Augmentation of t and b cell activation with specific cysteine protease linker sequences

Abstract

Provided herein are fusion proteins comprising antigens (e.g., cancer neoantigens) conjugated to localization domains and / or self-proteins through an amino acid linker sequence that is preferentially cleaved by S-cathepsin, and methods of use thereof.

Description

[0001] AUGMENTATION OF T AND B CELL ACTIVATION WITH SPECIFIC CYSTEINE PROTEASE LINKER SEQUENCES

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 595,844, filed November 3, 2023, the entire contents of which are incorporated by reference herein.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (M065670541WO00-SEQ-MAT.xml;

[0005] Size: 20,746 bytes; and Date of Creation: August 27, 2024) is incorporated by reference herein in its entirety.

[0006] BACKGROUND OF INVENTION

[0007] Somatic mutations are a common feature in many cancer types, including lung carcinomas, which feature considerable mutational burden (4.21 mutations / Mb of DNA) (1). Apoptotic or necrotic cancer cells release mutated proteins that can be taken up by antigen- presenting cells (APCs) and presented as neoantigen-derived peptides by MHC Class II (MHC- II) and MHC Class I (MHC-I) molecules to CD4+and CD8+T cells, respectively (2). Initial neoantigen presentation to antigen- specific CD8+or CD4+T cells via APCs in secondary lymphoid organs such as lymph nodes, results in robust activation and can drive tumor cell clearance (3,4,5).

[0008] Peptide-MHC-I or peptide-MHC-II complexes on the surface of APCs drive strong priming and activating signals for CD8+and CD4+T cells, respectively. CD4+T cells activation can be sensitive to antigen expression levels and subsequent intracellular antigen processing. Unlike proteasome-driven processing of peptides for MHC-I loading, peptide-MHC-II presentation occurs through a coordinated series of endosomic fusion events that are dependent on the activation state of the cell and the expression of factors that regulate presentation.

[0009] Antigen that is taken up by activated APCs through endocytic vesicles is subjected to a class of proteases, cathepsins, that stochastically fragment protein material into peptides suitable for loading onto MHC-II (6). Altering the nature of the up-taken protein has been shown to greatly influence antigen processing and the resultant CD4+T cell activation. SUMMARY OF INVENTION

[0010] Provided herein are fusion proteins designed to be preferentially cleaved by certain cathepsin proteases. When these fusion proteins are used, a robust expansion of antigen specific CD4+T cells is observed specifically when implementing an optimized S-cathepsin cleavage site as a linker that conjugates the domains of the fusion protein. Surprisingly, the addition of elements that improve MHC-II antigen presentation also resulted in improved endogenous CD4+and CD8+T cell responses at early and late tumor timepoints in both draining lymph nodes and in tumor-bearing lung in the tested model systems.

[0011] Also provided herein are fusion proteins that tether a non-immunogenic protein (or peptide) to a highly immunogenic CD4+T cell epitope. These fusion proteins generate a robust antibody response against the non-immunogenic protein (or peptide).

[0012] The optimized S-cathepsin linkers provided herein in conjugation with CD4+epitopes have profound implications for T cell and antibody-based vaccinations. mRNA vaccination benefit from the modular fusion protein design that allows for addition of these cleavage sites and is capable of inducing robust CD4+T cell response against known immunogenic peptides or strong antibody titer against any protein of interest including those previously considered non- immunogenic. This disclosure demonstrates that optimized S-cathepsin cleavage sites improve mRNA-based vaccination which in turn results in improved tumor clearance.

[0013] Thus, this disclosure provides in one aspect a fusion protein comprising a localization domain conjugated to an antigen (such as an antigenic peptide) through a linker that comprises an amino acid sequence of DVQAVG-GGDHDH (SEQ ID NO: 1) or a functional variant thereof.

[0014] This disclosure further provides in one aspect a fusion protein comprising a localization domain conjugated to a cancer neoantigen (including a neoantigenic peptide) through a linker that comprises an amino acid sequence of DVQAVG-GGDHDH (SEQ ID NO: 1) or a functional variant thereof.

[0015] The disclosure provides in another aspect a method of stimulating an immune response in a subject comprising administering to a subject in need thereof a fusion protein comprising a localization domain conjugated to a cancer neoantigen (or cancer neoantigenic peptide), through a linker that comprises an amino acid sequence comprising DVQAVGGGDHDH (SEQ ID NO: 1) or a functional variant thereof in an amount effective to stimulate an immune response to the cancer neoantigen. The fusion protein may be administered in its protein form. Alternatively, the subject may be administered an RNA vaccine comprising an RNA that encodes the fusion protein, optionally formulated with lipids, further optionally in a lipid nanoparticle.

[0016] In some embodiments, the immune response is a CD4+T cell response.

[0017] In some embodiments, the subject has cancer. In some embodiments, the subject has increased risk of developing cancer compared to a control population.

[0018] The disclosure provides in another aspect a fusion protein comprising a self-protein conjugated to a cancer neoantigen through a linker that comprises an amino acid sequence comprising DVQAVGGGDHDH (SEQ ID NO: 1) or a functional variant thereof.

[0019] In some embodiments, the self-protein is overexpressed in cancer cells compared to noncancer cells in the subject. In some embodiments, the self-protein is mesothelin. In some embodiments, the self-protein is mucin- 1. In some embodiments, the cancer neoantigen is a CD4+cancer neoantigen.

[0020] The disclosure provides in another aspect a nucleic acid, such as but not limited to an RNA, encoding any of the foregoing fusion proteins.

[0021] The disclosure provides in another aspect a composition comprising a nucleic acid, such as but not limited to an RNA, encoding any of the foregoing fusion proteins formulated in a lipid nanoparticle. In some embodiments, the composition is formulated for intramuscular administration.

[0022] The disclosure provides in another aspect a method of stimulating an immune response in a subject comprising administering to a subject in need thereof any of the foregoing fusion proteins, for example in protein form or as a nucleic acid that encodes the fusion protein, such as an RNA, in an amount effective to stimulate an immune response to the cancer neoantigen (including neoantigenic peptide) and / or the self-protein. The RNA many be administered as a vaccine that comprises the RNA optionally formulated with lipids, further optionally in a lipid nanoparticle.

[0023] The disclosure provides in another aspect a method of stimulating an immune response in a subject comprising administering to a subject in need thereof an mRNA encoding a fusion protein comprising a self-protein conjugated to an immunogenic antigen, through a linker that comprises an amino acid sequence comprising DVQAVGGGDHDH (SEQ ID NO: 1) or a functional variant thereof in an amount effective to stimulate an immune response to selfprotein. In some embodiments, the immune response to the self-protein comprises a B-cell immune response or an antibody response. In some embodiments, the immune response comprises a CD4+T cell response. In some embodiments, the subject has cancer. In some embodiments, the subject has increased risk of developing cancer compared to a control population.

[0024] Various embodiments relate equally to the foregoing aspects and these are recited below.

[0025] In some embodiments, the cancer neoantigen (including a cancer neoantigenic peptide) is a MHC Class II presented cancer neoantigen (including a MHC Class II presented neoantigenic peptide). Such peptides are presented to CD4+T cells and they may be referred to herein as CD4+T cell neoantigens (including neoantigenic peptides) or CD4+ neoantigens (including neoantigenic peptides). In some embodiments, the cancer neoantigen is a mutant KRAS neoantigen.

[0026] In some embodiments, the fusion protein may further comprise a MHC Class I presented cancer neoantigen. Such peptides are presented to CD8+T cells and they may be referred to herein as CD8+T cell neoantigens (including neoantigenic peptides) or CD8+neoantigens (including neoantigenic peptides).

[0027] In some embodiments, the localization domain is a truncated transferrin receptor comprising a transmembrane domain and / or a cytosolic domain.

