Generating neo-antigens by mRNA translational errors to be targeted by therapies

Abstract

Provided are methods for treating cancer by administrating to an individual who has the cancer an inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPPD) and an immune checkpoint inhibitor. Also provided are methods for promoting translation of proteins within cells such that proteins include one or more misincorporated amino acids to thereby develop new proteins that contain neoantigens. Vaccines that include the neoantigens are included. Methods for producing and detecting misincorporation of a tyrosine in proteins expressed by cells are also provided.

Description

[0001] Attorney Docket No.: 058636.00815

[0002] GENERATING NEO-ANTIGENS BY mRNA TRANSLATIONAL ERRORS TO BE TARGETED BY THERAPIES

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims priority to U.S. provisional application no. 63 / 699,007, filed September 25, 2024, the entire disclosure of which is incorporated herein by reference.

[0005] SEQUENCE LISTING

[0006] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy was created on September 25, 2025, is named “058636.00815.xml” and is 36,497 bytes in size.

[0007] RELATED INFORMATION

[0008] Current cancer therapies target the genetic drivers, overexpression, signatures, or mutations that are specifically found in the DNA of cancer cells. There is an ongoing need for developing new approaches to treatment of cancer and other disorders where new or enhanced immune response is produced. The present disclosure is related to this need.

[0009] BRIEF SUMMARY

[0010] The disclosure provides a method for treating cancer comprising administrating to an individual who has the cancer an inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPPD) and an immune checkpoint inhibitor. In an example, the HPPD inhibitor is nitisinone. In examples, the nitisinone is administered in combination, which may be a concurrent or sequential administration, with an immune checkpoint inhibitor that is any of anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-LAG-3 antibody, an anti- TIM-3 antibody, an anti-VISTA antibody, an anti-B7-H3 antibody, an anti-TIGIT antibody, or any combination thereof. In non-limiting examples, the individual has pancreatic adenocarcinoma (PDAC), kidney cancer, or liver cancer., or breast cancer, or lung cancer. It is expected the method will be applicable for treating other cancer types, particularly those that are characterized by having at least a primary tumor. In examples, administering a combination of nitisinone and an immune checkpoint inhibitor produces an anti-cancer result that is greater than using the nitisinone or the immune checkpoint inhibitor alone. The anticancer result may be a synergistic result. The method may be used for treating cancers that are initially resistant to immune checkpoint blockade therapy. In an example, administering nitisinone and an anti-PD-1 antibody results in greater inhibition of PDAC growth than using the nitisinone or the anti-PD-1 antibody alone.

[0011] In another but related aspect, the disclosure provides a method for promoting translation of proteins within cells such that the cells comprise one or more misincorporated amino acids, wherein at least some of the proteins that are translated with the one or more misincorporated amino acids comprise at least one segment that has a candidate immunogenic property. This method comprises providing a stimulus to the cells such that amino acid misincorporation occurs during translation. This method can be practiced by altering the amount of at least one amino acid that is available to the cells for use in translation, such as by increasing or decreasing the amount of at least one type of amino acid available to the cells for use in translation or altering the ratio of at least two different amino acids that are available to the cells for use in translation. In an example, altering the amount of the at least one amino acid comprises increasing the amount of tyrosine available for use in translation and / or altering of a phenylalanine-tyrosine ratio available to the cells for use in translation. In examples, at least one misincorporated amino acid comprise a tyrosine incorporated into the protein from a phenylalanine codon. In examples, a misincorporated amino acid is present in the segment of the protein that has a candidate immunogenic property. In examples, the disclosure further includes detecting the misincorporation of the tyrosine within a protein by detecting a change in a detectable signal. The detectable signal is detectable only when a tyrosine is misincorporated into the protein. This method can be performed in vitro or in vivo. In an example, the cells are present in an individual, and the stimulus comprises delivering to the cells an agent that promotes the amino acid misincorporation in the cells. In examples, cells that have the one or more misincorporated amino acids are cancer cells, and the at least one segment that has a candidate immunogenic property comprises a neoantigen. The disclosure further includes determining the amino acid sequence of the segment of the protein that has the candidate immunogenic property, and optionally testing the segment of the protein that has the candidate immunogenic property to determine whether or not the immunogenic property is present. In examples, the segment of the protein that has the candidate immunogenic property elicits an innate or adaptive immune response, or a combination thereof. In examples, a humoral immune response is elicited. In examples, the humoral immune response comprises antibody-dependent cellular cytotoxicity comprising antibodies directed to a cell surface protein comprising a misincorporated amino acid. In examples, an adaptive immune response is elicited and includes a cell-mediated immune response.

[0012] The disclosure includes an isolated or recombinantly produced protein or peptide comprising a misincorporated amino acid produced according to the described methods, as well as polynucleotides encoding the same. In an example, an mRNA encoding a protein or peptide identified by a described method is provided and may be used in methods of stimulating immune responses. As such, the disclosure provides a therapeutic or prophylactic composition comprising protein or an immunogenic segment of the protein, or a polynucleotide encoding the protein or peptide. In an example, the disclosure provides a method comprising introducing into cells an HPPD inhibitor such that a component of a major histocompatibility complex (MHC) is translated with a tyrosine incorporated into the MHC component from a phenylalanine codon. In examples, the MHC is a Human Leukocyte Antigen (HLA). In examples, a described method results in an HLA that can display an antigen that the HLA could not display without the misincorporated tyrosine.

[0013] BRIEF DESCRIPTION OF FIGURES

[0014] FIG. 1. Harnessing Tyr misincorporation to create non-genetic iTNEs for cancer therapy, (a) Overview of of exemplary methods of the disclosure, (b) Schematic of iTNEs driven by mutational burden or Phe-to-Tyr (FtY) misincorporation in tumor cells.

[0015] FIG. 2. TyrPhe imbalance increases FtY misincorporation, (a) EGFP fluorescence requires cyclization of Tyr66 during maturation. Green fluorescence is abolished by Y66F mutation, (b) Schematic of FtY reporter system. Mutation of critical Tyr to Phe blocks EGFP, mCardinal (mCard), and Katushka2S (K2S) fluorescence in cells growing in normal conditions and can only fluoresce in FtY-inducing conditions, (c) EGFP fluorescence of mCherry+ PDAC cell expressing empty vector (EV)-, EGFP WT-, and Y66F-IRES-mCherry grown in normal and TyrHi:PheLo conditions, (d) Percentage of EGFP+ cells from mCherry+ FtY reporter cells grown at various Tyr concentrations in the absence of Phe. (e) zsGreen+ PDAC FtY reporter (mCard-IRES-zsGreen) cells grown as described in d. (f) Time-course of zsGreen+ PDAC cells undergoing FtY misincorporation (%mCard+) in the indicated media conditions, (g) FtY misincorporation (%EGFP+) in mCherry+ PDAC cells grown in the indicated media conditions, (h) Effects of TyrPhe imbalances on the percentage of zsGreen+ PDAC cells undergoing FtY misincorporation (%mCard+).

[0016] FIG. 3. Tyr is directly misincorporating into Phe in FtY reporters, (a-e) Percentage of FtY misincorporating mouse PDAC (a-b), kidney (c) and liver (d) cancer, and neuroblastoma (e) cells grown as indicated, (f) Schematic of13Cg-Tyr labeling to measure FtY misincorporation (12Cg-F->13Cg-Y) and newly synthesized (12Cg-Y->13Cg-Y) proteins by proteomics, (g-h) Proteomics of K2S-FLAG reveal that 20% of newly synthesized proteins (g) and peptides (h) undergo FtY misincorporation in TyrHi:PheLoconditions.

[0017] FIG. 4. Consequence of FtY errors in PDAC. (a) Schematic of tRNA pulldown to measure charged AAs and RNA modifications, (b) Pulldown of charged tRNAPhefrom PDAC cells grown as indicated, (c) IVT assay of EGFP WT or FtY mRNA using the indicated conditions, (d) PUS siRNA screen in high Tyr. (e) FtY errors in cells transfected with indicated mRNAs, (f) Effects of pathway inhibitors on FtY errors, (g) Kinases predicted to phosphorylate RPSUD2. (h) Representative depiction of FtY-pathway. (i) Growth of PDAC cells, (j) Global and surface proteome of F-containing peptides. FtY peptides detected in normal conditions (black circles) can reflect low FtY events or FtY missense (het or homo) mutations in aneuploid PDAC cell lines.

