSFRVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRL

[0381] LRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTC

[0382] VEWLRRYLKNGNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQL NGEELIQDMELVETRPAGDGTFQKWASWVPLGKEQYYTCHVYHQGLPEPLTLRW EPPPSTVSN

[0383] CONSTANTS OF THE Ag PLATFORM OF THE PRESENT DISCLOSURE Sequence of N terminal linker single chain H2-Kb (with beta-2m) with murine lgG2c Fc domain followed by PADRE

[0384] - Protein Sequence (SEQ ID NO: 48)

[0385] GCGASGGGGSGGGGSIQKTPQIQVYSRHPPENGKPNILNCYVTQFHPPHIEIQMLK NGKKIPKVEMSDMSFSKDWSFYILAHTEFTPTETDTYACRVKHASMAEPKTVYWD RDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVD

[0386] DTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLG CYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADM AALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDSPKAHVTHHS RPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVETRPAGDGTFQKWASW VPLGKEQYYTCHVYHQGLPEPLTLRWEPPPSTVSNGGGSEPRVPITQNPCPPLKE CPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCWVDVSEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRWSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISK PRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNT ATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGKGG GGSGGGGSDIVMTQTPSSLAVSAGEKVTMSCKSSQSLLYSKNKKNYLAWYQQKP GQSPKLLISWASSRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCEQYYNIPYT FGGGTKLELKRGSAKFVAAWTLKAAA

[0387] - mRNA equivalent sequence with 3’untranslated region and polyA tail (SEQ ID NO: 49)

[0388] AUGGAGACAGAUACCCUGCUCCUGUGGGUCUUGCUCUUGUGGGUACCUGGC UCAACUGGATCTATTATTAATTTTGAGAAACTGGGUUGUGGGGCCUCAGGCGG CGGGGGUUCUGGCGGUGGGGGCAGUAUACAAAAAACGCCCCAGAUACAAGU AUACUCUAGACACCCGCCAGAAAAUGGGAAGCCCAAUAUCCUGAACUGCUAU GUGACACAGUUUCAUCCCCCACAUAUAGAGAUACAGAUGUUGAAAAACGGUA AAAAGAUCCCAAAAGUGGAAAUGUCCGACAUGAGCUUUAGUAAAGAUUGGUC CUUCUACAUACUUGCUCAUACGGAGUUCACGCCCACGGAAACCGAUACUUAC GCGUGCCGAGUGAAGCAUGCUUCCAUGGCAGAGCCCAAGACUGUUUACUGG GACAGAGAUAUGGGUGGGGGUGGGUCAGGUGGCGGAGGAUCAGGAGGCGG GGGCUCCGGAGGCGGCGGAUCUGGUCCUCAUUCUCUUAGAUACUUUGUUAC AGCGGUCUCUCGGCCCGGGCUGGGGGAGCCGAGAUAUAUGGAAGUAGGCUA UGUUGACGAUACCGAGUUUGUCCGAUUCGAUUCCGAUGCAGAAAAUCCGCGA UAUGAGCCUCGAGCGCGGUGGAUGGAACAAGAGGGUCCGGAAUAUUGGGAG

[0389] RECTIFIED SHEET (RULE 91.1 ) CGGGAGACUCAGAAAGCAAAGGGGAAUGAGCAAUCAUUUAGAGUUGACUUGA GAACGUUGUUGGGGUGUUAUAAUCAGAGCAAGGGCGGGUCACAUACGAUCC AGGUUAUCUCUGGAUGCGAGGUCGGCUCUGACGGUCGAUUGUUGCGAGGCU AUCAACAGUAUGCGUACGACGGUUGCGAUUAUAUUGCUCUCAACGAAGAUCU UAAAACGUGGACGGCAGCUGACAUGGCUGCCUUGAUUACUAAACACAAGUGG GAGCAAGCAGGAGAGGCCGAAAGAUUGCGGGCGUAUCUGGAGGGCACAUGU GUAGAAUGGCUUAGACGCUACCUCAAAAAUGGCAAUGCCACACUCCUUCGGA CCGACAGCCCAAAAGCGCACGUUACGCAUCACUCUAGGCCGGAAGACAAGGU