[0028] In some embodiments, the linker comprises an amino acid sequence of DVQAVGGGDHDH (SEQ ID NO: 1). In some embodiments, the linker consists of an amino acid sequence of DVQAVGGGDHDH (SEQ ID NO: 1). In some embodiments, the linker is a functional variant of SEQ ID NO:1. In some embodiments, the linker comprises an amino acid sequence of DVQAVGGGDHDH (SEQ ID NO: 1) with one amino acid substitution. In some embodiments, the linker comprises an amino acid sequence of XVQAVGGGDHDH (SEQ ID NO: 2), DXQAVGGGDHDH (SEQ ID NO: 3), DVXAVGGGDHDH (SEQ ID NO: 4), DVQXVGGGDHDH (SEQ ID NO: 5), DVQAVXGGDHDH (SEQ ID NO: 6), DVQAVGXGDHDH (SEQ ID NO: 7), DVQAVGGXDHDH (SEQ ID NO: 8), DVQAVGGGXHDH (SEQ ID NO: 9), DVQAVGGGDXDH (SEQ ID NO: 10), DVQAVGGGDHXH (SEQ ID NO: 11), or DVQAVGGGDHDX (SEQ ID NO: 12). In some embodiments, the linker comprises an amino acid sequence of DVQAXGGGDHDH, where X is selected from valine, isoleucine, leucine or methionine (SEQ ID NO: 13). In some embodiments, the linker comprises an amino acid sequence of DVQAVXGGDHDH, where X is selected from glycine or glutamine (SEQ ID NO: 14). In some embodiments, the linker comprises an amino acid sequence of DVQAVGXGDHDH, where X is selected from glycine, glutamine or serine (SEQ ID NO: 15). In some embodiments, the linker comprises an amino acid sequence of DVQAVGGGXHDH, where X is selected from proline, histidine or aspartate (SEQ ID NO: 16).

[0029] Various teachings herein are described in the context of cancer neoantigens but it is to be understood that such teachings apply to other antigens including antigenic peptides as such term is known in the art. Thus, the compositions and methods provided herein contemplate the use of antigens including antigenic peptides and in some instances immunogenic peptides in place of cancer neoantigens including neoantigenic peptides.

[0030] Various other aspects and embodiments of this disclosure are described in greater detail below.

[0031] BRIEF DESCRIPTION OF DRAWINGS

[0032] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying Figures, which are schematic and are not intended to be drawn to scale. In the Figures:

[0033] FIGs. 1A-1D show schematics and experimental data resulting from use of T cell focused vaccination. FIG. 1A shows a concept design for T cell focused vaccination. A localization element is tethered to a T cell antigen of interest. An optimized S-cathepsin protease linker sequence separates the two. FIG. IB shows a lenti-viral construct schematic for generating lung tumors that express known CD4+and CD8+T cell neoantigens. FIG. 1C shows the number of expanded OT-II T cells harvested from a tumor-draining lymph node derived from the indicated construct. FIG. D shows the proliferation index of transferred OT-II cells in terms of the number of divisions divided by the number of cells that went into division.

[0034] FIGs. 2A-2D show graphs depicting the number of tumor- specific CD4+and CD8+T cells, the percentage of CXCR3+, FOXP3" T cells, and the tumor area of lung tissue following vaccination in the model system of FIGs 1A-1D. FIG. 2A shows the number of tumor- specific CD4+T cells recovered from the tumor-draining lymph node at 5 weeks post tumor- initiation (from left to right, first bar indicates Endog, second bar indicates B-Cat, third bar indicates E- Cat, and fourth bar indicates S-Cat). FIG. 2B shows the percent of tumor- specific Thl cells (CXCR3+, FOXP3 ) in the tumor-bearing lung at the indicated timepoints (from left to right, for each timepoint, the first bar indicates Endog-ltgbl and the second bar indicates S-cat-ltgbl). FIG. 2C shows the number of tumor- specific CD8+T cell recovered from the tumor-bearing lung at 5 weeks post tumor initiation (from left to right, for each timepoint, the first bar indicates Endog-ltgbl and the second bar indicates S-cat-ltgbl). FIG. 2D shows the percent area of lung tissue that is tumor at the indicated timepoints (from left to right, for each timepoint, the first bar indicates Endog-ltgbl and the second bar indicates S-cat-ltgbl).

[0035] FIGs. 3A-3D show a schematic and the results of a model system developed to test the ability of fusion proteins to drive an immune response against a non-immunogenic protein. FIG. 3A shows the concept design for an antibody-focused vaccination. A non-immunogenic protein target is tethered to a T cell antigen of interest. An optimized S-cathepsin protease linker sequence separates the two. FIG. 3B shows the percent (left) and number (right) of GFP-specific B cells in the tumor-draining lymph node at 6 weeks post tumor initiation (from left to right, for each graph, the first bar indicates w / o S-cat and the second bar indicates w / S-cat). FIG. 3C shows the percent of GFP-specific B cells that express the indicated isotype in the setting with the S-cathepsin site (from top to bottom, the first bar indicates IgA, the second bar indicates IgG2c, the third bar indicates IgG2b, and the fourth bar indicates IgGl). FIG. 3D shows GFP antibody levels in the serum of naive mice 3-weeks post vaccination with purified GFP alone or GFP linked to a T cell antigen with the S-cathepsin linker sequence (GFP-Opt) (for each “GFP” and “GFP-Opt” condition, from top to bottom, the first bar indicates IgG2c and the second bar indicates IgG).

[0036] FIGs. 4A-4C show an mRNA vaccination construct and results relating to the use thereof. FIG. 4A shows a schematic of the self-replicating RNA (srRNA) lipid nanoparticle (ENP) system that allows for the amplification of the desired antigen constructs in transfected cells. Expression of a payload of interest is driven by a sub-genomic promoter. FIG. 4B shows the number of antigen-specific CD4+T cells in the spleens of naive mice 3 weeks post-ENP vaccination. S-cat-Mutant contains the mutant CD4+neoantigen with the S-cathepsin linker. S- cat-WT is an innocuous WT form of the antigen. Endog. Contains the CD4+neoantigen with its endogenous flanking residues instead of the S-cathepsin linker. FIG. 4C shows that vaccination with a CD8+neoantigen and the S-cat-Mutant CD4+neoantigen vaccine results in decreased tumor burden in mice 3 weeks post vaccination. DETAILED DESCRIPTION OF INVENTION

[0037] This disclosure provides fusion proteins for use in immunization of subjects through for example vaccination. The fusion proteins comprise at least two domains that are conjugated to each other using a linker that comprises a unique and optimized recognition and cleavage sequence specific for S-cathepsin. It has been found surprisingly that such optimized S- cathepsin sequences contribute to efficient and robust antigen processing in APCs and resultant presentation to CD4+and CD8+T cells. In some embodiments, the fusion proteins comprise a cancer neoantigen domain conjugated to a localization domain. The localization domain functions to traffic the fusion protein to particular regions of a cellular or extracellular environment. In some embodiments, the localization domain traffics the fusion protein (and thus the cancer neoantigen) to regions in which S-cathepsin is found. In this way, the fusion protein is designed to be cleaved preferentially in APC and by S-cathepsin, thereby enhancing antigen processing and presentation by APC. It is to be understood that other antigens including antigenic peptides may be used in the compositions and methods provided herein in place of a cancer neoantigen.

[0038] This disclosure further provides fusion proteins that comprise a non-immunogenic protein, such as a self-protein, and an immunogenic peptide, such as but not limited to a cancer neoantigen, conjugated to each other using a linker that comprises an optimized S-cathepsin recognition and cleavage sequence. These constructs are intended for use in stimulating a B cell (and thus antibody) response against the self-protein.

[0039] The fusion proteins of this disclosure may be administered in their protein form or in a nucleic acid form that encodes the protein form. In the latter embodiment, the nucleic acid may be an RNA provided in an RNA vaccine. The fusion proteins may also be used to drive a strong antibody titer against the protein of interest.

[0040] When used in a cancer setting, the fusion proteins of this disclosure resulted in improved endogenous CD4+and CD8+T cell response at early and late tumor timepoints in both the draining lymph node and in tumor-bearing lungs of animal subjects, ultimately resulting in improved tumor clearance.

