Shuffled polypeptide vaccines

Abstract

Provided herein are methods for generating shuffled polypeptide vaccines. Also provided are nucleic acids encoding the shuffled polypeptide and their use in cancer therapeutics.

Description

[0001] Attorney Docket No. 45817-0180W01 / MTX1503.20

[0002] SHUFFLED POLYPEPTIDE VACCINES CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U. S. Patent Application No. 63 / 738,582, filed on December 24, 2024, and U. S. Patent Application No. 63 / 755,534, filed on February 7, 2025, the entire contents of each of which are hereby incorporated by reference.

[0003] SEQUENCE LISTING

[0004] 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, created on December 22, 2025, is named 45817-0180W01_SL.xml and is 54,027 bytes in size.

[0005] BACKGROUND

[0006] In 2020, there were an estimated 19.3 million new cancer cases and 10 million cancer deaths worldwide (Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 2021;71(3):209-49). Recent advances in the field of cancer immunotherapy continues to augment the treatment landscape in multiple tumor subtypes across a broad spectrum of treatment settings. In the US, the estimated percentage of patients with cancer who are eligible for checkpoint inhibitor therapy greatly increased from 1.54% in 2011 to 43.63% in 2018 but only 12.46% of those patients responded to treatment, imparting a large unmet need for patients and concurrent opportunity to improve clinical benefit of immunotherapy across a broad array of solid tumor cancers (Haslam A, Prasad V. Estimation of the Percentage of US Patients With Cancer Who Are Eligible for and Respond to Checkpoint Inhibitor Immunotherapy Drugs. JAMA Network Open 2019;2(5):el92535-e).Attorney Docket No. 45817-0180W01 / MTX1503.20

[0007] SUMMARY

[0008] Provided herein are compositions comprising shuffled polypeptide vaccines, nucleic acids encoding the shuffled polypeptide vaccines, and methods for generating the nucleic acids that encode the shuffled polypeptide vaccines.

[0009] In some embodiments, provided herein are methods of generating a nucleic acid, the method comprising: (a) identifying at least 3 fragments of a target polypeptide generated by introducing breaks in the target polypeptide at break points, optionally wherein additionally one or more of the fragments are truncated to remove one or more amino acid residues that contribute to function of the target polypeptide, optionally wherein additionally one or more of the fragments are modified with one, two, or three amino acid residues to enhance immune recognition and response, optionally wherein additionally one or more of the fragments are truncated to remove one or more amino acid residues that are part of a sequence of amino acids that shares sequence identity to 9 or more consecutive amino acids of a portion of a naturally occurring polypeptide other than the target polypeptide, and optionally wherein additionally one or more of the fragments are modified to add 1-14 amino acids at the C-terminal end and / or the N-terminal end of the one or more fragments, wherein the 1-14 amino acids are identical to the 1-14 amino acids that were present on either side of the one or more break points prior to introducing breaks in the target polypeptide; (b) rearranging the fragments identified and optionally truncated and / or modified in step (a) in different permutations, thereby generating a plurality of different predicted shuffled polypeptides, wherein each of the plurality of predicted shuffled polypeptides retains in shuffled form at least 95% of the amino acid sequence from the target polypeptide; (c) comparing predicted 3-dimensial (3D) structures of each of the plurality of predicted shuffled polypeptides to a 3D structure of the target polypeptide; (d) selecting a shuffled polypeptide that is determined in step (c) to have the greatest structural deviation from the target polypeptide; and (e) creating a nucleic acid that encodes the shuffled polypeptide.

[0010] In some embodiments, the method comprises identifying the break points used in step (a) at least in part based on one or more of: (i) the identification and preservationAttorney Docket No. 45817-0180W01 / MTX1503.20

[0011] and / or amplification of one or more T cell epitopes of the target polypeptide, (ii) the identification and disruption of one or more regions in the target polypeptide that contribute to function of the target polypeptide, and (iii) the identification and disruption of one or more regions in the target polypeptide that share sequence identity with a portion of a naturally occurring polypeptide other than the target polypeptide.

[0012] In some embodiments, at least 4, 5, 6, 7, 8, 9, or 10 fragments are identified in step (a).

[0013] In some embodiments, each of the fragments is between 9 amino acids and 300 amino acids in length.

[0014] In some embodiments, the methods further comprises eliminating or minimizing one or more pseudo-epitopes present in the predicted shuffled polypeptides, wherein a pseudo-epitope is an epitope not present in the target polypeptide and created by rearrangement of the fragments.

[0015] In some embodiments, the plurality of different predicted shuffled polypeptides generated in step (b) exclude any permutations that contain one or more pseudo-epitopes that comprise nine or more consecutive amino acids in length and are identical with a portion of a naturally occurring polypeptide other than the target polypeptide.

[0016] In some embodiments, one or more of the fragments are truncated to remove one or more amino acid residues that associated with a biological function of the target polypeptide.

[0017] In some embodiments, one or more of the fragments are truncated to remove one or more amino acid residues that are part of a sequence of amino acids comprising 9 or more consecutive amino acids identical to a portion of a naturally occurring polypeptide other than the target polypeptide.

[0018] In some embodiments, one or more of the fragments are modified by adding 1-14 amino acids to the C-terminal end and / or the N-terminal end of the one or more fragments, wherein the added 1-14 amino acids are identical to the 1-14 amino acids that were present adjacent to the one or more break points prior to introducing breaks in the target polypeptide.

[0019] oAttorney Docket No. 45817-0180W01 / MTX1503.20

[0020] In some embodiments, one or more of the fragments are modified by adding 14 amino acids to the C-terminal end and / or the N-terminal end of the one or more fragments, wherein the added 14 amino acids are identical to the 14 amino acids that were adjacent to the one or more break points prior to introducing breaks in the target polypeptidee.

[0021] In some embodiments, all of the fragments are modified by adding 1-14 amino acids to the C-terminal end and / or the N-terminal end of all of the fragments, wherein the added 1-14 amino acids are identical to the 1-14 amino acids that were adjacent to each respective break point prior to introducing breaks in the target polypeptide.

[0022] In some embodiments, all of the fragments are modified by adding 14 amino acids to the C-terminal end and / or the N-terminal end of all of the fragments, wherein the added 14 amino acids are identical to the 14 amino acids that were adjacent to each respective break point prior to introducing breaks in the target polypeptide.