[0018] FIG, 5. High Tyr increases FtY errors and suppresses PDAC tumor burden in an immune-dependent manner, (a) Phe concentrations in >100 regions of the indicated mouse PDAC tumors, (b) Schematic of Tyr catabolism and effects of tyrosinemia type 1 (TT1) and NTBC indicated in red and blue, respectively, (c) NTBC increases plasma Tyr. (d) NTBC does not affect mouse weight, (e) Schematic of orthotopic PDAC xenografts treated with or without NTBC. (f) PDAC tumor volume as described in e. (g-h) Tumor burden of NTBC- treated mouse PDAC xenografts in C57BL / 6 (g) and athymic nude (h) mice, (i) Schematic of tumor digestion for FtY analysis of FtY reporter PDAC cells. Immune profiling will be performed on non-reporter cells, (j) CK19, cleaved caspase 3 (CC3), and CD8 staining of mouse PDAC tumors, (k) FtY errors of PDAC FtY reporter cells from control and NTBC- treated tumors. (I) Mouse PDAC FtY reporter tumor from NTBC-treated mice. Areas 1 and 2 reflect areas with and without FtY errors, respectively

[0019] FIG. 6. Targeting cell surface FtY epitopes in PDAC tumors, (a) Anti-HA antibody (PDB:5XCU) bound to HA peptide, (b) Schematic of cell surface (s)HA WT and FtY sequences, (c) Schematic of sHA WT and FtY reporter PDAC cells treated with chimeric mouse lgG2a anti-HA under normal and FtY conditions, (d) Cell surface staining of sHA(FtY) in control and high Tyr. (e) Percentage of sHA reporter cells stained with anti-HA from d. (f) ADC-treatment in FtY-promoting conditions (1000uM Tyr: 25uM Phe) as indicated, (g) in vitro assay of NK- or macrophage- driven ADCC, ADCP, or CDC assay targeting sHA WT and FtY expressing PDAC cells with chimeric lgG2a anti-HA grown in control and high Tyr. (h) sHA is not immunogenic in mouse PDAC tumors, (i) Schematic of orthotopic PDAC xenografts in C57BL / 6 or nude mice treated with or without NTBC, ICB (anti-PD-L1 and / or -CTLA-4) or anti-HA (brown arrows), (j) Structure of MHC-I peptide binding pockets A-F. Pocket B is a primary anchor site that binds position 2 (P2) of the peptide. F9 is highlighted as a residue that is important for P2 binding., (k) Schematic of peptides presented on MHC-I is altered upon FtY misincorporation. The amino acid sequence YPYDVDPDYA is SEQ ID NO:39.

[0020] FIG. 7. Targeting FtY cell surface proteins to induce antibody-dependent cellular toxicity (ADCC). Schematic of subcutaneous mouse PDAC (e.g. DB168) xenografts in athymic nude mice treated with or without NTBC or anti-HA (arrows) (left panel). Tumor volume measurements are shown on the right panel. Anti-HA treatment decreases tumor growth expressing the surface HA (sHA) wild-type (WT) reporter in both control and NTBC- treated mice. While anti-HA treatment (arrows) decreases sHA FtY reporter tumors only in mice treated with nitisinone (NTBC).

[0021] FIG. 8. FtY misincorporation of major histocompatibility complex I (MHC-I) in high Tyr conditions. Structure of human MHC-I (e.g. human leukocyte antigen A (HLA) showing surface Phenylalanine residues that can affect immunopeptide binding, (left). Detection of FtY misincorporation of specific HLA peptides using proteomics combined with13Cg-Tyr labelling (right). The amino acid sequences in FIG. 8 are RFIAVGYVDDTQFVRF (SEQ ID NO:41) and RFDSDAASQRM (SEQ ID NO:40).

[0022] FIG. 9. FtY misincorporation of affects the immunopeptidome presented by MHC. High Tyr causes FtY misincorporation in immunopeptides presented by MHC and detected using proteomics combined with13Cg-Tyr labelling, (left). Schematic of the different types of neo-antigens created by FtY misincorporation (right). The amino acid sequences in the left panel correspond to SEQ ID NOs 1-38, from bottom to top. The dots in the amino acid sequences represent amino acid flanking residues in the parent protein sequence. The amino acids to the right and the left of the dots are the amino acids immediately N- and C- terminal in the full protein sequence.

[0023] FIG. 10. Data showing other cancer cells exhibit FtY misincorporation upon TyrPhe imbalance. Percentage of FtY misincorporating cells in breast cancer (MDA-MB-231), endometrial cancer (HEC-1-A), and lung cancer (NCI-H441) cells grown as indicated.

[0024] FIG. 11. Schematic (left) and result (right) showing NTBC treatment improves antitumor effects of immune checkpoint blockade therapy. To obtain the results, C57BL / 6 mice were orthotopically injected with KrasG12D, TP53- / - mouse pancreatic adenocarcinoma (PDAC) cells into the pancreata. Mice were treated with or without nitisinone (NTBC) in the drinking water and treated with or without lgG2c (Fc-blocked) anti-PD1 every 3 days for 5- 6weeks. Pancreatic tumor weights were measured at the end of the experiment to assess the effects on tumor burden. As can be seen from the results, combining NTBC with an anti- PD1 antibody produces superior results relative to using NTBC or anti-PD1 alone. It is expected this approach can be extended to other immune checkpoint inhibitors when used in combination with NTBC.

[0025] DETAILED DESCRIPTION

[0026] Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0027] Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.

[0028] Although subject matter of this disclosure will be described in terms of certain examples, but other examples are also within the scope of this disclosure. Various changes may be made without departing from the scope of the disclosure. AH compositions of matter described herein can comprise or consist of any one or combination of composition components, and all steps may comprise or consist of the described steps. The steps may be performed sequentially, and one or more steps may be omitted.

[0029] In examples, the disclosure relates to methods of promoting antigen formation by modulating promoting translation of proteins within cells such that the proteins comprise one or more misincorporated amino acids. In examples, at least some of the proteins that are translated with one or more misincorporated amino acids comprise at least one segment that has a candidate immunogenic property. Promoting antigen formation is performed by providing a stimulus to the cells such that amino acid misincorporation occurs during protein translation, as described further herein.

[0030] “At least one segment” of a protein that has a candidate immunogenic property includes intact proteins, or fragments of such proteins. “Misincorporated” means an amino acid other than the amino acid assigned by a cognate codon is incorporated into the protein during translation. As a non-limiting example, a tyrosine is misincorporated into the protein from a phenylalanine codon.

[0031] A segment that has a candidate immunogenic property may range in the number of amino acids within the segment. In examples, a segment that has a candidate immunogenic properties comprises a candidate linear epitope. In examples, a protein that comprises at least one misincorporated amino may comprise or otherwise form part of a conformational epitope. In examples, the misincorporated amino acid is found in a described segment of a protein that comprises or consists of at least 2 consecutive amino acids, one of which is misincorporated. In examples, the segment comprises 3 or more amino acids. In examples, the segment is of sufficient length to be displayed in a major histocompatibility complex. In examples, the segment is of sufficient length to be displayed in any HLA context.

[0032] The type of protein that includes at least one misincorporated amino acid is not limited, and includes any protein produced by a cell, including without limitation surface displayed proteins, intracellular proteins that may be trafficked to any particular cellular location, including any organelle, or the cytoplasm, or the protein may be secreted. In examples, the type of protein that includes at least one misincorporated amino acid may be any major histocompatibility complex I (MHC-I) protein, including but not limited to a Human Leukocyte Antigen protein, as illustrated in Fig. 8. In examples, misincorporation of one ore more amino acids affects the immunopeptidome, resulting in the capability to display different types of neoantigens, as illustrated in Fig. 9. Fig. 9 illustrates MHC-I neoantigen presentation. It is expected a similar effect can be induced for any HLA type The disclosure includes eliciting a similar effect on MHC-II proteins. The disclosure includes isolated polypeptides and peptides, and isolated candidate immunogenic segments and proteins produced by a described method, and all polynucleotide sequences encoding them. The disclosure includes determining the sequences of described proteins comprising one or more misincorporated amino acids.

[0033] The disclosure provides vaccine formulations that contain the described immunogenic segments or polynucleotides that encode such segments, and methods of using the same, to stimulate an immune response against segments of the polypeptides that are translated with a misincorporated amino acid.

[0034] In examples, a candidate immunogenic property can be tested to confirm its immunogenic activity. The immunogenic activity can comprise stimulation of an innate immune response, a humoral immune response, a cell-mediated immune response, or a combination thereof. In examples, the humoral response comprises one or a combination of antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) comprising antibodies directed to a cell surface protein comprising a misincorporated amino acid, or complement-dependent cytotoxicity (CDC) The target of the immune response may be correlated with or causative of any disorder or condition for which a stimulated immune response could be of benefit, including but not necessarily limited to any type of cancer. In examples, the disclosure provides for production of Tumor neoepitopes (TNEs).

[0035] In examples, the stimulus that promotes amino acid incorporation comprises an alteration of the amount of at least one amino acid that is available to the cells for use in translation. In an example, alteration of the amount of the at least one amino acid comprises increasing or decreasing the amount of at least one type of amino acid available to the cells for use in translation, or altering the ratio of at least two different amino acids that are available to the cells for use in translation. In an example, alteration of the amount of the at least one amino acid comprises increasing the amount of tyrosine available for use in translation and / or altering of a phenylalanine-tyrosine ratio available to the cells for use in translation. In an example, at least one misincorporated amino acid comprise a tyrosine incorporated into the protein from a phenylalanine codon. In an example, the misincorporated amino acid is present in the segment of the protein that has the candidate immunogenic property.

[0036] The disclosure includes detecting the misincorporation a misincorporated amino acid, illustrated by detecting a misincorporated tyrosine within a protein by detecting a change in a detectable signal, said detectable signal being detectable only when a tyrosine is misincorporated into the protein. In examples which are illustrated by the description and figures herein, the detectable signal may be produced by a protein that comprises the misincorporated tyrosine. In examples, two proteins that produce different detectable signals may be used in a described method, such as for detecting the presence or absence of a protein that contains a misincorporated amino acid.