[0041] Fusion proteins

[0042] As used herein, a fusion protein comprising at least two domains covalently conjugated to each other through a linker that comprises an amino acid sequence of SEQ ID NO: 1 or a functional variant thereof. Fusion proteins may comprise two, three or more domains, provided at least two domains are conjugated to each other using the aforementioned linker, and preferably wherein the aforementioned linker is used to conjugate a cancer neoantigen to the remainder of the fusion protein (as described below).

[0043] In some embodiments, the fusion protein comprises a localization domain conjugated to a cancer neoantigen. Such fusion proteins may be used to direct the conjugate to particular regions of a cell including lysosomal compartments or extra-lysosomal regions such as the plasma membrane, or the cytosol, or the pericellular space. The localization domain is primarily responsible for trafficking the conjugate to such regions within cells such as antigen presenting cells (APCs). The conjugate may be directed to certain locations in order to enhance its cleavage by S-cathepsin enzymes in the cell. These conjugates are capable of triggering an immune response directed against the neoantigen.

[0044] In some embodiments, the fusion protein comprises a self-antigen conjugated to an immunogen. The self-antigen is a protein that exists naturally in a subject or a fragment including a peptide thereof. The self-antigen is not by itself immunogenic and thus would typically not give rise to an immune response in vivo. By conjugating it to an immunogen, such as for example a cancer neoantigen, it may be presented to the immune system and may itself trigger an immune response.

[0045] Such conjugates may further comprise a localization domain conjugated to either the self-antigen or the immunogen. In some embodiments, the same linker may be used to conjugate the localization domain to either the self-antigen or the immunogen. In other embodiments, a different linker is used to conjugate the localization domain to either the selfantigen or the immunogen.

[0046] The fusion proteins are associated with more robust antigen processing and downstream immune response induction. Antigen processing refers to the degradation of a protein into peptide fragments and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by cells, for example, APCs, to either CD4+or CD8+T cells.

[0047] The fusion proteins may be administered to a subject in their amino acid form which is engulfed by APCs and processing accordingly. Alternatively, the fusion proteins may be administered to a subject as a nucleic acid encoding such fusion protein. If the nucleic acid is an RNA such as a self-replicating RNA, it is translated by cells near the site of administration (e.g., muscle cells), secreted into the extracellular environment, scavenged by antigen-presenting cells from the extracellular environment or from dying transfected cells (such as muscle cells), processed and MHC presented by the antigen-presenting cells to CD4+T cells or CD8+T cells typically in lymph nodes. In still another alternative, APCs that artificially express the fusion proteins may be administered to a subject and they in turn present processed antigen to T cells within the subject. In yet another alternative, the engineered APCs may be cultured with T cells which are then stimulated and administered to the subject in an activated state.

[0048] The linkers used in the fusion proteins are preferentially cleaved by S-cathepsin (compared to other cathepsin enzymes such as cathepsin B or cathepsin L). S-Cathepsin is predominantly found in APCs, and therefore the fusion protein remains intact until it is taken up by antigen-presenting cells and cleaved by such cells.

[0049] The fusion proteins may be considered autologous fusion proteins (and the vaccines that comprise such fusion proteins may be considered autologous vaccines) if they carry one or more neoantigens that are known to be expressed by the cancer (including tumor) in the subject being treated with the vaccine. Alternatively, the fusion proteins may be considered allogeneic fusion proteins (and the vaccines that comprise such fusion proteins may be considered allogeneic vaccines) if they carry one or more neoantigens that may be expressed by the cancer type in the subject being treated with the vaccine but were not a priori identified in the cancer of the subject being treated.

[0050] The fusion proteins may be referred to herein as recombinant intending that they are made through genetic engineering. A recombinant entity such as a recombinant protein (or recombinant fusion protein) is one that does not occur in nature and instead is the result of a combination of entities such as amino acid or nucleic acid sequences which are not combined in nature. The fusion proteins provided herein are recombinant since they contain amino acid sequences (or protein domains) derived from different proteins fused together, e.g., by peptide bonds. An entity that is naturally occurring, on the other hand, intends that it may be found in nature. For example, a protein or nucleic acid that is present in an subject or a patient and can be isolated from such source and has not been intentionally modified in for example a laboratory setting by human hand is naturally occurring.

[0051] S-Cathepsin Cleaved Linkers

[0052] The fusion proteins of this disclosure comprise improved S-cathepsin cleavable linkers. An example of such a linker comprises an amino acid sequence of DVQAVG-GGDHDH (SEQ ID NO: 1). The amino acid sequence is read from its amino end to its carboxy end and the scissile bond cleaved by S-cathepsin is denoted by a hyphen. The six amino acids before the scissile bond may be denoted as the unprimed (or non-primed) positions and the six amino following the scissile bond may be denoted as the primed positions.

[0053] Without intending to be bound by any theory or mechanism of action, it is believed that the S-cathepsin linkers contemplated by this disclosure, including SEQ ID NO:1, are effective in antigen cleavage and presentation to MHC molecules because of their length and the positioning of the scissile bond relative to the linked domains. The linker is believed to be cleaved in a manner that does not impact the neoantigen (or the self-protein), thereby not negatively impacting the immune response directed to either.

[0054] Functional variants of SEQ ID NO:1 are also contemplated including those that differ from SEQ ID NO:1 by one, two, three or more amino acid substitutions at any position along the sequence. In some embodiments, the valine at the 5thposition is invariant, and any one of the amino acids in the prime positions (those following the scissile bond) may be substituted for another amino acid, including for example a conservative substitution amino acid.

[0055] In some embodiments, the amino acid substitutions are conservative substitutions such as the following:

[0056] Asp (D) may be substituted with Glu (E) or Asn (N);

[0057] Vai (V) may be substituted with He (I), Leu (L), Met (M), Phe (F), Ala (A), or Norleucine;

[0058] Gin (Q) may be substituted with Asn (N) or Glu (E);

[0059] Ala (A) may be substituted with Vai (V), Leu (L), or He (I);

[0060] Gly (G) may be substituted with Ala (A);

[0061] His (H) may be substituted with Asn (N), Gin (Q), Lys (K), or Arg (R).

[0062] The linker may be a functional variant of SEQ ID NO: 1 in which one or more of the amino acids may be substituted with another amino acid having common side-chain properties. For example, amino acids may be grouped according to the following common side-chain properties: a. hydrophobic: Norleucine, Met (M), Ala (A), Vai (V), Leu (L), lie (I); b. neutral hydrophilic: Cys (C), Ser (S), Thr (T), Asn (N), Gin (Q); c. acidic: Asp (N), Glu (Q); d. basic: His (H), Lys (K), Arg (R); e. residues that influence chain orientation: Gly (G), Pro (P); f. aromatic: Trp (W), Tyr (Y), Phe (F).

[0063] A functional variant of SEQ ID NO: 1 is a variant that is capable of being cleaved by S- cathepsin in a manner similar to SEQ ID NO:1. S-Cathepsin may cleave a functional variant with at least 60% of the activity with which it cleaves SEQ ID NO:1. S-Cathepsin may cleave the functional variant with at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%, or more of the activity with which it cleaves SEQ ID NO:1.

[0064] In some embodiments, the linker comprises an amino acid sequence of DVQAVGGGDHDH (SEQ ID NO: 1). In some embodiments, the linker consists of an amino acid sequence of DVQAVGGGDHDH (SEQ ID NO: 1). In some embodiments, the linker is a functional variant of SEQ ID NO:1. In some embodiments, the linker comprises an amino acid sequence of DVQAVGGGDHDH (SEQ ID NO: 1) with one amino acid substitution. In some embodiments, the linker comprises an amino acid sequence of XVQAVGGGDHDH (SEQ ID NO: 2), DXQAVGGGDHDH (SEQ ID NO: 3), DVXAVGGGDHDH (SEQ ID NO: 4), DVQXVGGGDHDH (SEQ ID NO: 5), DVQAVXGGDHDH (SEQ ID NO: 6), DVQAVGXGDHDH (SEQ ID NO: 7), DVQAVGGXDHDH (SEQ ID NO: 8), DVQAVGGGXHDH (SEQ ID NO: 9), DVQAVGGGDXDH (SEQ ID NO: 10), DVQAVGGGDHXH (SEQ ID NO: 11), or DVQAVGGGDHDX (SEQ ID NO: 12). In some embodiments, the linker comprises an amino acid sequence of DVQAXGGGDHDH, where X is selected from valine, isoleucine, leucine or methionine (SEQ ID NO: 13). In some embodiments, the linker comprises an amino acid sequence of DVQAVXGGDHDH, where X is selected from glycine or glutamine (SEQ ID NO: 14). In some embodiments, the linker comprises an amino acid sequence of DVQAVGXGDHDH, where X is selected from glycine, glutamine or serine (SEQ ID NO: 15). In some embodiments, the linker comprises an amino acid sequence of DVQAVGGGXHDH, where X is selected from proline, histidine or aspartate (SEQ ID NO: 16).