[0023] In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more predicted shuffled polypeptides are generated in step (b).

[0024] In some embodiments, when more than 10 fragments are identified in step (a), the method further comprises, prior to step (c), implementing a computational optimization method, such as a genetic algorithm, to select 10-20 predicted shuffled polypeptides that are compared in step (c), wherein the genetic algorithm selects predicted shuffled polypeptides with fewer pseudo-epitopes than predicted shuffled polypeptides that are not selected by the genetic algorithm.

[0025] In some embodiments, the shuffled polypeptide retains in shuffled form at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid residues from the target polypeptide.

[0026] In some embodiments, the target polypeptide is a naturally occurring human polypeptide.

[0027] In some embodiments, the naturally occurring human polypeptide is a tumor associated antigen.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0028] In some embodiments, the tumor associated antigen is MAGEA4, MAGEA6, MAGEC2, NY-ESO-1 (CTAG1B), PRAME, SSX1, or XAGE1B.

[0029] In some embodiments, the nucleic acid is a mRNA.

[0030] In some embodiments, a nucleic acid is generated by any one of the abovedescribed methods.

[0031] Also provided herein are encoding a shuffled polypeptide, wherein the shuffled polypeptide comprises at least three fragments derived from a target polypeptide, wherein the at least three fragments are a in the shuffled polypeptide in an order different from the order of the fragments in the target polypeptide, wherein the shuffled polypeptide retains in shuffled form at least 95% of the amino acid sequence from the target polypeptide, and wherein one or more of the fragments are optionally: (i) modified with one, two, or three residues to enhance immune recognition and response; (ii) truncated to remove one or more amino acid residues that contribute to function of the target polypeptide; (iii) truncated to remove one or more amino acid residues that are part of a sequence of amino acids that shares sequence identity to 9 or more consecutive amino acids of a portion of a naturally occurring polypeptide other than the target polypeptide; and (iv) modified by adding 1-14 amino acids to the C-terminal end and / or the N-terminal end of the one or more fragments, wherein the added 1-14 amino acids are identical to the 1-14 amino acids that were adjacent to the one or more break points prior to introducing breaks in the target polypeptide.

[0032] In some embodiments, the shuffled polypeptide comprises at least 4, 5, 6, 7, 8, 9, or 10 fragments derived from the target polypeptide.

[0033] In some embodiments, each of the fragments is between 9 amino acids and 300 amino acids in length.

[0034] In some embodiments, one or more of the fragments are truncated to remove one or more amino acid residues associated with a biological function of the target polypeptide.

[0035] In some embodiments, one or more of the fragments are truncated to remove one or more amino acid residues that are part of a sequence of amino acids comprising 9 orAttorney Docket No. 45817-0180W01 / MTX1503.20

[0036] more consecutive amino acids identical to a portion of a naturally occurring polypeptide other than the target polypeptide.

[0037] In some embodiments, one or more of the fragments are modified by adding 1-14 amino acids to the C-terminal end and / or the N-terminal end of the one or more fragments, wherein the added 1-14 amino acids are identical to the 1-14 amino acids adjacent to the one or more break points prior to introducing breaks in the target polypeptide.

[0038] In some embodiments, one or more of the fragments are modified by adding 14 amino acids to the C-terminal end and / or the N-terminal end of the one or more fragments, wherein the added 14 amino acids are identical to the 14 amino acids adjacent to the one or more break points prior to introducing breaks in the target polypeptide.

[0039] In some embodiments, each of the fragments are modified by adding 1-14 amino acids to the C-terminal end and / or the N-terminal end of each of the fragments, wherein the added 1-14 amino acids are identical to the 1-14 amino acids adjacent to each respective break point prior to introducing breaks in the target polypeptide.

[0040] In some embodiments, each of the fragments are modified by adding 14 amino acids to the C-terminal end and / or the N-terminal end of each of the fragments, wherein the added 14 amino acids are identical to the 14 amino acids adjacent to each respective break point prior to introducing breaks in the target polypeptide.

[0041] In some embodiments, the shuffled polypeptide retains in shuffled form at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid residues from the target polypeptide.

[0042] In some embodiments, the target polypeptide is a naturally occurring human polypeptide.

[0043] In some embodiments, the naturally occurring human polypeptide is a tumor associated antigen.

[0044] In some embodiments, the tumor associated antigen is MAGEA4, MAGEA6, MAGEC2, NY-ESO-1 (CTAG1B), PRAME, SSX1, or XAGE1B.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0045] In some embodiments, the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0046] In some embodiments, the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0047] In some embodiments, the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3.

[0048] In some embodiments, the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4.

[0049] In some embodiments, the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0050] In some embodiments, the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:6.

[0051] In some embodiments, the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:7.

[0052] In some embodiments, the tumor associated antigen is PD-L1.

[0053] In some embodiments, the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:24.

[0054] In some embodiments, the tumor associated antigen is a viral oncogenic protein. In some embodiments, the viral oncogenic protein is human papillomavirus (HPV) protein.

[0055] In some embodiments, the HPV protein is HPV16 E6 and / or HPV16 E7.

[0056] In some embodiments, the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:8.

[0057] In some embodiments, the naturally occurring human polypeptide is a tumor associated immune regulator.

[0058] In some embodiments, the tumor associated immune regulator is IDO1.

[0059] In some embodiments, the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:27.

[0060] In some embodiments, the nucleic acid is a mRNA.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0061] Also described herein are pharmaceutical compositions comprising one or more of the nucleic acids as described in any of the embodiments above.

[0062] In some embodiments, the pharmaceutical composition comprises at least 2, 3, 4, 5, or 6 different nucleic acids selected from the group consisting of: a first nucleic acid encoding a first shuffled polypeptide comprising at least 3 fragments derived from MAGEA4; a second nucleic acid encoding a second shuffled polypeptide comprising at least 3 fragments derived from MAGEA6; a third nucleic acid encoding a third shuffled polypeptide comprising at least 3 fragments derived from MAGEC2; a fourth nucleic acid encoding a fourth shuffled polypeptide comprising at least 3 fragments derived from NY-ESO-1 (CTAG1B); a fifth nucleic acid encoding a fifth shuffled polypeptide comprising at least 3 fragments derived from PRAME; a sixth nucleic acid encoding a sixth shuffled polypeptide comprising at least 3 fragments derived from SSX1; and a seventh nucleic acid encoding a seventh shuffled polypeptide comprising at least 3 fragments derived from XAGE1B.