[0037] In examples, a described method is performed in cells in vitro.

[0038] In examples, a described method is performed in vivo, such as in a non-human mammal, or in a human individual. As such, in one aspect, the disclosure provides for use of a stimulus that promotes misincorporation of amino acids in proteins in cells within an individual by delivering to the cells an agent that promotes the amino acid misincorporation in the cells. In a non-limiting example the agent is an inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPPD). In an example, the agent that promotes the described misincorporation is nitisinone.

[0039] The disclosure includes the following provisos that may apply but are not intended to be bnding: the agent is not an indoleamine 2,3-dioxygenase (IDO1) inhibitor; the agent is not a Tryptophan 2,3-dioxygenase (TDO) inhibitor; the individual treated according to this disclosure does not have a mutated gene that encodes TDO; the individual does not have neuroblastoma; the individual does not have colorectal cancer; the individual does not have an infection comprising Fusobacterium nucleatum ; and the individual does not have hereditary tyrosinemia type 1 (HT-1) or alkaptonuria (AKU).

[0040] The disclosure includes administrating to an individual who has cancer an inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPPD) and an immune checkpoint inhibitor. In an example, the HPPD inhibitor is nitisinone. The disclosure demonstrates that administration of an HPPD inhibitor, using nitisinone, can be combined with immune checkpoint inhibition to produce an anti-cancer effect that is improved relative to use of nitisinone or the immune checkpoint inhibitor alone (see, FIG. 11).

[0041] In general, the immune checkpoint inhibitor that is used in combination with promotion of amino acid misincorporation comprises an antibody wherein the term “antibody” includes intact antibodies, and antigen binding fragments thereof. In examples, the antibody may be an antigen-binding (Fab) fragment, an Fab’ fragment, an (Fab’)2 fragment, an Fd, an Fv, a dAb, a single-chain variable fragment (scFv), a camelid antibody, a single-chain Diabody (scDb), or a diabody (Db).

[0042] In some examples, the immune checkpoint inhibitor that is used in combination as described herein may be an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-VISTA antibody, an anti- B7-H3 antibody, an anti-TIGIT antibody, or any combination thereof. In examples, the anti- PD-1 antibody may be pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, or toripalimab. In some examples, the anti-PD-L1 antibody may be avelumab, atezolizumab, or durvalumab. In some examples, the anti-CTLA-4 antibody may be ipilimumab or tremelimumab. In an example, the anti-LAG-3 antibody may be relatlimab. In an example, the anti-TIM-3 antibody is INCAGN02390. In an example, the anti-VISTA KVA antibody is KVA12123. In an example, the anti-B7-H3 antibody is Enoblituzumab. In examples, the anti- TIGIT antibody is Vibostolimab, Etigilimab, or Tiragolumab. Combination of the antibodies may also be administered.

[0043] In examples, an effective amount of an HPPD inhibitor in combination with an immune checkpoint inhibitor is an amount of each agent that when used in combination reduces one or more signs or symptoms of a disease and / or reduces the severity of the disease. An effective amount may also inhibit or prevent the onset of a disease or a disease relapse. A precise dosage for each agent can be selected by the individual physician in view of the patient to be treated. Dosage and administration can be adjusted to provide sufficient levels of a described combination of agents to maintain the desired effect. Additional factors that may be taken into account include the severity and type of the disease state, age, weight and gender of the patient, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and / or tolerance / response to therapy.

[0044] The agents can be provided as pharmaceutical compositions and administered to an individual in need thereof using any suitable route, examples of which include intravenous, intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, and intra-tumoral, depending on the particular cancer being treated. The agents may be introduced as a single administration or as multiple administrations or may be introduced in a continuous manner over a period of time. The HPPD inhibitor and the immune checkpoint inhibitor may be administered concurrently or sequentially.

[0045] In examples, the cells that have the one or more misincorporated amino acids are cancer cells, and the segment that has a candidate immunogenic property comprises a neoantigen that is a candidate for use in prophylaxis or therapy of cancer. In non-limiting examples, the cancer cells are of renal cell carcinoma, breast cancer, prostate cancer, pancreatic cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, colon cancer, esophageal cancer, glioma, glioblastoma or another brain cancer, stomach cancer, bladder cancer, testicular cancer, head and neck cancer, melanoma or another skin cancer, any sarcoma, including but not limited to fibrosarcoma, angiosarcoma, osteosarcoma, and rhabdomyosarcoma, and any blood cancer, including all types of leukemia, lymphoma, and myeloma. In examples, the cancer cells are pancreatic cancer cells, kidney cancer cells, liver cancer cells, or neuroblastoma cells. Representative uses of promoting amino acid misincorporation as a strategy for treating cancer are provided for different cancer types (see Fig.3a-e and FIG. 10).

[0046] As discussed herein, for any candidate immunogenic segments produced according to a described method, the disclosure provides for determining the amino acid sequence of the segment of the protein that has the candidate immunogenic property, and testing the segment of the protein that has the candidate immunogenic property to determine whether or not the immunogenic property is present.

[0047] The disclosure includes therapeutic and prophylactic compositions comprising a protein or an immunogenic segment of a protein produced and identified as described herein. Any described protein or segment thereof can be combined with an immuno-effective amount of any adjuvant. The disclosure includes administering an effective amount of such compositions to an individual in need thereof.

[0048] The disclosure includes isolated or recombinantly produced proteins and segments thereof that have a misincorporated amino acid produced according to any described method.

[0049] The disclosure is illustrated by the following description which pertains to cancer and particularly pancreatic cancer, but is applicable to production, identification, and use of candidate immunogens discussed above for any other cancer or disorder or condition that would benefit from a stimulated immune response.

[0050] A. Pancreatic ductal adenocarcinoma

[0051] As discussed above, an aspect of this disclosure relates improving immune responses to cancer. Pancreatic ductal adenocarcinoma (PDAC) is a representative and non-limiting example of a type of cancer that may be treated using the described methods, and targeting other cancer types as discussed above is included in the disclosure.

[0052] PDAC is characterized by a microenvironment that is severely nutrient deprived, immunosuppressed, has low levels of immunogenic tumor neo-epitopes (iTNE), including neoantigens, and are insensitive to immune checkpoint blockade (<5% response rate). However, long-term survival studies revealed that neoantigens could stimulate T-cell killing in PDAC. Primary PDAC tumors enriched with immunogenic neoantigens have higher densities of activated CD8+ T-cells that are correlated with lower recurrence and increased patient survival. Although de novo neoantigens have significant potential in cancer immunotherapy, prior approaches rely on genetic alterations that may or may not be presented in PDAC cells. Without intending to be constrained by any particular theory, it is considered that mechanisms that regulate mRNA translational errors can be harnessed to create non- genetic iTNEs in PDAC tumors and sensitize them to immune-dependent therapies (Fig.1 ).

[0053] The mRNA translation error rate is estimated to be <1% for every codon because of the multiple checkpoints during mRNA translation, including tRNA selection, charging, editing / proofreading, and ribosomal decoding of mRNA. tRNA editing deficiencies and ribosomal mutations can increase the misincorporation frequency. Severe tryptophan- restriction was found to increase tryptophan to phenylalanine (Phe) misincorporation resulting in low frequency iTNEs. This indicates that mistranslation can globally give rise to TNEs without depending on the cancer genome. However, inhibiting tRNA synthetase editing does not provide sufficient tumor-specificity, and selective AA deprivation in tumor is difficult. Instead of severe depletion of a single AA, the disclosure reveals that AA imbalance can influence the mRNA translation error rate in PDAC tumors and other cancers.

[0054] The disclosure incldues elucidating the condition(s) and mechanism(s) that regulate the frequency of mRNA translational errors in PDAC, and to analyze their role during PDAC tumorigenesis. The disclosure relates in part to inducing tyrosine (Tyr)-misincorporation because it is the most effective for mediating molecular recognition of proteins, such as antibodies and biologies. The disclosure includes Phe-to-Tyr (FtY) reporters that fluoresce upon Tyr misincorporation during mRNA translation (Fig.2a-c). Without intending to be bound by any particular theory, it is considered that increasing FtY misincorporation events in cancer can increase the repertoire of cell surface iTNE in PDAC and other types of cancer as described herein, which can be harnessed to identify novel immune-dependent therapeutic avenues in the treatment of cancer patients.

[0055] PDAC is an aggressive disease with poor prognosis and is predicted to become the second leading cause of cancer-related deaths in the United States by 2030. Current standard of care for PDAC patients includes combined surgery, chemo- or radiation-therapy. Unfortunately, due to late detection, recurrence, and unsuccessful therapies, the overall 5- year survival is <12%. This unacceptable outcome reflects incomplete understanding of PDAC tumor biology, which has restricted the scope of current therapies.