[0065] Localization domains

[0066] As used herein, a localization domain is a protein (and more typically a protein fragment) that directs (or traffics or delivers) the fusion protein to an intracellular or extracellular region of interest. The localization domain may direct the fusion protein to a region where S-cathepsin is present, including predominantly present. The localization domain may direct the fusion protein to a lysosomal compartment, or to the cytosol, or to the plasma membrane, or to the pericellular space (defined as a narrow perimeter surrounding the outside of the cell). Localization domains may be truncated versions of proteins that are endogenously expressed in the subject to be treated. In some instances, the localization domain is a truncated version of an endogenously expressed protein that comprises a transmembrane domain.

[0067] As an example, a truncated transferrin receptor (or a fragment thereof) may be used to direct the fusion protein to early endosomes within antigen-presenting cells (APCs). As another example, lysosome-associated membrane protein 1 (LAMP-1) (or a fragment thereof) may be used to direct the fusion protein to lysosomes. In some embodiments, full length transferrin receptor or LAMP-1 may be used, while in other embodiments, the transmembrane region and / or cytosolic regions may be used. This may include use of the cytoplasmic tail of transferrin receptor or LAMP-1. Another example of a localization domain is invariant chain (li). In still another example, a signal peptide sequence derived from any endogenously secreted protein (e.g., CD8 leader signal peptide sequence) may be placed at the amino end of a fusion protein design to drive its secretion into the pericellular space. These are known in the art and described in Fernandes et al. Eur. J. Immunol., 2000, 30:2333-2343; Diebold et al. Gene Therapy, 2001, 8:487-493; and Jing et al. J Cell Biol 1990, 110:283-294.

[0068] Localization domains that direct the fusion protein to the cytosolic region of an APC tend to stimulate a CD8+T cell response (via MHC Class I presentation). Localization domains that direct the fusion protein to a membrane region of an APC tend to stimulate a C4+T cell response (via MHC Class II presentation).

[0069] In some embodiments, truncated version of the transferrin receptor that traffic the fusion protein to the early endosomes is used. One such example of such a truncated transferrin receptor is used in the Examples. Suitable versions of the truncated transferrin receptor may comprise a transmembrane domain or a cytosolic domain, or both. While not intending to be bound by any particular theory, the cytosolic domain of the transferrin receptor contains tyrosine motifs that, upon internalization, may be involved in directing the fusion protein to early endosomes.

[0070] Cancer Neoantigens

[0071] As used herein, cancer neoantigen means a class of cancer antigens which arise from cancer-specific (or tumor- specific) changes in host proteins. Neoantigens encompass, but are not limited to, tumor antigens which arise from and / or differ from a host protein by, for example, one or more amino acid substitutions in the protein sequence of the host protein, a frame shift mutation, a mis sense mutation, introduction of a splice site, a nonsense mutation, a fusion of two or more host proteins, an in-frame deletion, and / or an insertion. These mutated proteins occur in the cancerous cells and not in the normal noncancerous cells in the subject. In some instances, cancer neoantigens may also encompass endogenous retroviral proteins expressed by the cancer cells. Neoantigens may be uniquely expressed by cancer cells (such as tumor cells), intending that they are not expressed in normal non-cancer (or non-tumor) cells in the subject.

[0072] Neoantigens may be commonly expressed by (or commonly observed in) a certain type of cancer (e.g., lung cancer) across several subjects having such cancer. An example of such neoantigens are certain KRAS neoantigens which have been observed in 30-40% of human lung tumor sample from different patients. Neoantigens may be uniquely expressed in a single subject (and optionally in a certain cancer type within that subject). The art is familiar with both commonly expressed neoantigens. The art is also familiar with methods for identifying (by sequence) neoantigens expressed by cancer cells (or tumor cells) present in a subject having cancer. See for example Hu et al. Nature Medicine, 2021, 27:515-525, which describes a clinical trial in which patients were treated with neoantigen- vaccines. See also Ott et al. Nature, 2017, 547(7662):217-221 which describes neoantigens, neoantigen vaccines, and methods for obtaining and sequencing neoantigens from patients having cancer. See also US 11,650,211.

[0073] Neoantigens have been observed in a number of proteins, including but not limited to NRAS, KRAS, TP53, CDKN2A, NF1, PTEN, RAC1, ARID2, SLC38A4, DSG3, MLL, DDX3X, ALPK2, RPTN, IDH1, KEL, NGF, BRAF, ANO4, POTEG, TPTE, USH2A, XIRP2, THSD7B, CTNNB1, MPP7, PARM1, ACSBG1, among others. An example of a personal neoantigen long-peptide vaccine is NeoVax, which comprises about 20-30 neoantigenic peptides contiguously arranged and which has been used to mount immune responses in patients with high-risk melanoma and in patients with glioblastoma.

[0074] The fusion protein may comprise a full-length cancer neoantigen or a fragment thereof. Accordingly, the fusion proteins may comprise neoantigenic proteins or neoantigenic peptides, and any discussion herein relating to neoantigens applies equally to neoantigenic proteins and neoantigenic peptides, unless stated otherwise.

[0075] The length of these neoantigenic peptides or proteins may vary. For example, their length may range from about 4 or about 6 or about 8 amino acids or about 10 amino acids to about 20 amino acids or about 30 amino acids or about 40 amino acids or about 50 amino acids or about their full length. If the neoantigens are intended to be presented on MHC Class I in order to induce a CD8+T cell immune response, then the neoantigen may be about 22 amino acids or less in length, including from about 8 to about 22 amino acids in length, or from about 8 to about 15 amino acids in length, or 9 or 10 amino acids in length. If the neoantigens are intended to be presented on for MHC Class II in order to induce a CD4+T cell immune response, then the neoantigen may be about 40 amino acids or less in length, including from about 8 to about 40 amino acids in length, or from about 8 to about 24 amino acids in length, or from about 12 to about 19 amino acids, or from about 14 to about 18 amino acids in length.

[0076] The neoantigen may be referred to as a MHC Class I binding or CD8+T cell neoantigen intending that it is presented by MHC Class I to CD8+T cells. The neoantigen may be referred to as an MHC Class II binding or CD4+T cell neoantigen intending that it is presented by MHC Class II to CD4+T cells. Some fusion proteins comprise both types of neoantigens. In these situations, the neoantigens may be derived from the same host protein or they may be derived from different host proteins.

[0077] Examples of neoantigens are provided for example in US Patent No. 11,793,867, and these include full length versions of the following mutated proteins or fragments thereof that comprise the mutation: ABL1 (E255K) found in chronic myeloid leukemia (DML), acute lymphocytic leukemia (ALL) and gastrointestinal stromal tumors (GIST); ABL1 (E255V) found in CML, ALL, and GIST; ABL1 (M351T) found in CML, ALL, GIST; ABL1 (Y253H) found in CML, ALL and GIST; ALK (E1269A) found in non-small cell lung cancer (NSCLC); ALK (L1196M) found in NSCLC; BRAE (V600E) found in CRC, GBM, KIRP, LUAD, SKCM and THCA; and all remaining neoantigenic peptides provided in US 11,793,867, including particularly those in Tables 1 and 2, all of which are incorporated by reference herein. As used herein the terminology ABL1 (E255K) intends that the neoantigen is a mutant form of the ABL1 protein having an E to K amino acid substitution at position 255. Other examples of neoantigens are provided in US 9,637,794; Eritsch et al. Cancer Immunol Re, 2014, 2(6):522-529 (see Table 1 for a list of neoantigens and their sequences), van Buuren et al. Oncolmmunol, 2014, 3:e28836:l-6 (see Table 1 which includes CD8+neoantigenic peptides).