[0063] In some embodiments, the pharmaceutical composition comprises: a first nucleic acid encoding a first shuffled polypeptide comprising at least 3 fragments derived from MAGEA4; a second nucleic acid encoding a second shuffled polypeptide comprising at least 3 fragments derived from MAGEA6; a third nucleic acid encoding a third shuffled polypeptide comprising at least 3 fragments derived from MAGEC2; a fourth nucleic acid encoding a fourth shuffled polypeptide comprising at least 3 fragments derived from NY-ESO-1 (CTAG1B); a fifth nucleic acid encoding a fifth shuffled polypeptide comprising at least 3 fragments derived from PRAME; a sixth nucleic acid encoding a sixth shuffled polypeptide comprising at least 3 fragments derived from SSX1; and a seventh nucleic acid encoding a seventh shuffled polypeptide comprising at least 3 fragments derived from XAGE1B.

[0064] In some embodiments of the pharmaceutical composition: the first shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1; the second shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2; the third shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3;Attorney Docket No. 45817-0180W01 / MTX1503.20

[0065] the fourth shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4; the fifth shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5; the sixth shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6; and the seventh shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0066] In some embodiments, the pharmaceutical composition comprises at least 2, 3, 4, 5, or 6 of any one of the nucleic acids described above.

[0067] In some embodiments of the pharmaceutical composition, each of the nucleic acids are mRNAs.

[0068] In some embodiments, the pharmaceutical composition comprises a lipid nanoparticle.

[0069] In some embodiments, the lipid nanoparticle comprises:

[0070] (i) an ionizable lipid,

[0071] (ii) a phospholipid,

[0072] (iii) a structural lipid, and

[0073] (iv) a PEG-lipid.

[0074] In some embodiments, the lipid nanoparticle comprises a compound of Formula (IL*):

[0075]

[0076] or a salt thereof, wherein:

[0077] R1is -OH, -NRN-C4-IO cycloalkenyl optionally substituted with one or more oxo or -N(RNRN”)

[0078] R is H or Ci-6 alkyl;

[0079] R is H or Ci-6 alkyl;Attorney Docket No. 45817-0180W01 / MTX1503.20

[0080] R is H or C1-6 alkyl;

[0081] o is 1, 2, 3, or 4;

[0082] n is 4, 5, 6, 7, or 8;

[0083] m is 4, 5, 6, 7, or 8;

[0084] M is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R2;

[0085] M’ is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R3;

[0086] R2aR2b

[0087] R2is

[0088]

[0089] or -(Ci-6 alkylene)-(C3-8 cycloalkyl)-Ci-6 alkyl;

[0090] R2ais -H or Ci- to alkyl;

[0091] R2bis -H or Ci-io alkyl;

[0092] R2Cis Ci-8 alkyl or C2-8 alkenyl;

[0093] R3isR3aR3b

[0094]

[0095] R3ais H or Ci-10 alkyl;

[0096] R3bis H or C1-8 alkyl; and

[0097] R3Cis Ci-10 alkyl or C2-8 alkenyl.

[0098] In some embodiments, the lipid nanoparticle comprises (i) Compound 1-25, (ii) DSPC or DOPE, (iii) cholesterol, and (iv) PEG2000-DMG or the compound of Formula (PII).

[0099] In some embodiments, the lipid nanoparticle comprises (i) Compound 1-25, (ii) DSPC, (iii) cholesterol, and (iv) PEG2000-DMG.

[0100] In some embodiments, the lipid nanoparticle comprises a molar ratio of about 20-60% ionizable lipid: 5-25% phospholipid: 25-55% cholesterol: and 0.5-15% PEG lipid.

[0101] In some embodiments, the pharmaceutical composition is formulated for intravenous, subcutaneous, intramuscular, intranasal, intraocular, rectal, pulmonary or oral delivery.

[0102] Also provided herein are methods of generating an immune response in a human subject in need thereof, comprising administering to the human subject an effectiveAttorney Docket No. 45817-0180W01 / MTX1503.20

[0103] amount of one or more of the above-described nucleic acids or any one of the above described pharmaceutical compositions.

[0104] Also provided herein are methods of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of one or more of the above-described nucleic acids or any one of the above described pharmaceutical compositions. In some embodiments, the cancer is an advanced solid tumor malignancy. In some embodiments, the cancer is melanoma, non-small cell lung cancer, hepatocellular carcinoma, urinary bladder cancer, bladder cancer, head and neck squamous cell carcinoma, esophageal carcinoma, breast cancer, colon / rectal adenocarcinoma, gastric carcinoma, ovarian carcinoma, cervical carcinoma, endometrial carcinoma, or renal cell carcinoma. In some embodiments, the method comprises further administering an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is nivolumab, relatlimab, or a combination thereof.

[0105] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0106] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0107] DESCRIPTION OF DRAWINGS FIG. 1A is a schematic of the methods described herein. FIG. 1B is an optional alternate method of the one shown in FIG. 1A.

[0108] FIG. 2 is a schematic showing mRNA (ISH) and protein (IHC) expression levels of the target mRNAs in a variety of tumor types.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0109] FIG. 3 are schematics confirming protein expression of the target mRNAs in multiple tumor types, including melanoma and NSCLC.

[0110] FIG. 4 are graphs showing intensity of epitopes derived from the mRNAs detected by mass spectrometry immunopeptidomics (IP -MS) across each evaluated HLA allele. Each panel represents one of the antigen targets, while each dot represents a peptide identification in a monoallelic A549 expressing the HLA allele annotated on the x-axis. Filled circles: mRNA-encoded target antigen identifications; open circles: mRNA-encoded pseudo epitope.

[0111] FIG. 5 are graphs showing luciferase activity for the indicated mRNA targets (top) and the associated cell viability (bottom). FIG. 5 discloses SEQ ID NOS: 47-51 (top and bottom), respectively, in order of appearance.