[0056] PDACs are characterized by a complex desmoplastic stroma that is poorly vascularized and severely deprived of nutrients, such as amino acids (AAs). Furthermore, most PDAC tumors harbor oncogenic mutations in KRAS and TP53, have low levels of immunogenic tumor-neoantigens, and are insensitive to current immunotherapies in the clinic. Although PDAC cells can utilize metabolic crosstalk (e.g. stellate cells, nerves, etc) and scavenging pathways (e.g. autophagy, etc), certain AAs can still remain limiting in different regions of the tumors. Nutrients are important for fueling various metabolic pathways required for growth and imbalances can affect multiple cellular processes, including the ability to synthesize proteins. Because PDAC tumor cells need to make proteins for growth, adaptation, and avoidance of the immune system, targeting specific mRNA translational processes may provide a therapeutic index for treating cancer patients. mRNA TRANSLATION, AA IMBALANCES, and ERRORS

[0057] Cancer cells contains ~6 billion proteins and makes -120 million new proteins per hour to maintain essential functions. In cells, messenger RNA (mRNA) is used as a template to make proteins through a series of elegant and well-ordered reactions by the ribosomes, transfer RNAs (tRNAs), and accessory proteins, that work together in a process known as mRNA translation. The ribosome is a large macromolecular complex of polypeptides and ribosomal (r)RNA that binds mRNA and tRNAs to synthesize proteins. tRNAs are structural RNA molecules that are necessary to interpret and read the genetic code on mRNA to make proteins by correctly delivering a covalently linked (a.k.a “charged tRNA”) amino acid (AA) to the ribosome. In order to accurately translate mRNA into functional protein, these steps are highly regulated to minimize errors (e.g. mischarging, misincorporation, mis-decoding, etc) during mRNA translation.

[0058] The error rate for mRNA translation is estimated to be 104-103, or <1% for every codon. This low error rate is regulated by multiple checkpoints during mRNA translation, including tRNA selection, charging, editing / proofreading, and ribosomal decoding. tRNA editing deficiencies and ribosomal mutations can increase the frequency of AA misincorporation during mRNA translation. Severe Trp-deprivation has been reported to cause Trp to phenylalanine (Phe) misincorporation (WtF) and created iTNE at a low rate. Whether WtF misincorporated TNEs can be targeted in cancer has previously remained to be elucidated. Nonetheless, this means that mistranslation can create TNEs without altering the genome of cancer cells. However, inhibiting tRNA synthetase editing alone does not provide sufficient tumor-specificity for patient treatment, and severe deprivation of a single AA in a tumor is difficult. It also is not clear whether there are other pathways and mechanisms that can regulate mRNA translation errors. By understanding the conditions and mechanisms that trigger mRNA translation errors, the present disclosure includes perturbing mRNA translation in cancers to generate non-genetic iTNEs and sensitize them to immune- dependent therapies.

[0059] Increasing mRNA translational errors by severe-deprivation of a single AA, but not the other AAs, in tumors is difficult. Intead of severe Trp-deprivation being solely attributed as the cause for WtF misincorporation, an alternative explanation could be an AA imbalance between Trp and Phe. This AA imbalance suggests that the relative abundance or scarcity of specific AAs could influence the fidelity of mRNA translation, resulting in the misincorporation of AAs. Without intending to be bound by any particular interpretation, it is considered that increasing the availability of certain AAs, such as Tyr, can cause an AA imbalance and increase misincorporation in PDAC and other cancers.

[0060] CANCER IMMUNOTHERAPY AND NEO-ANTIGENS

[0061] Cancer immunotherapy works by stimulating the body’s anti-tumor immune response. Immune checkpoint blockade (ICB) therapies (e.g. anti-PD1 , -CTLA4, and others as discussed above) has revolutionize the treatment and outcome of cancer patients by inhibiting immunosuppressive pathways in tumors and sensitizing them to immune-mediated cell death. Unfortunately, clinical trials have found that ICB therapy has limited efficacy in many solid tumors, including PDAC. Some tumors with high mutational burden (e.g. melanoma) are more responsive to ICB because of immunogenic tumor-neoantigen presentation on major histocompatibility complex I (MHC-I) that are recognized by the immune system to trigger cell death. Whereas ICB-insensitive tumors can lack sufficient immunogenic tumor-neoantigens or escape immune-survalience. However, neoantigens are important player in PDAC therapy as they were found to stimulate T-cell killing in long-term survivors.

[0062] Tumor neo-epitopes (TNE) are new protein sequences that can arise in cancer cells (e.g. mutations, viral proteins, etc) and are found on the cell surface or presented as peptides by MHC-I. Immunogenic TNEs (iTNEs) presented on the cell surface can be detected by biologies (e.g. antibodies, nanobodies, bispecific antibodies, recombinant protein, etc) that are recognized by phagocytic cells (e.g. macrophages, natural killer (NK), dendritic cells, etc) or specific-TCRs (T-cell receptors) on T-cells. However, not all missense mutations are immunogenic or can be distinguished from non-mutated versions by biologies and TCRs. Although, iTNEs have potential in clinical therapies, the heterogeneity of cancer and scarcity of iTNEs prove to be major challenges. New iTNEs can be created by radiation to induce mutations or viral delivery of foreign genes in cancer cells. However, these methods can cause irreversible genetic changes in non-cancerous tissues that lead to increased risks of unwanted side effects, such as new cancerous lesions. Therefore, the present disclosure provides different methods of generating iTNEs without altering the genome in a radiation and virus free manner.

[0063] Antibodies and similar biologies belong to a class of molecules called immunoglobulins that recognize and bind to specific targets to neutralize or eliminate them. Immunoglobulins have two domains, a constant and variable domain. The constant region determines the class of immunoglobulin (e.g. IgG, IgA, etc) and provides structural stability and mediates effector functions, such as activating complement and binding Fc receptors. The variable domain (a.k.a. Fab, Fragment antigen-binding) consists of unique protein sequences that are responsible for the specificity and strength of antigen recognition and binding. Directed evolution of variable domains to create stronger and specific binders to a target revealed high enrichment of Tyr. The physiochemical properties of Tyr (e.g. hydrogen bonding, Van der Waals, and pi-stacking) makes it effective at mediating molecular recognition. Therefore, without intending to be constrained by any particular interpretation, it is considered that increasing Tyr misincorporation will increase the availability of iTNE that can be recognized by immune-dependent therapies.

[0064] PDAC tumors are characterized by a nutrient poor environment, have low levels of iTNEs, and are insensitive to immune checkpoint blockade (ICB). However, immunogenic neoantigens can stimulate T-cell killing and decrease relapse in long-term PDAC survivors. The disclosure demonstrates that increasing FtY misincorporation in PDAC generates non- genetic iTNEs that can be targeted by immune-dependent therapies to improve the outcome of PDAC patients, and other cancers as described herein. The present disclosure provides molecular reporters and techniques to elucidate the effects of the AA environment on the frequency of FtY misincorporation in PDAC, assesses the frequency and impact of FtY misincorporation during PDAC tumorigenesis, and provides an approach comprising combining FtY misincorporation with immune-dependent therapies in PDAC tumors.

[0065] Neoantigens can stimulate T-cell killing of tumors and increase long-term survival of PDAC patients. However, the number of neoantigens is limited to mutational burden and susceptible to genetic heterogeneity in PDAC. The present disclosure provides for generating non-genetic immunogenic tumor neo-epitopes (iTNEs) in PDAC and other cancers as described above via describing the conditions and mechanisms that can increase the Phe to Tyr (FtY) misincorporation in tumors. The disclosure includes targeting FtY iTNEs with immune-dependent therapies during PDAC tumorigenesis. Although as aspect of this disclosure relates to PDAC, the described effects of FtY misincorporation also can be applied to the treatment of other cancers, as demonstrated on the figures that accompany this disclosure.

[0066] The disclosure relates in part to the impact of AA imbalance on the FtY misincorporation and the generation of non-genetic iTNE on PDAC tumorigenesis (Fig.1 ), the analysis of environmental conditions that affect the frequency of mRNA translation errors in PDAC cells; (2) assessment of mRNA translational errors generating appreciable amounts of iTNEs, and (3) FtY misincorporated iTNEs as targets to improve the outcome and survival of PDAC patients. Aspects of the disclosure relate to use of human and mouse PDAC cell lines (e.g. RATU-8902, DB168); for convenience, descriptions below refer generically to “PDAC cells”.