[0078] Self-Antigens Also provided herein are fusion proteins comprising a non-immunogenic self-protein conjugated to an immunogenic protein, peptide or epitope. The self-protein is conjugated to the immunogenic protein, peptide or epitope through a linker comprising an amino acid sequence of SEQ ID NO:1 or a functional variant thereof. These fusion proteins may be used to stimulate an immune response against the self-protein, including stimulating a B cell (or antibody) response against the self-protein. Self-proteins are typically non-immunogenic in a host (due to their “self’ nature). By conjugating a self-protein (or fragment thereof) to an immunogenic protein, peptide or epitope, an immune response may be stimulated towards the self-protein (or fragment thereof).

[0079] Self-antigens are conjugated to an immunogenic peptide or an immunogenic epitope in certain fusion proteins of this disclosure. An immunogenic peptide or an immunogenic epitope is a peptide or an epitope that stimulates an immune response. It may do so by binding an MHC molecule and inducing a cell-mediated or humoral response, for example, a cytotoxic CD8+T cell, a helper CD4+T cell, and / or a B cell (antibody) response. Immunogenic peptides are capable of binding to an appropriate MHC molecule and thereafter inducing an immune response.

[0080] In some embodiments, the immunogenic peptide or immunogenic epitope is a cancer neoantigen, such as a cancer neoantigenic peptide or a cancer neoantigenic epitope, or an artificially designed CD4+epitope that binds with high affinity to MHC Class II.

[0081] When conjugated to a self-protein, the immunogenic peptide or immunogenic epitope binds to MHC Class II and stimulates a CD4+T cell immune response that in turn facilitates a B cell (antibody) response.

[0082] Self-proteins may be virtually any protein that is normally present in a subject, and that typically is not immunogenic in that subject. In some embodiments, the self-protein is expressed in over-expressed in a tumor (and may be referred to as a tumor- associated antigen or TAA) although it may not be mutated in sequence. Examples of such proteins are disclosed in US 9,617,321. In these situations, the fusion proteins are used to break self-tolerance against such TAA, thereby treating the tumor.

[0083] As used herein, self-protein in relation to a particular subject means a protein that is encoded by the genome of said subject and that is under normal conditions, i.e., non- pathological conditions, optionally expressed in certain normal tissue types or at certain developmental stages of said subject. Preferably, it does not include proteins with acquired mutations. A tumor-associated antigen that is a self-protein in a subject includes a tumor- associated antigen that is or was expressed in said subject under normal conditions in certain tissues or at a certain developmental stage and is abnormally or aberrantly expressed in tumorigenic tissue of said subject, preferably in the same form and / or with the same structure.

[0084] As used herein, self-tolerance refers to an immune system that does not respond to (or mount an immune response against) a self-protein. Normally, self-tolerance is developed early by developmental events within the immune system that prevent, in particular, the subject’s own T cells and B cells from reacting with the subject’s own tissues. Breaking self-tolerance refers to a process that causes the immune system of a subject to recognize and mount an immune response to a self-protein. For example, by linking a strong CD4+T cell antigen to a self-antigen preferentially expressed by transformed cells in various forms of cancer, such as Mesothelin (Msln) or Mucin- 1 (Muc-1) (see Li et al. Mol Cancer Ther 2008, 7(2):286-296; Lohmueller et al. Sci Rep, 2016, 6:31740; and Chen et al. Int J Mol Sci 2021, 22(12):6567), it may be possible to break self-tolerance by linking B cell recognition of the self-antigen with CD4+T cell activation driven by the attached highly immunogenic epitope. Clinically this may result in high antibody titers against the self-antigen and potential activation of self-reactive T cells against the targeted self-protein. Subsequent anti-tumor antibody-mediated effector functions may result from breaking tolerance against a self-antigen.

[0085] Methods of Use

[0086] The fusion proteins of this disclosure may be administered to a subject in need thereof to stimulate a desired and specific immune response. If the subject has cancer or is at elevated risk of developing cancer (such as for example a subject positive for BRCA gene mutations), then a fusion protein comprising a cancer neoantigen and a localization domain, linked together with an optimized S cathepsin cleavage sequence is used. The fusion protein may be administered as a protein or as a nucleic acid that encodes the protein. The fusion protein may be administered through a variety of administration routes, including but not limited to intramuscular administration. Intramuscular administration is typically used for administration of an RNA vaccine or a protein-based vaccine. Other administration routes are however also contemplated provided they allow the administered protein or the expressed protein to be taken up by APCs. The fusion protein, in either form, may be administered once or more than once. If more than once, the administrations may be regularly spaced (e.g., weekly, monthly, etc.) or alternatively they may be administered on an as needed basis.

[0087] The subject may also receive a secondary agent such as an immune checkpoint inhibitor such as an CTLA-4 inhibitor (e.g., Ipilimumab (Yervoy)), or a PD-1 inhibitor (e.g., nivolumab (Opdivo) or pembrolizumab (Keytruda)).

[0088] Vaccines

[0089] The fusion proteins may be used as part of a vaccine. They may be presented in either their amino acid form or in a nucleic acid form that codes for the protein. As a vaccine, the fusion proteins may be used to induce immunity for prophylactic or therapeutic benefit (e.g., in a subject having or at elevated risk of developing cancer). In some embodiments, the vaccine may be a personalized cancer vaccine intending that it comprises (or encodes) a cancer neoantigen or a plurality of cancer neoantigens that are expressed in the subject to be treated.

[0090] A vaccine composition may comprise a pharmaceutically acceptable excipient. Excipient refers to substances in a pharmaceutical formulation that are not active ingredients such as, e.g., carriers, diluents, binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffers, flavoring agents, or colorants.

[0091] Aspects of the present disclosure relate to use of the antigen-based vaccine in subject having cancer or at elevated risk of developing cancer. A vaccine may include or be directed against one or more disease- specific neoantigens (or corresponding nucleic acids encoding them). In some embodiments, the antigen-based vaccine contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, or more neoantigens.

[0092] Nucleic acid formulations

[0093] In certain embodiments, the fusion protein may be administered as an RNA vaccine. RNA vaccines typically comprise one or more RNA that encode a protein of interest (e.g., the fusion protein of this disclosure) in a formulation (e.g., a lipid nanoparticle) and optionally in one or more excipients. The RNA may be a single- stranded mRNA polynucleotide. In other embodiments, the fusion protein may be administered as a DNA polynucleotide encoding an RNA that itself encodes the fusion protein. Upon administration, the nucleic acids enter cells in the vicinity of the site of administration. If the administration is muscular, the nucleic acids (e.g., RNA) enters muscle cells and / or tissue-resident dendritic cells (e.g., tissue-resident APCs). If taken up by muscle cells, those cells must also secrete the fusion protein and / or degrade sufficiently to release the fusion protein, which is then taken up by APCs. The APCs in turn process the fusion protein, including cleaving it at the S cathepsin cleavage site to release the cancer neoantigen, and then present the cancer neoantigen in the context of MHC Class I or II to CD8+or CD4+T cells respectively. This leads to an induction of cytotoxic T-lymphocyte (CTL) immune responses or memory T-cell-dependent immune responses against cancer cells expressing the cancer neoantigens.

[0094] A nucleic acid (e.g., RNA) vaccine may comprise one or more neoantigens (or neoantigen epitopes) (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28, 29, 30, or more neoepitopes). These may be encoded on a single RNA or they may be encoded on a plurality of RNA.

[0095] In some embodiments, the RNA also includes a 5' mRNA cap analogue, a 5' UTR, a signal sequence, a domain to facilitate antigen expression, a 3' UTR, and / or a poly A tail.