[0112] FIG. 6 is a graph (left) showing T-cell IFNy and TNF production was observed in primary human T-cells in response to all antigens (3 not shown) and a table (right) showing in vitro expansion of patient derived primary human T-cells with mRNA target OLPs resulting in antigen-specific T cell responses.

[0113] FIG. 7 is a graph (left) showing a summary of T cell responses across all antigens and HLA class I alleles in donors tested, displayed as fold change above control and graphs (right) showing antigen specific IFNy T-cell response across donors evaluated for reactivity against target antigens.

[0114] FIG. 8 are graphs showing levels of IFNy ELISpot responses for splenocytes of HLA-A2.1 (CB6F1) mice vaccinated with Composition A and HLA-A*02:01 (CB6F1) transgenic mice vaccinated with lipid nanoparticle (LNP) Formulated NTFIX Control mRNA.

[0115] FIG. 9 is a schematic showing the first study design described in Example 6. FIG. 10 are graphs showing the change in mean body weight of the mice (left), mean body weight (right), and spleen weight (bottom right).

[0116] FIG. 11 are graphs showing T cell proportions.

[0117] FIG. 12 are graphs showing that the constructs could generate HPV16 E6-E7 epitope specific CD8 T cell responses.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0118] FIG. 13 are graphs showing that the constructs could generate HPV16 E6-E7 epitope specific CD8 T cell responses.

[0119] FIG. 14 is a schematic showing the second study design described in Example 6. FIG. 15 is a graph showing that the shuffled HPV16 E6-E7 proteoform vaccine was more effective in delaying tumor growth (left) and the equation used to calculate tumor volume (right).

[0120] FIG. 16 is a survival graph showing that the shuffled HPV16 E6-E7 proteoform vaccine also increased overall survival as compared to the WT vaccine.

[0121] FIG. 17 are graphs showing tumor volume prior to treatment (left) and 16 days post tumor implant.

[0122] FIG. 18 are graphs showing HPV16 E7 epitope specific T cells were present in tumors of WT vs. shuffled vaccine treated mice.

[0123] FIG. 19 are graphs showing PD1, TIM3, KLRG1, and CD 127 expression in WT and shuffled vaccine treated mice.

[0124] FIG. 20A is an overview and model of protein designs to validate full-length translation for PD-L1. FIG. 20B is a schematic showing expected flow cytometry results. FIG. 20C are graphs showing that WT PD-L1 is localized at the cell surface whereas Shuffled PD-L1 is intracellularly localized.

[0125] FIG. 21A is a schematic showing the assay principle for testing functionality of Shuffled PD-L1. PD-L1 exerts its immunosuppressive effect by binding to cells expressing the cognate receptor PD1. A protein possessing the extracellular domain (EC) of PD1 fused to an antibody Fc domain is used as a reagent to probe this interaction. FIG.

[0126] 21B is a flow cytometry graph showing functional disruption of Shuffled PD-L1. FIG. 21C is a schematic showing the assay principle for testing functionality of IDO 1 is tested. IDO1 catalyzes the conversion of L-tryptophan to N-formyl-L-kynurenine. FIG. 21D is a graph showing functional disruption of Shuffled IDO1.

[0127] FIG. 22A are graphs showing epitopes presented by HLA-A*02:01 in Mono-Allelic A549 Cells Transfected with WT (top) or Shuffled PD-L1 (bottom). FIG. 22B areAttorney Docket No. 45817-0180W01 / MTX1503.20

[0128] graphs showing epitopes presented by HLA-A*02:01 in Mono- Allelic A549 Cells Transfected with WT (top) or Shuffled IDO1 (bottom).

[0129] FIG. 23 is a graph showing mRNA-dose-dependent activation of Jurkat reporter cells expressing a PD-L1 -specific T cell receptor.

[0130] FIG. 24A is a schematic showing the first study design described in Example 10. FIG. 24B is a graph showing the effect of WT and shuffled mRNAs on tumor growth.

[0131] FIG. 24C is a graph showing the effect of WT and shuffled mRNAs on survival. FIG. 25A is a schematic showing the second study design described in Example 10.

[0132] FIG. 25B is a graph showing the effect of control, WT, and shuffled mRNAs on CD8 T cell responses.

[0133] FIG. 25C are graphs showing levels of IFNy ELISpot responses for splenocytes mice vaccinated with control mRNA, WT mRNA, or shuffled TAA mRNA.

[0134] DETAILED DESCRIPTION

[0135] In aspects, the invention relates to methods for improving efficacy of cancer therapy using off the shelf (OTS) vaccines. The methods described herein allow for the production of vaccines that increase both the number and antitumor activity of a subject's T cells, such that the subject can mount an effective T cell response that recognizes tumor associated antigens such as cancer testis antigens (CTAs). CTAs are tumor antigens that are normally expressed in the testes but are aberrantly expressed in several cancers. The CTA vaccines described herein are designed such that multiple CTAs are targeted, thus potentially providing a clinical benefit for subjects with a variety of solid-tumor malignancies. In some aspects, the cancer vaccines may help to prevent the patient's cancer from recurring by instructing their immune system to better identify cancerous tissue derived from the original cancer lesion.

[0136] In other aspects, the methods involve improving other anti-cancer therapies such as checkpoint inhibitor therapies. Checkpoint inhibitor efficacy may be driven by blockingAttorney Docket No. 45817-0180W01 / MTX1503.20

[0137] the negative signals generated by engagement of these inhibitory receptors on T cells with their ligands on tumors and other immune cells, especially antigen presenting cells. The loss of inhibition following checkpoint blockade allows the subjects' T cells to recognize neoantigens as foreign. Combining the cancer vaccines of the invention with checkpoint inhibitor therapy, particularly in cancer patients with unresectable solid tumors, leads to T cell-mediated destruction of the tumor cells by increasing both the number and antitumor activity of a subject's T cells that recognize tumor associated antigens. In a newly diagnosed subject, the checkpoint therapy, such as pembrolizumab (an anti-PD-1 antibody), may begin as soon as possible. However, in many cases, subjects often do not respond to checkpoint therapies alone and a combination therapy is needed. Accordingly, in some instances, a checkpoint inhibitor may be administered together with the vaccine.