[0067] Elucidating the conditions and effects of FtY misincorporation in PDAC

[0068] Aspects of this disclosure relate to Tyr-misincorporation because it is effective at mediating molecular recognition of proteins. Because of the similarities of Phe and Tyr and their codons (TT[T / C] vs T[A / C]T), we generated Phe-to-Tyr (FtY) fluorescent protein (FP) e.g. EGFP and other reporters that only fluoresce upon Tyr misincorporation. We found that FtY reporters are not fluorescent under normal growth conditions, but will fluoresce in high Tyr and low Phe (TyrHi:PheLo) (Fig.2a-c). This indicates that FtY misincorporation can occur in response to Tyr: Phe imbalance. High Tyr (300-2000pM) is physiologically relevant as it is achievable in human patients treated with an HPD (hydroxyphenylpyruvate dioxygenase) inhibitor, nitisinone (NTBC, a.k.a. Orfadin), which blocks Tyr catabolism. Experimental Design / Methods i) Conditions that promote FtY misincorporation

[0069] The disclosure includes modulating conditions that may affect the FtY misincorporation frequency in PDAC and other cancers as described herein, as follows. a) TyrPhe imbalance: Because FtY reporters activate in the TyrHi:PheLo, this suggests that the TyrPhe ratio can affect the FtY errors in PDAC cells. The disclosure includes expressiong FtY reporters in human and mouse PDAC cells to assess whether TyrPhe imbalances can increase FtY errors by fluorescent microscopy and flow cytometry. FtY reporter PDAC are cultured in different concentrations of Tyr and Phe that are physiologically relevant in the human body (e.g. tumors, plasma, and NTBC-treatment). High Tyr: Phe would increase FtY misincorporation in PDAC cells, but suppressed when sufficient Phe is present. The described data found that TyrHi:PheLocan increase FtY errors in PDAC cells over time (Fig.2d-f). Furthermore, high Tyr equally drives FtY misincorporation in both Phe codon (TT[T / C]) reporters, indicating that specific Phe codons do not affect FtY errors (Fig.2f). Although the high TyrPhe ratio increases FtY misincorporation, the effect size also is affected by Phe concentration (>100|JM, Fig.2g-h). This suggests that TyrPhe imbalance can drive FtY misincorporation in PDAC cells, and in other cancer cells as described herein. Because PDAC tumors are severely AA-deprived, specifically increasing Tyr availability has the potential to induce FtY misincorporation in PDAC cells. Furthermore, the described data reveal that other cancer cell types also increase FtY misincorporation in high Tyr conditions (Fig.3a-e and Fig. 10). Tyr misincorporation using FtY reporters in normal and tumor mouse pancreatic organoids can also be assessed. b) Proteomics: The disclosure includes determining if FtY reporters are undergoing FtY misincorporation by using proteomics to identify Phe sites that become Tyr, which has an extra oxygen (+16m / z). To initiate experiments, PDAC are switched to media containing13Cg- Tyr to separate FtY misincorporation (9+16 = +25m / z) from the background oxidation (+16m / z) of peptides. FtY reporters (e.g. Katushka2S-FLAG) are purified with anti-FLAG magnetic beads, trypsin digested, and analyzed by liquid chromatography tandem-mass spectrometry (LC-MS / MS) (Fig.3f). The disclosure includes assessing13Cg-Tyr incorporation into12C-Tyr and12C-Phe sites to measure newly synthesized and FtY misincorporated peptides, respectively. Incorporation of13Cg-Tyr into12Cg-Tyr-containing peptides reflects newly synthesized peptides. Whereas,13Cg-Tyr incorporation into12Cg-Phe-containing peptides represents newly FtY misincorporated peptides. Based on these measurements, the disclosure provides for calulating the FtY misincorporation rate of newly synthesized Phe (F)-containing peptides. The described data reveal that every newly synthesized F- containing peptide has a 20% risk of undergoing FtY misincorporation in high Tyr conditions (Fig.3g-h). This also reveals that there are no specific differences in the FtY misincorporation error rate between the two Phe codons in Katushka2S-FLAG. ii) Mechanisms of FtY misincorporation: Because13Cg-Tyr is incorporated into the FtY reporter, this means that FtY misincorporation is caused by errors in tRNA charging or ribosomal decoding, and not due to post-translational oxidation of Phe (Fig.3f-h). This disclosure includes assessing whether high Tyr-induced FtY misincorporation is caused by tRNA mischarging of Phe tRNAs (tRNAPhe) with Tyr or decoding errors by the ribosome with Tyr charged tRNATyr(Tyr-tRNATyr). a) tRNA mischarging: We have tested if high Tyr affects tRNA charging of tRNAPheusing biotinylated anti-sense oligo and metabolomics (Fig.4a). We found that high Tyr did not significantly change tRNAPheor tRNATyrlevels, or the mischarging of tRNAPhewith Tyr (Fig.4b). This indicates that high Tyr likely increases ribosomal decoding errors of Phe codons by tRNATyrto drive FtY misincorporation in PDAC cells. b) in vitro translation assay (IVT): To undergo FtY misincorporation, PDAC cells may need to activate signaling cascades to regulate RNA modification or post-translation modifications (PTMs) in response to TyrHi:PheLoconditions. The disclosure includes performing IVT assay using extracts from PDAC cells grown in physiological and TyrHi:PheLoconditions while varying the concentrations of Tyr and Phe used in the assay. Protein synthesis of FtY reporter mRNA are quantified by kinetic fluorescent readings and wild-type (WT) reporter mRNA is used as a positive control. The presence of FtY fluorescence determines Tyr: Phe imbalance, modifications, or both can be used for FtY misincorporation. The described data reveals that high Tyr alone did not induce FtY reporter fluorescence using commercial IVT kits or in-house physiological or high Tyr extracts (Fig.4c). Given that only AAs and mRNA are added to high Tyr extracts in IVT assays, this indicates that Tyr: Phe imbalance alone is not sufficient at inducing FtY misincorporation and suggests that post- transcriptional mRNA modifications that occur in high Tyr are important, which can lead to ribosomal or mRNA decoding errors. Therefore, the disclosure includes assessing the FtY errors using modified mRNA identified below in IVT assays. c) Ribosome accuracy, RNA modifications or PTMs: Cancer cells can alter RNA modifications in response to stress. Ribosomal decoding errors may reflect changes in RNA modifications (rRNA, tRNA, mRNA). The disclosure includes biochemical assays to elucidate the role of rRNA, tRNA, and mRNA modification in FtY misincorporation. c.i) Pseudouridine synthases (PUS): There are ~13 PUS’ that can modify uridine (U) to pseudouridine (^PU) in mRNAs, tRNAs, rRNAs to regulate RNA stability, splicing, or basepairing. Because ^PU modifications can affect mRNA translation fidelity, the disclosure includes assessing if PUS can regulate FtY misincorporation using an arrayed siRNA screen. The described results from a siRNA screen revealed that RPLISD2 and DKC1 knockdown (KD) did not affect wild-type (WT) fluorescence intensity, but decreased and increased FtY misincorporation intensity in PDAC cells, respectively (Fig.4d). RPLISD2 modifies mRNA, while DKC1 uses a H / ACA snoRNA to specifically modify rRNA. This suggests that loss of ^PU modifications can affect the decoding fidelity of mRNA or ribosome. Consistent with this, PDAC cells transfected with in vitro transcribed ^PU and ml ^PU FtY mCardinal mRNA significantly increased FtY misincorporation compared to II and methoxyll (5moU) mRNA (Fig.4e). Because ^PU, ml^PU, and 5moU can all increase mRNA stability, this suggests that ^PU and ml^PU promote FtY errors by affecting ribosomal-mRNA decoding. This can be validated using sgRNA and sg / siRNA-resistant cDNAs of WT or catalytically inactive (Cl) RPUSD2 and DKC1. c.ii) Effects of RNA modifications: tRNATyr, rRNA, and FtY reporter (EGFP, K2S, or mCard) mRNA are enriched using anti-sense oligos from cells grown in control and high Tyr and spiked into IVT assay (e.g. Fig.4c). Purified tRNAs, rRNAs, or mRNAs are prepared for direct RNA oxford nanopore sequencing (ONT) or digested into nucleosides to measure RNA modification(s) changes by LC-MS (Fig.4a). The disclosure also includes assessing changes to RNA modifications in control or high Tyr with or without depletion of RPUSD2 or DKC1. It is expected that tRNA, mRNA, or rRNA from high Tyr cells will have differential ^PU modifications that are dependent on RPUSD2 or DKC1. c.iii) Biochemical signaling: Because low Phe can activate the two major AA sensing pathways, GCN2 and mTOR, the disclosure includes assessing their role in regulating FtY errors. FtY reporter cells grown in control or high Tyr are treated with GCN2iB, Torinl , GSK2606414, or ISRIB to inhibit GCN2, mTOR, PERK or p-elF2a, respectively. The described data found that GCN2 inhibition delays, but did not block, FtY errors in PDAC cells (Fig.4f). This suggests that GCN2 substrate(s) is important for early FtY errors, but not completely necessary for FtY misincorporation in PDAC cells. elF2a is phosphorylated by all integrated stress response kinases (ISRKs), and inhibition with ISRIB weakly suppressed FtY errors in PDAC cells (Fig.4f). These results can be supported using sgRNA, shRNA or cDNA mutants of GCN2, PERK, or elF2aS51Ain PDAC cells, WT will is used as a control. The described results suggest that another pathway is contributing to FtY errors.