[0390] UACCCUCCGAUGCUGGGCCCUUGGAUUCUAUCCAGCAGACAUAACCCUCACC UGGCAGCUUAACGGCGAAGAGCUGAUACAGGAUAUGGAACUCGUGGAGACUA GACCAGCAGGCGAUGGUACGUUCCAAAAAUGGGCUUCAGUUGUCGUACCGU UGGGCAAGGAACAGUAUUAUACGUGCCACGUAUAUCACCAGGGAUUGCCUGA ACCUUUGACCCUGAGGUGGGAACCUCCACCUAGUACCGUCUCUAAUGGGGG UGGCUCAGAACCUCGGGUCCCUAUUACCCAAAACCCCUGCCCGCCAUUGAAG GAAUGCCCUCCUUGUGCUGCACCGGACUUGUUGGGCGGUCCAAGUGUUUUU AUAUUCCCGCCGAAAAUAAAAGACGUCUUGAUGAUCUCACUCAGUCCAAUGG

[0391] UCACCUGCGUCGUUGUCGAUGUUAGCGAAGAUGACCCUGACGUUCAGAUUA GCUGGUUUGUUAACAACGUCGAGGUCCAUACGGCACAAACACAAACUCACAG

[0392] GGAAGACUACAACAGUACGCUCCGGGUGGUGUCAGCGCUCCCCAUACAGCAC CAAGAUUGGAUGUCAGGUAAGGAGUUUAAGUGUAAGGUCAAUAAUAGAGCCU UGCCGUCCCCGAUUGAAAAGACUAUCAGCAAACCUCGGGGGCCCGUUAGAGC CCCACAAGUCUACGUUCUGCCACCCCCGGCUGAGGAAAUGACGAAAAAGGAA UUUAGUCUCACAUGUAUGAUUACAGGGUUCCUUCCUGCUGAAAUAGCUGUUG ACUGGACGUCUAACGGCAGGACCGAGCAAAAUUAUAAAAAUACUGCCACCGU CUUGGAUAGUGAUGGCAGUUAUUUUAUGUAUAGUAAGCUCAGGGUGCAAAAA UCUACAUGGGAGCGCGGCAGCUUGUUCGCGUGUAGUGUUGUCCACGAGGGG

[0393] CUGCAUAAUCACCUGACGACUAAAACUAUUUCCCGCUCUCUGGGAAAAGGAA G U GCGAAAU U UG U GGCCGC U U GGACAU UGAAGGCAGCAGCGTAGT GAUG U A CAAGUAGUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUU CUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

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[0423] Although various embodiments of the disclosure have been described and illustrated, it will be apparent to those skilled in the art in light of the present description that numerous modifications and variations can be made. All publications and the priority document are hereby incorporated by reference. The scope of the invention is defined more particularly in the appended claims.

[0424] RECTIFIED SHEET (RULE 91.1 )

Claims

CLAIMSWhat is claimed is:1 . A single chain polypeptide comprising a major histocompatibility complex I (MHCI) molecule and a T helper epitope linked to the C-terminus of the MHCI molecule.

2. The single chain polypeptide of claim 1 , wherein the single chain polypeptide further comprises an Fc domain of an immunoglobulin linked to the C-terminus of the MHCI molecule.

3. The single chain polypeptide of claim 2, wherein the T helper epitope is linked to the C-terminus of the Fc domain (MHCI-Fc-Th).

4. The single chain polypeptide of claim 2, wherein the T helper epitope is placed between the C-terminus of the MHCI molecule and the Fc domain (MHCI-Th-Fc).