[0096] In some embodiments, the manufacture of an RNA vaccine involves identifying somatic mutations in a tumor in the subject using next-generation sequencing (NGS) and then predicting immunogenic neoantigen epitopes ( or "neoepitopes") based on such sequence information. The RNA vaccine targeting the selected neoepitopes may then be manufactured on a per-patient basis. The art is familiar with identifying such cancer- or tumor- specific neoantigens and formulating them in an RNA vaccine. See for example US 10,155,031; US 10,738,355; and US 2019 / 0083593.

[0097] In some embodiments, the RNA comprises a 5' cap. The basic mRNA cap structure is known to contain a 5'-5' triphosphate linkage between 2 nucleosides ( e.g., two guanines) and a 7-methyl group on the distal guanine, i.e., m7GpppG. Exemplary cap structures can be found, e.g., in U.S. Pat. Nos. 8,153,773 and 9,295,717 and Kuhn, A. N. et al. (2010) Gene Then 17:961- 971. In some embodiments, the 5' cap is a beta-S-ARCA cap. The S-ARCA cap structure includes a 2'-0 methyl substitution (e.g., at the C2' position of the m7G) and an S-substitution at one or more of the phosphate groups. In some embodiments, the RNA comprises synthetic cap analogues that stabilize mRNA and increase protein translation via binding to eukaryotic translation initiation factor 4E (EIF4E). The RNA may further comprise regulatory elements in the 5’ untranslated region (UTR) and the 3’ UTR and these in turn also stabilize the RNA thereby increasing protein translation. The RNA may comprise a poly(A) tail that stabilizes the RNA and also increases protein translation. In still other embodiments, the RNA may comprise modified nucleosides that decrease innate immune activation and increase translation. The RNA may also be codon optimized, leading to increased translation.

[0098] In some embodiments, the RNA vaccine is formulated in a lipid nanoparticle or liposome. In some embodiments, a lipid nanoparticle formulation for the RNA may be used to in a variety of administration routes including but not limited to intramuscular.

[0099] In one embodiment, the nanoparticles comprise at least one lipid. In one embodiment, the nanoparticles comprise at least one cationic lipid. The cationic lipid can be monocationic or polycationic. Any cationic amphiphilic molecule, e.g., a molecule which comprises at least one hydrophilic and lipophilic moiety is a cationic lipid within the meaning ascribed herein. In one embodiment, the positive charges are contributed by the at least one cationic lipid and the negative charges are contributed by the RNA.

[0100] In one embodiment, the nanoparticles comprise at least one helper lipid. The helper lipid may be a neutral or an anionic lipid. The helper lipid may be a natural lipid, such as a phospholipid or an analogue of a natural lipid, or a fully synthetic lipid, or lipid-like molecule, with no similarities with natural lipids. In one embodiment, the cationic lipid and / or the helper lipid is a bilayer forming lipid.

[0101] In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadeceny 1-3- trimethylammonium propane (DOTMA) or analogs or derivatives thereof and / or l,2-dioleoyl-3- trimethylammonium-propane (DOTAP) or analogs or derivatives thereof.

[0102] In one embodiment, the at least one helper lipid comprises l,2-di-(9Z-octadecenoyl)-sn- glycero-3-phosphoethanolamine (DOPE) or analogs or derivatives thereof, cholesterol (Choi) or analogs or derivatives thereof and / or l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or analogs or derivatives thereof.

[0103] In one embodiment, the molar ratio of the at least one cationic lipid to the at least one helper lipid is from 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1:1. In one embodiment, in this ratio, the molar amount of the cationic lipid results from the molar amount of the cationic lipid multiplied by the number of positive charges in the cationic lipid. RNA vaccines, including RNA modifications and lipid nanoparticle compositions, are known in the art and reference may be made for example to US 9,950,065; US 10,106,800; US 10,576,146; US 10,717,982; US 10,729,784; US 10,808242; US 11,020,477; 11,173,120; US 11,684,577; US 11,771,653; US 20170166905; US 20170273907; US 2020 / 0085974; US20210251898, US20220125899; US20220347307.

[0104] Nucleic acid as used herein refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) including mRNA and in vitro transcribed RNA. The nucleic acids encoding the fusion proteins provided herein may be recombinantly produced or chemically synthesized such as by a nucleic acid synthesizer. The nucleic acid may be presented as a single- stranded or doublestranded and linear or covalently circularly closed nucleic acid. A nucleic acid may be isolated intending that it (i) was amplified in vitro, for example via am amplification reaction such as polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis. A nucleic may be introduced into (i.e., transfected into) cells, for example in the form of RNA which can be prepared by in vitro transcription from a DNA template. The RNA may be modified through use of stabilizing sequences, capping, and polyadenylation. The vaccines provided herein may comprise a single RNA encoding a single fusion protein, or they may comprise a plurality of RNA each encoding a single fusion protein, or they may comprise a single RNA encoding a plurality of fusion proteins, or they may comprise a plurality of RNA each encoding a plurality of fusion proteins. As used herein, a plurality means 2 or more, including for 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0105] RNA refers to a polynucleotide that comprises (and preferably is composed entirely of) ribonucleotide residues. A ribonucleotide refers to a nucleotide with a hydroxyl group at the 2'- position of a beta-D-ribofuranosyl group. It may be double-stranded RNA, single-stranded RNA, isolated RNA such as partially or completely purified RNA, synthetic RNA, and recombinantly generated RNA such as modified RNA which differs from naturally occurring RNA by addition, deletion, substitution and / or alteration of one or more nucleotides and / or incorporation of non-naturally occurring ribonucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotide residues of the RNA. Nucleotides in RNA molecules can also comprise nonstandard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA. The RNA in other embodiments is not chemically modified. In some embodiments, the RNA only comprises standard nucleotides, such as naturally occurring nucleotides. RNA may include and / or may be mRNA (messenger RNA) that may be (but is not exclusively) produced from a DNA template and encodes a peptide or protein such as a fusion protein of this disclosure. Typically, an mRNA comprises a 5’-UTR, a protein coding region, and a 3’-UTR. mRNA typically has a limited half-life in cells and in vitro. The art is familiar with in vitro transcription methodology and commercially available kits for generating RNA. In some embodiments, the RNA is a self-replicating RNA, such as single stranded self-replicating RNA. One example of a self-replicating RNA is single stranded RNA of positive sense. The self-replicating RNA may be viral RNA or RNA derived from viral RNA. The self-replicating RNA may alphaviral genomic RNA or may be derived from alphaviral genomic RNA. In one embodiment, the self-replicating RNA is a viral gene expression vector.

[0106] The stability and translation efficiency of RNA may be modified as required. For example, RNA may be stabilized and its translation increased by one or more modifications having a stabilizing effects and / or increasing translation efficiency of RNA.

[0107] These various nucleic acid, including RNA, teachings, including modifications to RNAs are known in the art and reference can be made to published PCT application PCT / EP2006 / 009448 and US 10,729,784.

[0108] Subject

[0109] The term "subject" refers to a human or non-human subject. A subject includes, but is not limited to, a mammal including but not limited to a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline. The subject may be a companion animal such as a dog or cat, or an agricultural animal such as a cow, pig, goat, etc., or it may be a show animal such as a horse. The subject may also be a laboratory animal such as a mouse, rat or rabbit.

[0110] Cancer Types

[0111] Certain fusion proteins of this disclosure may be used to induce a cancer- specific (or tumor- specific) immune response in a subject having cancer or in a subject at elevated risk of developing cancers. Such cancers may be solid tumor cancers or non- solid tumor cancers such as leukemia. Cancers include, but are not limited to, B cell cancers, (e.g., multiple myeloma, Waldenstrom's macroglobulinemia), melanomas, bone cancer, bladder carcinoma, breast cancer, laryngeal cancer, lung cancer (e.g., small cell lung carcinoma), colorectal cancer, prostate cancer (e.g., metastatic, hormone refractory prostate cancer), pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, neuroblastoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, testicular cancer, retinoblastoma; esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic and the like. Other non-limiting examples of types of cancers include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, and the like.