[0070] Because FtY errors was higher in PDAC cells transfected with ^PU, but not U, FtY mCardinal mRNA (Fig.4e), this indicates that (1) RNA modifications are occurring on mRNA, (2) PUS’ cannot efficiently modify unmodified cytosolic mRNA, and (3) PUS enzymes are active in the nucleus, perhaps co-transcriptionally. Otherwise, U- or 5moU-FtY mCardinal mRNA should have reached similar levels of FtY errors. Consistent with this, CDK and DHODH inhibitors that are known to decrease transcription blocked FtY errors in PDAC cells. Furthermore, RPLISD2 is predicted to be phosphorylated by ISRKs and CDKs, such as GCN2 and CDKs (Fig.4g). The disclosure includes immunoprecipitating endogenous or FLAG-tagged RPLISD2 in PDAC cells grown in control or high Tyr, with or without GCN2 or CDK inhibitor to measure phosphorylation differences by immunoblot and phosphoproteomics. RPLISD2 Ser-to-Ala phospho-mutants will be used to assess FtY errors in RPLISD2 KO cells. The disclosure also includes determining RPLISD2 nuclear localization in PDAC cells grown control and high Tyr and the effects of CDK or GCN2 inhibitors by immunofluorescence microscopy or cellular fractionation. It is expected that high TyrPhe increases mRNA pseudouridylation of newly transcribed transcripts in a RPUSD2-dependent manner, which contributes ribosomal decoding errors that increase FtY misincorporation in PDAC cells (Fig.4h). iii) Biological effects of FtY misincorporations: High Tyr-induced FtY misincorporations could directly or indirectly affect PDAC cancer biology. The disclosure includes assessing the effects of FtY misincorporation on PDAC: a) Cell Growth: The disclosure includes monitoring if activation of the FtY reporter is associated with changes in cell proliferation or death. PDAC cells expressing FtY reporters will be grown in physiological and high Tyr conditions that induce FtY misincorporation for several days. Cell proliferation will be measured using live-cell and fluorescent microscopy (Cytation C10) and cell counting. Furthermore, viability will be assessed by flow cytometry using Annexin V and DAPI staining. Because -20% of every Phe codon can undergo FtY misincorporation (Fig.3f-h), it was predicted that high Tyr will not change proliferation or death. The described data show that high Tyr did not affect the growth of PDAC cells under similar Phe levels (Fig.4i). This indicates that high Tyr and FtY misincorporation does not decrease, but more importantly does not increase, the growth rate of PDAC cells. b) Proteome: The disclosure includes identifying FtY misincorporation of endogenous proteins in PDAC cells grown in high Tyr, particularly for cell surface proteins that can act as iTNEs. The disclosure includes evaluating the effects of FtY misincorporations on the proteome by global and cell surface proteomics. PDAC are grown in control and high Tyr media containing13Cg-Tyr to identify FtY misincorporated peptides (Fig.3f). Proteins are extracted from PDAC cells by TCA precipitation and cell surface proteins are enriched by cell surface-biotinylation and streptavidin pulldown. Proteins are trypsin-digested into peptides for LC-MS / MS proteomics. Percentage of newly synthesized (12Cg-Y->13Cg-Y) and FtY misincorporation (12Cg-F->13Cg-Y) peptides of a specific protein are calculated. It is expected that the percentage of newly synthesized proteins and FtY misincorporation will vary in the proteome due to different synthesis rates, and half-lives. Because we detected13Cg-Tyr labelling of the FtY reporter, we expect that a fraction of the global, cell surface antigens, and MHC-I antigens on PDAC cells will contain FtY errors. The described data revealed that high Tyr induced a FtY misincorporation frequency of >20% of newly synthesized proteins in both global and cell surface proteome (Fig.4j).

[0071] Differences in FtY error rate in high Tyr can occur between cell lines (Fig.3a-e). This would suggest differences in genetic mutations or expression could regulate FtY misincorporation in cancer, which can reveal biomarkers and identify FtY-inducing drug targets. In this scenario, the disclosure includes bioinformatically determining pathway enrichment of cancer cells exhibiting high- and low- FtY errors and validation using sgRNAs or cDNAs. It may be difficult to detect FtY misincorporation for most proteins by proteomics due to low coverage of low abundant proteins. Nonetheless, it is more likely that abundant proteins are presented by MHC-I. The disclosure also includes fractionating peptides and running longer gradients to obtain better coverage by LC-MS / MS proteomics to measure FtY misincorporation of F-containing peptides. Although examples of this disclosure related to ^Pll modifications on mRNA, other RNA Il-specific modification can contribute to FtY error rates. The disclosure includes detecting these modifications by direct RNA ONT sequencing or by LC-MS / MS of partial RNase-digested RNA fragments near the Phe / Tyr codon.

[0072] 2. Assessing the effects of FtY misincorporation during PDAC tumorigenesis.

[0073] The ability to increase the number and repertoire of immunogenic tumor neoepitopes (iTNEs) using non-genetic altering processes, like mRNA translation errors, can significantly enhance immune-dependent cancer therapies. FtY misincorporation maximally increases in conditions of high Tyr and normal Phe (<100pM) in vitro (Fig.2g-h). Because more than 50% of PDAC tumors regions have <100pM Phe (Fig.5a), without intending to be bound by any particular theory, it is considered that increasing Tyr availability (>300pM) can induce FtY misincorporation in tumors. Nitisinone (NTBC) is an FDA-approved HPD (hydroxyphenyl pyruvate dioxygenase) inhibitor that blocks upstream Tyr catabolism to increase long-term survival in tyrosinemia type I (TT1) patients. However, NTBC treatment also elevates plasma Tyr to superphysiological levels (>300pM) in patients and mice (Fig.5b- c). Therefore, NTBC-treatment provides a means to increase Tyr availability in regions with low Phe during PDAC tumorigenesis.

[0074] Experimental Design / Methods i) Effects of NTBC on PDAC tumorigenesis: NTBC treatment can increase plasma Tyr to high levels (>300pM) in humans and mice. We also confirmed that NTBC-treatment is not toxic in mice (Fig.5d). The disclosure includes assessing NTBC on FtY misincorporation and PDAC tumorigenesis in immune-competent and -deficient mice. All mice will be placed on vehicle or 16mg / L NTBC in the drinking water, and blood is collected from the retro-orbital capillaries every two weeks to measure plasma Tyr and NTBC levels by LC-MS (Fig.5e). a) Immune-competent mice: The disclosure includes orthotopically injecting mouse PDAC cells (e.g. DB168) with or without FtY reporters into the pancreata, which reflects the unique microenvironment of the pancreas, of C57BL / 6 (immune-competent) mice (10 mice / group; Fig.5e). Tumors are monitored and measured weekly by ultrasound for 3-5 weeks, and collected for histological, molecular, and biochemical analysis at the end of the experiment or when mice reach clinical morbidity (e.g. weight loss). Before tumor harvesting, mice are injected with EF5 to measure tumor hypoxia. Tumor sections are stained for markers of proliferation (p-Histone H3, etc), death (Cleaved caspase 3, etc), arrest (p21 , etc), hypoxia (EF5, etc), blood vessels (CD31, etc), nutrient deprivation (p-elF2a, etc) by immunohistochemistry (IHC). With 10 mice / group, we have -80% power (p<0.05, 1-sided Fischer exact test) to detect two-fold differences in tumor size, survival or any specific IHC stain. FtY misincorporation is assessed by flow cytometry or fluorescent microscopy. The described data shows that NTBC treatment increased plasma Tyr in C57BL / 6 mice (~800pM) and decreased mouse PDAC (non-reporter) tumors (Fig.5c, f-g). Although bulk or interstitial fluid measurement have been used to quantify the average AA availability in tumors, these methods cannot detect regionalized AA differences. Therefore, the disclosure includes measuring AAs from -100 pieces / tumor (e.g. Fig.5a). This suggests that NTBC can decrease PDAC tumor burden in immune-competent mice, potentially by increasing Tyr and FtY errors in tumors. b) Immune-deficient mice: To assess the immune-dependent effects of NTBC and in PDAC tumors, non-reporter and FtY reporter mouse and human PDAC are orthotopically injected into the pancreas of athymic nude (T-cell deficient) mice (10 mice / group; Fig.5e). PDAC tumors are monitored, measured, and prepared for downstream analysis. FtY misincorporation of PDAC cells expressing FtY reporters is determined. Because high Tyr induces FtY misincorporation and does not affect growth in vitro, the disclosure shows NTBC effects on PDAC tumor burden is dependent on the immune system and will not affect tumor growth in nude mice. The described data reveal that NTBC-treatment in nude mice had no significant effect on the tumor burden of mouse (non-reporter) PDAC cells (Fig.5h). This indicates that the anti-tumor effect of NTBC in C57BL / 6 mice is dependent on the immune system, more specifically T-cells. c) Immune profiling: Because the described data found that NTBC decreased PDAC tumor burden in an immune-dependent manner (Fig.5g-h), this suggests that there will be differences in the tumor immune microenvironment. Immune profiling of (non-reporter only) tumors are assessed by tissue staining (e.g. IHC / IF) and flow cytometry. For flow cytometry, a piece of the tumor is digested into single cells and stained with cell-specific markers. Cells are stained with anti-CD45 (immune cells), -CD8 (killer T), -F4 / 80 (macrophage), and other markers, to elucidate the immune profile of NTBC-treated (non-reporter) PDAC tumors (Fig.5i). The staining of sections reveal spatial localization of immune cells either on the periphery or within the tumor. The described data reveal higher CD8+ cells in NTBC-treated PDAC tumors (Fig.5j). To elucidate the immune effects of NTBC-treatment on (non-reporter) PDAC tumors, the disclosure includes depletion of specific immune cell populations in C57BL / 6 mice using anti-mouse immune-specific antibodies (e.g. anti-CD8) and measureing effects on tumor burden, proliferation and death. Depletion of specific immune cell populations suppresses the anti-tumor effects of NTBC treatment in C57BL / 6 mice and reflect the observations in nude mice (Fig.5h). ii) Assessing FtY misincorporation in tumors: Because NTBC treatment increases plasma Tyr (~800pM, Fig.5c), the disclosure includes analyzing if PDAC tumors have increased FtY misincorporation events that could create iTNEs: a) Flow cytometry: A portion of the PDAC tumors expressing FtY reporters from C57BL / 6 and athymic nude mice are digested into single cells, stained for CD45 and cell viability, to measure the percentage of live cells that have undergone FtY misincorporation at the time of harvesting (Fig.5i). The disclosure includes gating on DAPIne9;CD45ne9;zsGreenPoscells and measuring the fluorescence of FtY reporters upon NTBC-treatment (10 mice / group). It A portion of the PDAC cells in the tumor will activate the FtY reporter in mice treated with NTBC. The described data revealed that NTBC can increase FtY misincorporation in PDAC tumors in mice (Fig.5k). This indicates that high plasma Tyr increases FtY misincorporation in PDAC, and that tumor Phe levels cannot completely suppress FtY misincorporation in PDAC. b) Spatial distribution of FtY errors: Another portion of PDAC tumors from C57BL / 6 and nude mice is fixed in PFA + 2% L-lysine, equilibrated in sucrose, and embedded in OCT (optimal cutting temperature) to preserve the fluorescence of FtY reporter and internal control (10 mice / group). This is relevant because staining with anti-RFP or -GFP would not measure FtY misincorporation (e.g. fluorescence) of the reporter. It is expected some regions of the PDAC tumor in NTBC-treated mice will have fluorescent FtY reporter, a sign of FtY misincorporation. The described data show that NTBC treatment can increase FtY misincorporation in different regions of mouse PDAC tumors (Fig.5l). To avoid immune clearance, sections from nude mice are stained for blood vessels (CD31), nutrient deprivation (p-elF2a) and hypoxia (EF5) to mark regions and relative proximity to FtY misincorporating PDAC cells. It is expected that areas of FtY misincorporation would depend on TyrPhe imbalances, which may be related to blood vessel proximity, nutrient deprived, or hypoxic regions of the tumor.