5. The single chain polypeptide of claim 1 , wherein the polypeptide is a multivalent single chain polypeptide comprising two or more p-MHCI molecules, a Fc domain of an immunoglobulin linked to C-terminus of the two or more p-MHCI molecules and the T helper epitope linked to the C-terminus of the Fc domain.

6. The single chain polypeptide according to any one of claim 1 to 5, wherein the single chain polypeptide further comprises a single-chain variable fragment (scFv).

7. The single chain polypeptide according to any one of claims 1 to 6, wherein the MHCI molecule is a human leukocyte antigen (HLA) class I molecule.

8. The single chain polypeptide according to any one of claims 1 to 7, wherein the MHCI molecule is a peptide MHCI (p-MHCI) molecule.

9. The single chain polypeptide of claim 8, wherein the p-MHCI is a peptide self-major histocompatibility complex I (psMHCI).

10. The single chain polypeptide according to any one of claims 1 to 9, wherein the single chain polypeptide is presented in higher order multivalent displays on synthetic nano-particles and / or nanofibers.

11. The single chain polypeptide of claim 1 , wherein the single chain polypeptide comprises (i) SEQ ID NO: 17 linked to SEQ ID NO: 21 , or (ii) SEQ ID NO: 25 linked to SEQ ID NO: 21.

12. The single chain polypeptide of claim 2, wherein the single chain polypeptide comprises SEQ ID NO: 17 linked to SEQ ID NOs: 19, 20 and 21 , or (ii) SEQ ID NO: 25 linked to SEQ ID NOs: 19, 20 and 21.

13. An engineered nucleic acid polynucleotide encoding the single chain polypeptide according to any one of claims 1 to 12.

14. The engineered nucleic acid polynucleotide of claim 13, wherein the nucleic acid polynucleotide is a deoxyribonucleic acid (DNA) polynucleotide or a messenger ribonucleic acid (mRNA) polynucleotide.

15. An immunogenic composition comprising antigens (Ags), wherein each Ag comprises a single chain polypeptide comprising a peptide major histocompatibility complex I (p-MHCI) molecule and a T helper epitope linked to the C-terminus of the major histocompatibility complex I (MHCI) molecule, wherein the peptide is known to be displayed on the MHCI molecule.

16. The immunogenic composition of claim 15, wherein the single chain polypeptide further comprises an Fc domain of an immunoglobulin (p-MHCl-Fc-Th) linked to the C-terminus of the MHCI molecule.

17. The immunogenic composition of claim 16, wherein the T helper epitope is linked to the C-terminus of the Fc domain.

18. The immunogenic composition of claim 17, wherein the T helper epitope is placed between the C-terminus of the MHCI molecule and the Fc domain.

19. The immunogenic composition of claim 15, wherein the single chain polypeptide is a multivalent fused polypeptide comprising two or more p-MHCI molecules, a Fc domain of an immunoglobulin linked to C-terminus of the two or more p-MHCI molecules and the T helper epitope linked to the C-terminus of the Fc domain.

20. The immunogenic composition according to any one of claims 15 to 19, wherein the single chain polypeptide further comprises a single-chain variable fragment (scFv).

21. The immunogenic composition according to any one of claim 16 to 20, wherein the immunogenic composition is provided in the form of a messenger ribonucleic acid (mRNA) vaccine composition comprising: (a) an mRNA polynucleotide comprising an open reading frame encoding the Ags; and (b) an agent used to introduce the mRNA into cells.

22. The immunogenic composition according to any one of claims 15 to 20, wherein the antigens are presented in higher order multivalent displays on synthetic nanoparticles, nano-fibers, viral vectors, liposomes and / or nucleic acid origami.

23. The immunogenic composition according to any one of claim 15 to 22 wherein immunogenic composition further comprises an adjuvant.

24. The immunogenic composition according to any one of claims 15 to 23, wherein the MHCI molecule is a human leukocyte antigen (HLA) class I molecule.

25. The immunogenic composition according to any one of claims 15 to 24, wherein the p-MHCI is a peptide self-major histocompatibility complex I (psMHCI).