[0138] The use of nucleic acid technology (e.g., mRNAs) allows for induced production of a broad array of secreted, membrane-bound, and intracellular proteins in humans.

[0139] Antigen-encoded mRNA is an attractive technology platform for tumor associated antigen vaccination as an mRNA vaccine can deliver multiple tumor associated antigens in a single molecule, a vaccine can be rapidly manufactured, and the tumor associated antigens are endogenously translated and enter into the natural cellular antigen processing and presentation pathway. Specifically, mRNA antigen-specific therapies that encode tumor-associated antigens can be used to induce a tumor antigen-specific T-cell response. mRNA encoding for cancer antigens is formulated in an LNP that delivers antigen mRNA primarily to the cytosol of APCs. Once in the cytosol, the mRNA is translated by host cell machinery, processed by proteasomal enzymes, and loaded onto HLA molecules for presentation to T cells. Tumor antigen-specific T cells recognize their cognate peptides-HLA ligands on APCs, leading to T-cell activation, proliferation, and migration to tumor sites. Primed cytotoxic CD8+ T cells recognize target peptide-HLA on tumor cells and initiate tumor cell killing. This mRNA-based vaccine technology overcomes the challenges commonly associated with DNA- based vaccines, such as risk of genome integration or the high doses and devices needed for administration (eg, electroporation).Attorney Docket No. 45817-0180W01 / MTX1503.20

[0140] Each nucleic acid cancer vaccine can include multiple nucleic acids encoding full-length or near full length tumor associated antigens (in shuffled form). The tumor associated antigens are encoded in whole protein (or near whole protein) form rather than as epitopes, thereby maximizing the HLA class I and II alleles on which they can be presented to the immune system. Additionally, the tumor associated antigens are encoded in a shuffled format such that the tumor associated antigen retains the epitopes necessary for T cell recognition but do not retain functional activity.

[0141] Thus, embodiments of the present disclosure provide nucleic acid (RNA, such as mRNA) vaccines that include one or more nucleic acids having one or more open reading frames encoding tumor associated antigens such as CTAs. As provided herein, nucleic acid cancer vaccines encoding tumor associated antigens having different properties may be used to induce a balanced immune response, comprising cellular and / or humoral immunity. Methods of treating a patient having a solid tumor malignancy with a cancer vaccine having a maximized anti-cancer efficacy for a given set of epitopes is also provided.

[0142] Methods for Generating a Shuffled Polypeptide

[0143] As shown in FIGs. 1A and 1B, provided herein are methods for generating a shuffled polypeptide vaccine for use in eliciting an immune response against a target polypeptide (e.g., MAGEA4, MAGEA6, MAGEC2, NYES01, PRAME, SSX1, and XAGE-1B).

[0144] Once a target polypeptide is identified, breakpoints are identified in silico in the target polypeptide. The break points are identified at least in part based on the identification and preservation of one or more T cell epitopes of the target polypeptide (so as to elicit an effective immune response against the target polypeptide), the identification and disruption of one or more regions in the target polypeptide that contribute to function of the target polypeptide (so as to reduce the likelihood that the vaccine produces a functional protein), modification with one, two, or three amino acid residues to enhance immune recognition and response, and / or the identification andAttorney Docket No. 45817-0180W01 / MTX1503.20

[0145] disruption of one or more regions in the target polypeptide that share sequence identity to 9 or more consecutive amino acids of a portion of a naturally occurring polypeptide other than the target polypeptide (so as to avoid an off-target immune response). Epitopes can be identified using a database such as, e.g., Lonza Epibase, antitope, and NetMHCPan. Regions of the target polypeptide that contribute to function of the target polypeptide can be identified using databases such as, e.g., InterPro and NCBI Conserved Domains Database. Regions in the target polypeptide that share sequence identity with a portion of a naturally occurring polypeptide other than the target polypeptide can be identified by searching and comparing regions of sequences in the target polypeptide to the human proteome database.

[0146] Once breakpoints are identified, predicted fragments are generated in silico. At least 3 or more (e g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) fragments can be generated. The fragments can vary in length depending on the size of the target protein and the number of fragments generated. For example, in some embodiments, each of the fragments is between 9 amino acids and 300 amino acids in length (e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278,Attorney Docket No. 45817-0180W01 / MTX1503.20

[0147] 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, or more).

[0148] The predicted fragments are then analyzed to determine if any amino acids in the predicted fragment contribute to function of the target polypeptide. If such amino acids are present, then the fragments can be truncated at the C-terminal end and / or the N-terminal end of the fragment to remove one or more amino acid residues that contribute to function of the target polypeptide. The predicted fragments are also analyzed to determine whether the fragment contains any sequence of amino acids that shares sequence identity with a portion of a naturally occurring polypeptide other than the target polypeptide (as outlined in FIG. IB). If there is any identity to a naturally occurring polypeptide, the fragment can be truncated at the C-terminal end and / or the N-terminal end of the fragment to remove one or more of those amino acids. In some instances, the step of identifying fragments can also include modification to a fragment by adding 1-14 amino acids at the C-terminal end and / or the N-terminal end of the one or more fragments, wherein the 1-14 amino acids are identical to the 1-14 amino acids that were present on either side of the one or more break points prior to introducing breaks in the target polypeptide.

[0149] Once the fragments are identified, the fragments are rearranged in silico to generate a plurality of different predicted shuffled polypeptides. Each of the plurality of predicted shuffled polypeptides retains in shuffled form at least 95% of the amino acid sequence from the target polypeptide. Each of the plurality of predicted shuffled polypeptides can be analyzed to identify any pseudo-epitopes (i.e., epitopes that are not present in the target polypeptide but are created by rearranging the fragments) present in the predicted shuffled polypeptides. The presence of pseudo-epitope is determined with in silico binding prediction (such as NetMHCpan 4.1) against common HLA alleles in populations. In some cases, the plurality of different predicted shuffled polypeptides generated are limited to exclude (i.e., eliminate or minimize) any permutations that contain one or more pseudo-epitopes that are nine amino acids in length and are identical with a portion of a naturally occurring polypeptide other than the target polypeptide (asAttorney Docket No. 45817-0180W01 / MTX1503.20