[0112] Treatment

[0113] As used herein, treating refers to reducing, preventing, or ameliorating a disorder and / or symptoms associated therewith (e.g., a neoplasia or tumor). A subject may be treated after the onset, or suspected onset, of a disease (e.g., cancer). Treatment may be evidenced by a reduction in the severity of a disease or disorder in a patient, e.g., extending the life or prolonging the survivability of a patient with the disease, or delaying its recurrence, e.g., lengthening the period of remission in a patient who had suffered from the disease. In the case of a subject having a tumor, treatment may be evidenced by a reduction in tumor mass or volume, or a reduction is size or number of distal (or metastatic) lesions. Treatment does not require that the disorder, condition, or symptoms associated therewith be completely eliminated. As used herein, preventing refers to avoiding or delaying the onset of a disease or condition or of symptoms associated with a disease or condition in a subject that has not developed such symptoms at the time agent of interest is administering.

[0114] The following examples are provided to illustrate specific instances of the practice of the present disclosure and are not intended to limit the scope of the invention. As will be apparent to one of ordinary skill in the art, the present invention will find application in a variety of compositions and methods.

[0115] EXAMPLES

[0116] Example 1.

[0117] We developed optimal synthetic amino acid sequences for cathepsin B, L, and S. These sequences were then inserted between a localization protein element (also referred to as a localization domain herein) and a known CD4+T cell neoantigen peptide (FIG. 1A). The goal of this construct design was to generate a CD4+T cell response against a membrane-localized neoantigen with the cathepsin linker sites acting as a method to augment presentation by antigen-presenting cells. To test this, a construct (FIG. IB) was generated and then integrated into lentiviral particles and delivered to a genetically engineered mouse model of lung adenocarcinoma (LU AD) driven by Cre inducible activation of an oncogenic Kras allele (KrasG12D / +) concurrent with homozygous loss of the tumor-suppressor gene Trp53 (p53fl / fl) (8). This model (hereby KP) faithfully captures the pathological course of LU AD progression seen in humans (9). Upon intratracheal delivery, lung tumors are initiated and simultaneously express T cell neoantigens to elicit a CD8+and CD4+T cell response. To test the efficacy of the cathepsin linker sites, tumors were initiated in mice that either lacked a cathepsin linker sequence or had an optimized site for B, L, or S cathepsin. Five weeks after tumor initiation, one million CFSE-labeled and congenically marked (CD45.1+) splenocytes were transferred from an OT-II transgenic mouse that produces CD4+T cells specific to the neoantigen of interest. Ten days after transfer, the mice were sacrificed and the tumor-draining lymph node was collected to assess how well the transferred CD4+T cells proliferated in different tumor contexts. All three of the cathepsin linker sequences led to greater in vivo proliferation of transferred CD4+T cells (FIGs. 1C-1D) with the S-cathepsin sequence condition demonstrating the most pronounced expansion and number of cells. From this data, it was reasoned that the artificially designed S- cathepsin linker sequence augmented the ability of APCs to process the tumor-derived fusion protein and optimally present a CD4+neoantigen of interest.

[0118] This concept was further expanded upon by studying how these linker sites altered the endogenous CD4+and CD8+T cells in the model of lung adenocarcinoma. Utilizing a similar construct depicted in FIG. IB, tumors were initiated in KP mice with or without various cathepsin linker sequences. At 5 weeks and 10 weeks post-tumor initiation, the mice were sacrificed and the tumor-bearing lung was collected for T cell phenotyping. Like the transfer assays, it was found that the S-cathepsin linker site produced a superior expansion of tumorspecific CD4+T cells (FIG. 2A). Furthermore, the phenotype of the tumor- specific CD4+T cells in the setting containing the S-cathepsin cleavage sites were more effector-like and had markers of a conventional Thl subset (CXCR3+, FOXP3-) that are desired for an anti-tumor immune response (FIG. 2B). A concordant increase in tumor- specific CD8+T cells was also observed (FIG. 2C). This improved T cell response led to a demonstrable decrease in overall tumor burden at various timepoints through tumor progression (FIG. 2D).

[0119] Example 2.

[0120] In addition to studying the role of the optimized cathepsin site in generating a robust anti-tumor T cell response, we also explored its ability to promote a B cell driven antibody response. Activation of B cells requires CD4+T cell help. We reasoned that linking a CD4+T cell neoepitope to any protein target of interest that a B cell may recognize, may greatly boost B cell activation and therefore antibody titers. Further activation could be provided by inserting the S-cathepsin linker sequence between the protein target of interest and the CD4+antigen (FIG. 3A). With this novel concept design, high-affinity, class-switched antibodies could be generated against any non-immunogenic protein. This concept was tested by generating a similar construct as the one described in FIG. IB that resulted in tumor expression of a cell surface form of eGFP. A CD4+T cell neoantigen was tethered to the C-terminal end of eGFP either with or without an S-cathepsin linker. The presence of the S-cathepsin led to a dramatic expansion of eGFP-specific B cells in the tumor draining lymph node germinal centers of tumor-bearing mice (without S- cathepsin: 950 vs. with S-cathepsin linker: 75,000) (FIG. 3B, Right). Furthermore, these recently activated eGFP-specific B cells exhibited signs of class-switching to higher affinity IgG antibodies that are more effective in eliciting a potent effector immune response (FIG. 3C). Finally, when naive mice were vaccinated with whole eGFP antigen or eGFP linked to a CD4+neoepitope containing the S-cathepsin linker, antibody titers in the peripheral blood were notably higher in mice that contained the CD4+neoepitope and the S-cathepsin linker (FIG. 3D). These data demonstrate that by linking a CD4+neoepitope to an antigen of interest, potent antibody titers can be generated. The presence of an artificial S-cathepsin linker sequence improves the processing of these antigen elements which in turn augments B cell activation and subsequent antibody production.

[0121] Example 3.

[0122] The clinical potential of the optimized S-cathepsin linker sites prompted us to test them in an mRNA vaccination setting. A self-amplifying mRNA vaccine platform (FIG. 4A) was utilized. With this approach, a modest amount of mRNA carrying a payload of interest can be packaged into lipid nanoparticles and delivered to mice via an intramuscular injection into the hind leg (10). Embedded within the mRNA construct, a sub-genomic promoter drives the expression of the same neoantigens expressed by cancer cells in the mouse model. The efficacy of the S-cathepsin sites in promoting a CD4+T cell response was tested by vaccinating naive mice with various mRNA payloads based on the concept design described in FIG. 1A. Three weeks post mRNA-LNP vaccination of naive mice, the population of antigen- specific CD4+T cells in the spleen (FIG. 4B) was assessed. The presence of the S-cathepsin linker sequence was required to generate a meaningful population of antigen- specific CD4+T cells in this setting. These studies indicate that the optimized S-cathepsin linker containing CD4+T cell vaccine likely controls and reduces tumor-burden (FIG. 4C).

[0123] In summary, the addition of the novel S-cathepsin sequence provided herein in fusion proteins greatly improved vaccination strategies. Specifically, by separating engineered fusion proteins with the highly specific S-cathepsin sequence, desired peptide-antigens can be processed by antigen-presenting immune cells such as dendritic cells in peripheral lymphoid tissue. It was shown that the design is amenable for eliciting both a CD4+T cell response as well as a B cell and subsequent antibody response. The implications of these results are profound as mRNA vaccines designed to combat various forms of cancer are extensive and in need of optimization. The design presented herein is highly modular and conserved from mouse to human which ensures a practical efficacy that can be applied to most vaccination strategies. Utilization of the cleavage sequence in a generalized fusion protein design will dramatically alter the landscape for mRNA-based vaccination therapies.

[0124] REFERENCES

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[0134] 10) Li Y, Su Z, Zhao W, Zhang X, Momin N, Zhang C, Wittrup KD, Dong Y, Irvine DJ, Weiss R. Multifunctional oncolytic nanoparticles deliver self-replicating IL- 12 RNA to eliminate established tumors and prime systemic immunity. Nat Cancer. 2020;l(9):882-93. Epub 20200810. doi: 10.1038 / s43018-020-0095-6. PubMed PMID: 34447945; PMCID: PMC8386348.