[0075] The disclosure includes assessing the impact of high Tyr by NTBC treatment on PDAC tumorigenesis. Because NTBC elevates plasma Tyr, anti-tumor effects of NTBC may be caused by downstream Tyr-dependent metabolites, such as dopamine. However, this is unlikely because we did not detect significant changes by LC-MS. It is possible that there is heterogeneity in response, and that not all PDAC cells will respond in a similar manner to NTBC and high Tyr. PDAC cells may undergo transient FtY misincorporation during tumorigenesis. However, this is addressed because we detected FtY misincorporation in PDAC tumors (Fig.5k-I). Although fluorescent proteins in PDAC tumors are not immunogenic, immune profiling can be be performed on non-reporter PDAC tumors to avoid potential immunogenic effects of the fluorescent proteins.

[0076] 3. Elucidating the effects of FtY-directed Targeted therapies in PDAC tumors

[0077] High Tyr can increase the FtY misincorporation for every Phe codon during mRNA translation and can generate iTNEs that can be targeted by the immune system (Fig.3f-h, 4f-g). As a representative example, the disclosure includes assessing the efficacy of targeting FtY iTNEs to improve the anti-tumor immune response in PDAC. Mice are treated with NTBC to induce FtY misincorporation (Fig.5k-I), and blood is collected every two weeks to measure plasma Tyr by LC-MS as described in (Fig.5c, e).

[0078] Experimental Design / Methods i) Cell surface HA FtY reporter: There are currently no biologies that can be used to specifically target FtY iTNEs on the cell surface of PDAC tumors. The disclosure includes a cell surface FtY system that can be used with commercially available reagents. a) Cell surface HA(FtY): Hemagglutinin (HA: YPYDVPDYA (SEQ ID NO:39)) is used to tag proteins that can be recognized by anti-HA antibodies. The disclosure includes use of commercial mouse monoclonal anti-HA antibody that is dependent on the presence of Y8 in HA and does not bind to the HA FtY reporter (HA(FtY)), where Y8 was mutated to Phe (YPYDVPDFA) (SEQ ID NO:39) (Fig.6a-e). We made a cell surface HA(FtY) reporter by fusing a signal sequence (SS) to HA(FtY) containing a transmembrane domain (referred to sHA(FtY); Fig.6b). As a control, use sHA(WT) can be used. sHA is detected by immunoblot and flow cytometry staining using anti-HA antibody from PDAC cells grown with or without high Tyr. We found that mouse lgG2a anti-HA antibody only recognizes sHA(FtY) when cells undergo FtY misincorporation in high Tyr conditions (Fig.6c-e). This system allows us to test the efficacy of targeting cell surface FtY iTNEs in PDAC tumors in vitro and in vivo models. b) Targeting of sHA(FtY): To test the efficacy of targeting FtY iTNEs, sHA(WT)- and sHA(FtY)- expressing PDAC mCherry+ cells are treated with mouse lgG2a anti-RSV (control) and anti-HA conjugated with lysosomal cleavable anti-mitotic inhibitor (VcMMAF (Monomethyl Auristatin F)) for antibody drug-conjugate (ADC) therapy in normal or high Tyr, with different Phe concentrations. The described data reveal that ADC treatment decreased growth of sHA(WT & FtY)-expressing mCherry+ cells cultured in high TyrPhe FtY conditions (Fig.6f). sHA-expressing PDAC cells are co-cultured with macrophage or natural killer (NK) cells in normal or FtY-conditions (Fig.6g). Co-cultures are treated with mouse lgG2a anti-HA to induce antibody-dependent cell-mediated cytotoxicity (ADCC) or cellular phagocytosis (ADCP). mCherry+ PDAC cells are monitored by live fluorescent cell imaging. Anti-HA, but not control, treatment promotes immune cell-driven clearance of sHA(WT)-expressing PDAC cells, and only sHA(FtY) grown in FtY inducing conditions. ii) Antibody-directed therapies against sHA PDAC tumors: Antibody-directed therapies mainly kill tumor cells by three important mechanisms carried out by monocytes, macrophages, natural killer, neutrophils, dendritic cells, or eosinophils and induce ADCC, ADCP, or complement-dependent cytotoxicity (CDC). These can lead to tumor clearance by the innate immune system or trigger priming and expansion of CD8+ cytotoxic T-cells to enhance tumor cell killing. The disclosure includes determining whether antibody-directed therapies can be used to target cell surface FtY iTNEs in PDAC cells to enhance tumor clearance in mice. a) Immune-competent mice: To assess the efficacy of targeting FtY iTNEs in PDAC tumors, mouse PDAC cells expressing sHA(WT) or sHA(FtY) are injected into the pancreata of C57BL / 6 mice with or without NTBC-treatment. sHA alone does not elicit an immune response or PDAC tumor suppression compared to naive mouse PDAC cells (Fig.6h). Therefore, sHA is a suitable system to test the efficacy of targeting FtY iTNEs in PDAC tumors in mice. Tumors are allowed to establish and grow for 1-week before initiating treatment with chimeric mouse lgG2a anti-RSV (control) or anti-HA injections into the mice every 3 days (10 mice / group; Fig.6i). PDAC tumors are monitored and harvested after 3 weeks post-injection for tumor burden, histology, molecular, and biochemical analysis as described above. Chimeric mouse lgG2a anti-HA treatment will decrease sHA(WT) tumor burden, but will only significantly decrease sHA(FtY) PDAC tumors in NTBC-treated mice compared to untreated controls. This would indicate that NTBC is sufficient to increase FtY misincorporation in PDAC cells to generate FtY-specific iTNEs that can be targeted with biologies to enhance immune clearance. b) Immune-deficient mice: To assess the contribution of T-cells during anti-HA treatment, the disclosure includes orthotopically injecting mouse and human PDAC cells expressing sHA(WT) and sHA(FtY) into pancreata of athymic nude mice treated with or without NTBC, anti-HA, or both (10 mice / group; Fig.6i). Tumors are monitored by ultrasound to measure tumor growth, and harvested to measure effects on tumor burden, histology, molecular and biochemical analysis as described above. Similar tumor reduction by anti-HA treatment between C57BL / 6 and athymic nude mice indicates that anti-tumor effects of this antibody is not dependent on T-cells. Whereas partial efficacy would signal that anti-tumor effects of anti-HA treatment requires T-cells. iii) FtY misincorporation and MHC-I: Data presented herein show that FtY misincorporated proteins in high Tyr (Fig.3f-h; see also FIG. 8). FtY misincorporation may affect the repertoire of peptides presented by major histocompatibility complex I (MHC-I), especially of highly abundant proteins. There also is the possibility that FtY misincorporation can affect the peptide binding groove of MHC-I and alter peptide repertoire on MHC-I. For example, FtY misincorporation at Phe9 (F9) in the primary peptide anchor site of pocket B can potentially alter the preference of peptides that are bound and stabilized by MHC-I (Fig.6j-k). MHC-I loaded with new or mutant peptides (pMHC-l) can be recognized as “nonself’ by T-cell receptors (TCRs) and initiate cytotoxic T-cell killing of cancer cells. However, tumor cells can suppress cytotoxic T-cell killing by expressing “don’t kill me” signals (PD1 , CTLA-4, etc), which can be inhibited by ICB (e.g. anti-PD1 , -CTLA4, etc). The disclosure includes assessing the effects of FtY misincorporation on pMHC-l presentation and T-cell killing in PDAC tumors, and combining of FtY misincorporation with immune checkpoint blockade (see, Fig. 11). a) MHC-I stability: The disclosure includes knockdown (KD) of B2m and MHC-I (H2- K1) with shRNAs to decrease pMHC-l presentation in mouse (non-reporter) PDAC cells and inject them into the pancreas of C57BL / 6 mice treated with vehicle or NTBC (10mice / group). KD is the preferred method because complete B2m or MHC-I KO will result in loss of “missing-self” recognition and killing by NK cells. Tumors are monitored, harvested, and analyzed as described herein. If pMHC-l is required for the anti-tumor effects of NTBC, then B2m- and H2-K1-KD are expected to restore PDAC tumor burden in NTBC treated mice. The disclosure also includes performing TCR (V(D)J))-sequencing from non-reporter tumors and blood to identify enriched TCRs that may target FtY misincorporating PDAC cells upon NTBC treatment. b) Immune checkpoint blockade (ICB): To assess whether FtY misincorporation can alter pMHC-l repertoire (FtY-pMHC-l) and improve ICB and cytotoxic T-cell killing, the disclosure includes establishing PDAC tumors in C57BL / 6 mice with mouse (non-reporter) PDAC cells, treated with or without NTBC (40 mice / group). After 1 week of tumor formation, mice are separated into different groups treated with isotype control, anti-PD-L1, anti-CTLA- 4, or both (10 mice / group), Fig.6i). Tumors are monitored and harvested to measure effects on tumor burden, histological, molecular, and biochemical analysis as described above . It is expected that FtY misincorporation will increase the repertoire of FtY-pMHC-l and sensitize PDAC tumors to ICB.