[0150] shown in FIG. IB). Additionally, when more than 10 fragments are identified, the method further comprises optionally implementing a computational optimization method, such as a genetic algorithm, to select 10-20 predicted shuffled polypeptides, wherein the computational optimization method selects predicted shuffled polypeptides with relatively fewer pseudo-epitopes. Computational methods are provided for optimizing the arrangement of a plurality of polypeptide sequences within a construct to reduce in silico predicted binders arising at peptide junctions (i.e., pseudo-epitopes). The methods define an objective function based on predicted binding to one or more HLA alleles and iteratively explore alternative peptide orderings using one or more optimization strategies. Candidate arrangements are evaluated and selectively modified based on objective function values, with the search process guided toward arrangements exhibiting reduced predicted junction binding. Suitable optimization strategies include, but are not limited to, stochastic, heuristic, evolutionary, or combinatorial optimization algorithms, such as genetic algorithms, simulated annealing, or related approaches, thereby identifying an optimized peptide sequence arrangement relative to alternative permutations.

[0151] Once the plurality of shuffled polypeptides is generated, 3-dimensional (3D) structures of each of the plurality of predicted shuffled polypeptides is created. The predicted 3-dimensional (3D) structures of each of the plurality of predicted shuffled polypeptides is then compared to a 3D structure of the target polypeptide. A shuffled polypeptide is selected based on it having the greatest structural deviation from the target polypeptide. That is, the shuffled polypeptide that is selected has a predicted 3D structure that differs the most from the 3D structure of the target polypeptide as compared to any of the other predicted 3D structures of each of the plurality of predicted shuffled polypeptides. Once the polypeptide is selected, a nucleic acid (e.g., mRNA) that encodes the shuffled polypeptide is created.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0152] Shuffled Polypeptides

[0153] Target polypeptides used in the methods and compositions described herein include tumor associated antigens. Tumor associated antigens, such as a CT As, are dysregulated molecules found on cancer cells that trigger an immune response. Unlike tumor-specific antigens, these antigens are also present (though often at lower levels or different locations) on some normal cells such as a CTAs. For example, CTA expression is limited to male germ cells in healthy adults, but ectopic expression has been observed in tumor cells of multiple types of human cancer. Since male germ cells are devoid of HLA-class I molecules and cannot present antigens to T cells, CTAs are generally considered neoantigens when expressed in cancer cells and have the capacity to elicit immune responses that are strictly cancer-specific. CTAs for use with the compositions and methods described herein may be any such CTA known in the field including, but not limited to, MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA5, MAGEA6, MAGEA8, MAGEA9, MAGEA1O, MAGEA1 1, MAGEA12, BAGE, BAGE2, BAGE3, BAGE4, BAGE5, MAGEB1, MAGEB2, MAGEB5, MAGEB6, MAGEB3, MAGEB4, GAGE1, GAGE2A, GAGE3, GAGE4, GAGES, GAGE6, GAGE7, GAGES, SSX1, SSX2, SSX2b, SSX3, SSX4, CTAG1B, LAGE-lb, CTAG2, MAGECI, MAGEC3, SYCP1, BRDT, MAGEC2, SPANXA1, SPANXB1, SPANXC, SPANXD, SPANXN1, SPANXN2, SPANXN3, SPANXN4, SPANXN5, XAGE1D, XAGE1C, XAGE1B, XAGE1, XAGE2, XAGE3, XAGE- 3b, XAGE-4 / RP11-167P23.2, XAGE5, DDX43, SAGE1, ADAM2, PAGES, CT16.2, PAGE1, PAGE2, PAGE2B, PAGE3, PAGE4, LIPI, VENTXP1, IL13RA2, TSP50, CTAGE1, CTAGE-2, CTAGE5, SPA17, ACRBP, CSAG1, CSAG2, DSCR8, MMAlb, DDX53, CTCFL, LUZP4, CASC5, TFDP3, JARID1B, LDHC, M0RC1, DKKL1, SPO11, CRISP2, FMR1NB, FTHL17, NXF2, TAF7L, TDRD1, TDRD6, TDRD4, TEX15, FATE1, TPTE, CT45A1, CT45A2, CT45A3, CT45A4, CT45A5, CT45A6, H0RMAD1, H0RMAD2, CT47A1, CT47A2, CT47A3, CT47A4, CT47A5, CT47A6, CT47A7, CT47A8, CT47A9, CT47A10, CT47A11, CT47B1, SLCO6A1, TAG, LEMD1, HSPB9, CCDC110, ZNF165, SPACA3, CXorf48, THEG, ACTL8, NLRP4, COX6B2, LOC348120, CCDC33, LOC196993,Attorney Docket No. 45817-0180W01 / MTX1503.20

[0154] PASD1, LOC647107, TULP2, CT66 / AA884595, PRSS54, RBM46, CT69 / BC040308, CT70 / BI818097, SPINLW1, TSSK6, ADAM29, CCDC36, LOC440934, SYCE1, CPXCR1, TSPY3, TSGA1O, HIWI, MIWI, PIWI, PIWIL2, ARMC3, AKAP3, Cxorf61, PBK, C21orf99, OIP5, CEP290, CABYR, SPAG9, MPHOSPH1, ROPN1, PLAC1, CALR3, PRM1, PRM2, CAGE1, TTK, LY6K, IMP-3, AKAP4, DPPA2, KIAA0100, DCAF12, SEMG1, POTED, POTEE, POTEA, POTEG, POTEB, POTEC, POTER, GOLGAGL2 FA, CDCA1, PEPP2, OTOA, CCDC62, GPATCH2, CEP55, FAM46D, TEX14, CTNNA2, FAM133A, LOC130576, ANKRD45, ELOVL4, IGSF11, TMEFF1, TMEFF2, ARX, SPEF2, GPAT2, TMEM108, NOL4, PTPN20A, SPAG4, MAEL, RQCD1, PRAME, TEX101, SPATA19, ODF1, ODF2, ODF3, ODF4, ATAD2, ZNF645, MCAK, SPAG1, SPAG6, SPAG8, SPAG17, FBXO39, RGS22, cyclin Al, C15orf60, CCDC83, TEKT5, NR6A1, TMPRSS12, TPPP2, PRSS55, DMRT1, EDAG, NDR, DNAJB8, CSAG3B, CTAG1A, GAGE12B, GAGE12C, GAGE12D, GAGE12E, GAGE12F, GAGE12G, GAGE12H, GAGE12I, GAGE12J, GAGE13, LOC728137, MAGEA2B, MAGEA9B / LOC728269, NXF2B, SPANXA2, SPANXB2, SPANXE, SSX4B, SSX5, SSX6, SSX7, SSX9, TSPY1D, TSPY1E, TSPY1F, TSPY1G, TSPY1H, TSPY11, TSPY2, XAGE1E, XAGE2B / CTD-2267G17.3, and / or variants thereof.