[0135] EQUIVALENTS

[0136] The foregoing written specification is considered to be sufficient to enable one ordinarily skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as mere illustrations of one or more aspects of the invention. Other functionally equivalent embodiments are considered within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.

[0137] Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0138] All references, patents and patent applications that are recited in this application are incorporated by reference herein only for the subject matter that they are relied upon as indicated herein.

Claims

What is claimed is:CLAIMS1. A fusion protein comprising a localization domain conjugated to a cancer neoantigen through a linker that comprises an amino acid sequence of DVQAVGGGDHDH (SEQ ID NO: 1) or a functional variant thereof.

2. The fusion protein of claim 1, wherein the linker comprises an amino acid sequence of DVQAVGGGDHDH (SEQ ID NO: 1).

3. The fusion protein of claim 1, wherein the linker consists of an amino acid sequence of DVQAVGGGDHDH (SEQ ID NO: 1).

4. The fusion protein of claim 1, wherein the linker is a functional variant of SEQ ID NO: 1.

5. The fusion protein of claim 1, wherein the linker comprises an amino acid sequence of DVQAVGGGDHDH (SEQ ID NO:1) with one amino acid substitution.

6. The fusion protein of claim 1, wherein the linker comprises an amino acid sequence of XVQAVGGGDHDH (SEQ ID NO: 2), DXQAVGGGDHDH (SEQ ID NO: 3), DVXAVGGGDHDH (SEQ ID NO: 4), DVQXVGGGDHDH (SEQ ID NO: 5), DVQAVXGGDHDH (SEQ ID NO: 6), DVQAVGXGDHDH (SEQ ID NO: 7), DVQAVGGXDHDH (SEQ ID NO: 8), DVQAVGGGXHDH (SEQ ID NO: 9), DVQAVGGGDXDH (SEQ ID NO: 10), DVQAVGGGDHXH (SEQ ID NO: 11), or DVQAVGGGDHDX (SEQ ID NO: 12).

7. The fusion protein of claim 1, wherein the linker comprises an amino acid sequence of DVQAXGGGDHDH, where X is selected from valine, isoleucine, leucine or methionine (SEQ ID NO: 13).

8. The fusion protein of claim 1, wherein the linker comprises an amino acid sequence of DVQAVXGGDHDH, where X is selected from glycine or glutamine (SEQ ID NO: 14).

9. The fusion protein of claim 1, wherein the linker comprises an amino acid sequence of DVQAVGXGDHDH, where X is selected from glycine, glutamine or serine (SEQ ID NO: 15).

10. The fusion protein of claim 1, wherein the linker comprises an amino acid sequence of DVQAVGGGXHDH, where X is selected from proline, histidine or aspartate (SEQ ID NO: 16).

11. The fusion protein of any one of the foregoing claims, wherein the cancer neoantigen is a MHC Class II presented cancer neoantigen.

12. The fusion protein of any one of the foregoing claims, wherein the cancer neoantigen is a mutant KRAS neoantigen.

13. The fusion protein of any one of the foregoing claims, further comprising a MHC Class I presented cancer neoantigen.

14. A composition comprising an RNA encoding the fusion protein of any one of the foregoing claims formulated in a lipid nanoparticle.

15. The composition of claim 14, formulated for intramuscular administration.

16. A method of stimulating an immune response in a subject comprising administering to a subject in need thereof an RNA encoding the fusion protein of any one of claims 1-15, in an amount effective to stimulate an immune response to the cancer neoantigen.

17. A method of stimulating an immune response in a subject comprising administering to a subject in need thereof an RNA encoding a fusion protein comprising(a) a localization domain conjugated to(b) a cancer neoantigen, through a linker that comprises an amino acid sequence comprising DVQAVGGGDHDH (SEQ ID NO: 1) or a functional variant thereofin an amount effective to stimulate an immune response to the cancer neoantigen.

18. The method of claim 16 or 17, wherein the immune response is a CD4+T cell response.

19. The method of any one of claims 16-18, wherein the subject has cancer.

20. The method of any one of claims 16-19, wherein the subject has increased risk of developing cancer compared to a control population.

21. A fusion protein comprising a self-protein conjugated to a cancer neoantigen through a linker that comprises an amino acid sequence comprising DVQAVGGGDHDH (SEQ ID NO: 1) or a functional variant thereof.

22. The fusion protein of claim 21, wherein the self-protein is overexpressed in cancer cells compared to non-cancer cells in the subject.

23. The fusion protein of claim 21, wherein the self-protein is mesothelin.

24. The fusion protein of claim 21, wherein the self-protein is mucin- 1.

25. The fusion protein of any one of claims 21-24, wherein the cancer neoantigen is a CD4+cancer neoantigen.

26. The fusion protein of any one of claims 21-25, wherein the linker comprises an amino acid sequence of DVQAVGGGDHDH (SEQ ID NO: 1).

27. The fusion protein of any one of claims 21-25, wherein the linker consists of an amino acid sequence of DVQAVGGGDHDH (SEQ ID NO: 1).

28. The fusion protein of any one of claims 21-25, wherein the linker is a functional variant of SEQ ID NO: 1.

29. The fusion protein of any one of claims 21-25, wherein the linker comprises an amino acid sequence of DVQAVGGGDHDH (SEQ ID NO:1) with one amino acid substitution.

30. The fusion protein of any one of claims 21-25, wherein the linker comprises an amino acid sequence of XVQAVGGGDHDH (SEQ ID NO: 2), DXQAVGGGDHDH (SEQ ID NO: 3), DVXAVGGGDHDH (SEQ ID NO: 4), DVQXVGGGDHDH (SEQ ID NO: 5), DVQAVXGGDHDH (SEQ ID NO: 6), DVQAVGXGDHDH (SEQ ID NO: 7), DVQAVGGXDHDH (SEQ ID NO: 8), DVQAVGGGXHDH (SEQ ID NO: 9), DVQAVGGGDXDH (SEQ ID NO: 10), DVQAVGGGDHXH (SEQ ID NO: 11), or DVQAVGGGDHDX (SEQ ID NO: 12).

31. The fusion protein of any one of claims 21-25, wherein the linker comprises an amino acid sequence of DVQAXGGGDHDH, where X is selected from valine, isoleucine, leucine or methionine (SEQ ID NO: 13).

32. The fusion protein of any one of claims 21-25, wherein the linker comprises an amino acid sequence of DVQAVXGGDHDH, where X is selected from glycine or glutamine (SEQ ID NO: 14).

33. The fusion protein of any one of claims 21-25, wherein the linker comprises an amino acid sequence of DVQAVGXGDHDH, where X is selected from glycine, glutamine or serine (SEQ ID NO: 15).

34. The fusion protein of any one of claims 21-25, wherein the linker comprises an amino acid sequence of DVQAVGGGXHDH, where X is selected from proline, histidine or aspartate (SEQ ID NO: 16).

35. A composition comprising an RNA encoding the fusion protein of any one of claims 21-34 formulated in a lipid nanoparticle.

36. The composition of claim 35, formulated for intramuscular administration.

37. A method of stimulating an immune response in a subject comprising administering to a subject in need thereof an mRNA encoding a fusion protein of any one of claims 21-34, in an amount effective to stimulate an immune response to self-protein.

38. A method of stimulating an immune response in a subject comprising administering to a subject in need thereof an mRNA encoding a fusion protein comprising(a) a self-protein conjugated to(b) an immunogenic antigen, through a linker that comprises an amino acid sequence comprising DVQAVGGGDHDH (SEQ ID NO: 1) or a functional variant thereof in an amount effective to stimulate an immune response to self-protein.

39. The method of claim 37 or 38, wherein the immune response to the self-protein comprises a B-cell immune response or an antibody response.

40. The method of any one of claims 37-39, wherein the immune response comprises a CD4+T cell response.

41. The method of any one of claims 37-40, wherein the subject has cancer.

42. The method of any one of claims 37-40, wherein the subject has increased risk of developing cancer compared to a control population.

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