[0079] The disclosure includes assessing efficacy of targeting NTBC-induced FtY iTNEs during PDAC tumorigenesis. High TyrPhe may suppress cytotoxic activity of macrophages or NK cells during co-culture assays or in vivo. The disclosure includes ADC-target therapies against FtY iTNEs. In an example where antibody treatment does not target sHA to elicit an immune response and tumor regression, the disclosure includes ADC-therapy to deliver VcMMAF to target FtY PDAC tumors in vivo. High autophagy may suppress pMHC-l and iTNE presentation and prevent tumor suppression. In this example, the disclosure includes blocking autophagy using hydroxychloroquine to increase cell surface FtY-pMHC-l to increase responsiveness to ICB. In addition, ICB may not synergize with FtY-pMHC-l in PDAC tumors due to the inability of the immune system to generate a strong or specific response against FtY peptides in mice.

[0080] The ability to generate immunogenic tumor neo-epitopes (iTNEs) without relying on genetic mutations can expand the range and opportunities of potential targets for immune- dependent therapies in the treatment of cancer. Without intending to be bound by any particular interpretation, it is considered that increasing the mRNA translational errors will generate sufficient levels of iTNEs that can be targeted during tumorigenesis. We found that TyrPhe imbalances can increase the Phe to Tyr (FtY) misincorporation rate in PDAC cells. The disclosure includes description of the TyrPhe imbalances on FtY misincorporation rate, mechanisms and changes to the proteome, and assessment of the effects of TyrPhe imbalance on PDAC tumorigenesis, and testing the efficacy of targeting FtY iTNEs to improve PDAC treatment. Based on the foregoing, the disclosure includes development of FtY-specific biologies (e.g. monobodies, bivalent antibodies, etc), tumor vaccines and engineered-immune cells that target endogenous FtY misincorporated proteins to accelerate the treatment and improve the survival of human patients with PDAC and other cancers. The disclosure therefore includes a described neoantigen peptide in a vaccine formulation. In examples, the vaccine formulation includes an immunologically effective amount of an adjuvant and a described peptide. In examples, the vaccine formulation may comprise an isolated mRNA encoding a described protein or segment thereof comprising a misincorporated amino acid. The mRNA may include modifications to the mRNA backbone and / or the nucleotides.

[0081] Although neo-antigens are associated with long-term survival of cancer patients, many PDAC tumors are resistant to immunotherapies, like immune checkpoint blockade (ICB). The ability to non-genetically generate iTNEs specifically in tumor cells may provide a significant benefit for cancer treatments. The disclosure thus includes new therapeutic avenues, which may be considered a new paradigm, “misincorporation-aided therapies”, in cancer treatments. The disclosure includes using a combination of the amino acid misincorporation and ICB to treat cold tumors, and tumors that are resistant to immune checkpoint blockade.

[0082] The disclosure is illustrated by the foregoing disclosure. The disclosure includes variations of the examples that do not materially affect the operation of the described approaches.

Claims

What is claimed is:

1. A method for treating cancer, the method comprising administrating to the individual an inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPPD) and an immune checkpoint inhibitor.

2. The method of claim 1 , wherein the HPPD inhibitor is nitisinone.

3. The method of claim 2, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-VISTA antibody, an anti-B7-H3 antibody, an anti-TIGIT antibody, and a combination thereof.

4. The method of claim 3, wherein the immune checkpoint inhibitor is the anti-PD-1 antibody.

5. The method of claim 4, wherein the individual has pancreatic adenocarcinoma (PDAC), kidney cancer, or liver cancer.

6. The method of claim 5, wherein the immune checkpoint inhibitor is the anti-PD-1 antibody and the cancer is the PDAC.

7. The method of claim 6, wherein administering the nitisinone and the anti-PD-1 antibody results in greater inhibition of PDAC growth than using the nitisinone or the anti-PD- 1 antibody alone.

8. A method for promoting translation of proteins within cells, the proteins comprising one or more misincorporated amino acids, wherein at least some of the proteins that are translated with the one or more misincorporated amino acids comprise at least one segment that has a candidate immunogenic property, the method comprising providing a stimulus to the cells such that amino acid misincorporation occurs during translation.

9. The method of claim 8, wherein the stimulus comprises an alteration of the amount of at least one amino acid that is available to the cells for use in translation.

10. The method of claim 9, wherein the alteration of the amount of the at least one amino acid comprises increasing or decreasing the amount at least one type of amino acid available to the cells for use in translation or altering the ratio of at least two different amino acids that are available to the cells for use in translation.

11. The method of claim 10, wherein the alteration of the amount of the at least one amino acid comprises increasing the amount of tyrosine available for use in translation and / or altering of a phenylalanine-tyrosine ratio available to the cells for use in translation.

12. The method of any one of claims 8-11 , wherein the at least one misincorporated amino acid comprise a tyrosine incorporated into the protein from a phenylalanine codon.

13. The method of claim 12, wherein the misincorporated amino acid is present in the segment of the protein that has the candidate immunogenic property.

14. The method claim 13, further comprising detecting the misincorporation of the tyrosine within a protein by detecting a change in a detectable signal, said detectable signal being detectable only when a tyrosine is misincorporated into the protein, and wherein the detectable signal is optionally produced by a protein that comprises the misincorporated tyrosine.

15. The method of claim 14, wherein the method is performed in cells in vitro.

16. The method of claim 14, wherein the method is performed in cells in an individual, and wherein the stimulus comprises delivering to the cells an agent that promotes the amino acid misincorporation in the cells.

17. The method of claim 16, wherein the agent comprises nitisinone.

18. The method of 17, wherein the individual does not have tyrosinemia or alkaptonuria.

19. The method of claim 18, wherein the cells that have the one or more misincorporated amino acids are cancer cells, and wherein the at least one segment that has a candidate immunogenic property comprises a neoantigen, and wherein optionally the cancer cells are pancreatic cancer cells, kidney cancer cells, or liver cancer cells.

20. The method claim 19, further comprising determining the amino acid sequence of the segment of the protein that has the candidate immunogenic property.

21. The method of claim 20, further comprising testing the segment of the protein that has the candidate immunogenic property to determine whether or not the immunogenic property is present.

22. The method of claim 21 wherein the immunogenic property comprises stimulation of an innate or adaptive immune response, or a combination thereof.

23. The method of claim 22, wherein the adaptive immune response comprises a humoral immune response.

24. The method of claim 23, wherein the humoral immune response comprises antibodydependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) comprising antibodies directed to a cell surface protein comprising a misincorporated amino acid, or complement-dependent cytotoxicity (CDC).

25. The method of claim 16, wherein the adaptive immune response comprises a cell- mediated immune response.

26. An isolated or recombinantly produced protein comprising a misincorporated amino acid produced according to any one of claims 8-11 , or a polynucleotide encoding the protein or the immunogenic segment thereof.

27. A therapeutic or prophylactic composition comprising the protein of claim 26, or an immunogenic segment of the protein.

28. A method comprising introducing into cells an inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPPD) such that a component of a major histocompatibility complex (MHC) is translated with a tyrosine incorporated into the MHC component from a phenylalanine codon.

29. The method of claim 28, wherein the HPPD inhibitor is nitisinone.

30. The method of claim 29, wherein the MHC is a Human Leukocyte Antigen (HLA).

31. The method of claim 30, wherein the HLA can display an antigen that the HLA could not display without the misincorporated tyrosine.

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