26. A method of inducing polyclonal T cell receptor like (TCRL) antibodies (Abs) in a subject, the method comprising, administering a subject the immunogenic composition according to any one of claim 15 to 24, wherein the peptide is a target moietyrecognized by the induced polyclonal Abs, and wherein the MHCI is a self-MHCI of the subject’s species.

27. The method of claim 26, wherein the subject is a cancer patient, and the peptide is a cancer-associated antigen presented on cancer cells, and wherein the cancer is a hematological malignancy or a solid tumor cancer.

28. The method of claim 26, wherein the subject is infected by a pathogen and the peptide is derived from the pathogen.

29. The method according to any one of claims 26 to 28, wherein the immunogenic composition is administered in the form of a messenger ribonucleic acid (mRNA) vaccine composition comprising; (a) an mRNA polynucleotide comprising an open reading frame encoding the Ag; and (b) a transfection agent used to introduce the mRNA into cells.

30. The method according to any one of claims 26 to 29, wherein the subject is a human.

31. The immunogenic composition according to any one of claims 15 to 24 for use in inducing polyclonal T cell receptor like (TCRL) antibodies (Abs) in a subject, wherein the peptide is a target moiety recognized by the induced polyclonal Abs and wherein the MHCI is a self-MHCI of the subject’s species.

32. The immunogenic composition for use of claim 31 , wherein the subject is a cancer patient, the peptide is a tumor-associated antigen presented on cancer cells, and wherein the cancer is a hematological malignancy or a solid tumor cancer.

33. The immunogenic composition for use according to any one of claim 31 to 32, wherein subject is infected by a pathogen and the peptide is derived from the pathogen.

34. The immunogenic composition for use according to any one of claim 31 to 33, wherein the immunogenic composition is in the form of a messenger ribonucleic acid (mRNA) vaccine composition comprising; (a) an mRNA polynucleotide comprising an open reading frame encoding the Ag; and (b) an agent used to introduce the mRNA into cells.

35. The immunogenic composition for use according to any one of claims 31 to 34, wherein the subject is a human.

36. An in vitro method of raising polyclonal T cell receptor like (TCRL) antibodies (Abs), the method comprising, contacting B cells with the immunogenic composition according to any one of claim 16 to 25, wherein the peptide is a target moiety recognized by the induced polyclonal Abs, and wherein the MHCI is a self-MHCI of the B cell’s species.

37. The method of claim 36, wherein the peptide is a cancer-associated antigen presented on cancer cells, and wherein the cancer is a hematological malignancy or a solid tumor cancer.

38. The method of claim 36, wherein the peptide is derived from a pathogen.

39. The method according to any one of claims 36 to 38, wherein the immunogenic composition is provided in the form of a messenger ribonucleic acid (mRNA) composition comprising: (a) an mRNA polynucleotide comprising an open reading frame encoding the Ag; and (b) an agent used to introduce the mRNA into cells.

40. The method according to any one of claims 36 to 39, wherein the subject is a human.

41. A method of delivering the single chain polypeptide of claim 1 to a target cell, the method comprising (a) fusing to the single chain polypeptide a ligand that recognizes a marker on the surface of the target cell to form a fusion polypeptide, and (b) contacting the target cell with the fusion polypeptide.

42. The method of claim 41 , wherein the ligand is a single chain variable fragment (scFv) that contains the antigen-binding domains of the heavy (VH) and light (VL) chains of an antibody that recognizes the marker on the surface of the target cell.

43. The method according to any one of claims 41 and 42, wherein: - the target cell is a follicular dendritic cell (FDC) and the marker on the surface of theFDC is a complement receptor 1 (CD35), or- the target cell is a dendritic cell and the marker on the surface of the dendritic cell is CD205, or- the target cell is a B cell, and the marker on the surface of the B cell is a major histocompatibility complex (II).

44. A nanoparticle carrying multiple copies of the single chain polypeptide according to any one of claims 1 to 12.

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