[0155] In some embodiments, the shuffled polypeptides are as follows:

[0156] MAGEA4:

[0157] MLGVMGVYDGREHTVYGEPRKLLTQDWVQENYLEYRQVPGSNPARYEFLWGP RALAETSYVKVLEHVVRVNARVRIAYPSLREAALLEEEEGVMSSEQKSQHCKPE EGVEAQEEALGL VGAQ APTTEEQEAAVS S S SPL VPGTLEEVP AAES AGPPQ SPQG ANKVDELAHFLLRKYRAKELVTKAEMLERVLGTIAMEGDSASEEEIWEEGNNQI FPKTGLLIIVLGTIAMEGDSASESALPTTISFTCWRQPNEGSSSQEEEGPSTSPDAES LFREALSNKVDELAHFLLRKYAAESAGPPQSPQGASALPTTISFTCWRQRAKELV TKAEMLERVIKNYKRCFPVIFGKASESLKMIFGIDVKEVDPASNTYTLVTCLGLS YDGLLGNNQIFPKTGLLIIMEGDSASEEEIWEELGVMGVYDGREHTV (SEQ ID NO:1).Attorney Docket No. 45817-0180W01 / MTX1503.20

[0158] MAGEA6:

[0159] VIRK VAKL VHFLLLKYRAREP VTKAEMLGS VVGNWQ YFFP VIF SKASD SLQLVF GIELMPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLM EVDPIGHVYIFATCLGLSYDGLLGDNQIMPKTGFLIIILAIIAKEGDCAPEEKIWEEL SVLEVFEGREDSIFGDPKKLLTFPDLESEFQAALSRKVAKLVHFLLLKYKASDSL QLVFGIELMEVDPIGHVYIFATGEVPAAESPDPPQSPQGASSLPTTMNYPLWSQSY EDSSNQEEEGPSTFPDLESEFQAALSGREDSIFGDPKKLLTQYFVQENYLEYRQEA ASSSSTLVEVTLGEVPAAESPDPPQSTQYFVQENYLEYRQVPGSDPACYEFLWGP RALIETSYVKVLHHMVKISGGPRISYPLLHEWALREGEE (SEQ ID NO:2).

[0160] MAGEC2:

[0161] MLTKVWVQGHYLEYREVPHSSPPYYEFLWIEVGPDHFCVFANTVGLTDEGSDD EGMPENSLLIIILSVIFIKGNCASEEVIWEVLNAVGVYAGREHFVYGEPRELLTKV W VQGHYLEYREEAS S AS STL YLVF SP S SF ST S S SLILGEVPHS SPP YYEFLWGPRA HSESIKKKVLEFL AKLNNT VP S SFP S WYKD ALKD VEERVQ ATIDT ADD AT VMAS E SL S VMS SNVSF SEGLPD SE S SF T YTLDEK VAEL VEFLLLK YRAREFMELLF GL AL

[0162] lEVGPDHFCVFANTEKVAELVEFLLLKYEAEEPVTEAEMLMIVIKYKDYFPVILK RAREFMELLFGLALSPSSFSTSSSLILGGPEEEEVPSGVIPNLTESIPSSPPQGPPQGP SQSPLSSCCSSFSWSSFSEESSSQKGEDTGTCQGLPDSESSFTYTLDMPPVPGVPFR NVDNDSPTSVELEDWVDAQHPTDEEEEEASSASSTLYLVF (SEQ ID NO:3).

[0163] NY-ESO-1 (CTAG1B):

[0164] MYLAMPFATPMEAELARRSLAQDAPPLPVDHRQLQLSISSCLQQLSLLMWITQC FLPVFLAQPPSGQRRSLLMWITQVTVSGNILTIRLTAADHRQLQLSISSCLQARRS LAQDAPPLPVPGVLLKEFTVSGNILTIRLTAAMQAEGRGTGGSTGDADGPGGPGI PDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGC CRCGARGPESRLLEFYLAMPFATPMEAEL (SEQ ID NO:4).Attorney Docket No. 45817-0180W01 / MTX1503.20

[0165] PRAME:

[0166] MLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKA VLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAA QPMTCLQ AL YVD SLFFLRGRLDQLLRHVMNPLETL SITNCRL SEGD VMHLSQ SP S VSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLS HCSQLTTLSFYGNSISISALQSLLQHLIMERRRLWGSIQSRYISMSVWTSPRRLVEL AGQ SLLKDE AL AIAALELISIS ALQ SLLQHLIGL SNLTHVLYP VPLCELGRP SMVW LSANPCPHCGDRTFYDPEPILCPCFMPNLLKDEALAIAALELLPRELFPPLFMAAF LRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMI NLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQDGLSTEAEQPFIPVEVLVDLFL KEGACDELFSYLIEQYIAQFTSQFLSLQCLQALYVDSLFFLRGLSNLTHVLYPVPL ESYED IHGTLHLERLAYLHAR (SEQ ID NO: 5).

[0167] SSX1:

[0168] MERKQLVIYEEISDPEEDDEMNGDDTFAKRPRDDAKASEKRSKAFDDIATYFSK KEGRLHRIIPKIMPKKPAEDENDSKGVSEASGPQNDGKQLHPPGKANISEKINKRS GPKRGKHAWTHRLRHNRRIQVEHPQMTFGRLHRIIPKIMPKKGPKRGKHAWTH RLRERKQLVIYEEISDPWKKMKYSEKISYVYMKRNYKAMTKLGFKVTLPPFMC NKQATDFQGNDFDNDHNRRIQVEHPQMTFKAFDDIATYFSKKEWKKMKYSEKI SYVY (SEQ ID NO:6).

[0169] XAGE1B:

[0170] MVKVKIIPKEEHCKMPEAGEEQPQVMESPKKKNQQLKVGILHLGSISQTPGINLD LGSGVKVKIIPKEEHCKMRQKKIRIQLRSQCATWKVICKSCISQTPGINLDLGSGK NQQLKVGILHLGSRQKKIRIQLRSQCA (SEQ ID NO:7).

[0171] Additional target polypeptides used in the methods and compositions described herein include other tumor associated antigens such as CD2, CD 19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB,Attorney Docket No. 45817-0180W01 / MTX1503.20

[0172] 5T4, AGS-5, AGS-16, Angiopoietin 2, B2M, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, Carcinoetnbryonic antigen, CTLA4, Cripto, ED-B, ErbBl, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, Fibronectin, Folate Receptor, Ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gplOO, gpA33, GPNMB, ICOS, IGF1R, Integrin av, Integrin av0, LAG-3, Lewis Y, Mesothelin, c-MET, MN Carbonic anhydrase IX, MUC1, MUC16, Nectin-4, NKGD2, NOTCH, 0X40, OX40L, PD-1, PD-L1, PSCA, PSMA, RANKL, R0R1, R0R2, SLC44A4, Syndecan-1, TACI, TAG-72, Tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, and VEGFR-3.

[0173] Additional target polypeptides used in the methods and compositions described herein include viral proteins such as viral oncogenic proteins (e.g., HPV16 E6 and E7 proteins).

[0174] Additional target polypeptides used in the methods and compositions described herein include tumor associated immune regulators. A tumor associated immune regulator refers to any molecule, cell, or factor within the tumor microenvironment that actively modulates the immune response to cancer, often shifting it from anti-tumor (killing cancer) to pro-tumor (helping cancer grow / hide) by suppressing immune cells like T cells, promoting inflammation, or fostering angiogenesis. Examples include, IDO1, TGF-p, and IL-10.

[0175] Other Components of the Polypeptides

[0176] Some aspects of the present disclosure provide fusion proteins. In some embodiments, a nucleic acid (e g., an mRNA) encodes a fusion protein. Thus, an encoded protein may include two or more proteins (e.g., protein and / or protein fragment) j oined together with or without a linker.

[0177] In some embodiments, a signal peptide is fused to one or more of the shuffled proteins described herein. Signal peptides, comprising the N-terminal 15-60 amino acids of proteins, are typically needed for the translocation across the membrane on the secretory pathway and, thus, universally control the entry of most proteins both inAttorney Docket No. 45817-0180W01 / MTX1503.20

[0178] eukaryotes and prokaryotes to the secretory pathway. In eukaryotes, the signal peptide of a nascent precursor protein (pre-protein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing. ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by an ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor. A signal peptide may also facilitate the targeting of the protein to the cell membrane.

[0179] A signal peptide may have a length of 15-60 amino acids. For example, a signal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, a signal peptide has a length of 20-60, 25-60, 30-60, 35- 60, 40-60, 45- 60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15-20 amino acids.

[0180] Signal peptides from heterologous genes are known in the art and can be tested for desired properties and then incorporated into a protein or nucleic acid of the disclosure. In some embodiments, a signal peptide used in the present disclosure comprises MLRLLLRHHFHCLLLCAVWATPCLA (SEQ ID NO: 10). In some embodiments, a signal peptide used in the present disclosure comprises MKAILVVLLYTFTTANA (SEQ ID NO: 11).

[0181] In some embodiments, fusion proteins comprising a shuffled protein and another protein further are linked to one another through a linker.

[0182] In some embodiments, the linker is a GS linker. GS linkers are polypeptide linkers that include glycine and serine amino acids repeats. They comprise flexible and hydrophilic residues and can be used to perform fusion of protein subunits without interfering in the folding and function of the protein domains, and without formation of secondary structures. In some embodiments, an mRNA encodes a fusion protein that comprises a GS linker that is 3 to 20 amino acids long. For example, the GS linker mayAttorney Docket No. 45817-0180W01 / MTX1503.20

[0183] have a length of (or have a length of at least) 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, a GS linker is (or is at least) 15 amino acids long (e.g., GGSGGSGGSGGSGGG (SEQ ID NO: 12)). In some embodiments, a GS linker is (or is at least) 8 amino acids long e.g., GGGSGGGS (SEQ ID NO: 13)). In some embodiments, a GS linker is (or is at least) 7 amino acids long (e.g., GGGSGGG (SEQ ID NO: 14)). In some embodiments, a GS linker is (or is at least) 4 amino acid long (e.g., GGGS (SEQ ID NO:15)). In some embodiments, the GS linker comprises (GGGS)n (SEQ ID NO: 16), where n is any integer from 1-5. In some embodiments, a GS linker is (or is at least) 4 amino acid long (e.g., GSGG (SEQ ID NO:17)). In some embodiments, the GS linker comprises (GSGG)n (SEQ ID NO:18), where n is any integer from 1-5. In some embodiments, a linker is a glycine linker, for example having a length of (or a length of at least) 3 amino acids (e.g., GGG). In some embodiments, the linker comprises GGGG (SEQ ID NO: 19). In some embodiments, the linker comprises GGPG (SEQ ID NO:20). In some embodiments, a protein encoded by an mRNA includes two or more linkers, which may be the same or different from each other.

[0184] The linker may be, for example, a cleavable linker or protease-sensitive linker. In some embodiments, the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof (see, e.g., WO 2017 / 127750). This family of self-cleaving peptide linkers, referred to as 2A peptides, has been described in the art (see, e.g., Kim, J. H. et al. PLoS ONE 201 l;6:el8556). In some embodiments, the linker is an F2A linker.

[0185] The skilled artisan will appreciate that other art-recognized linkers may be suitable for use in the constructs of the disclosure (e.g., encoded by the nucleic acids of the disclosure).

[0186] In some embodiments, the shuffled proteins may contain a stabilization domain. Protein stabilization domains are protein sequences or structures that can enhance the stability of a protein to various environmental stresses, such as temperature, pH, and proteolysis.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0187] Protein Synthesis

[0188] Any one of the proteins described herein may be synthesized using methods known in the art. For example, the proteins may be synthesized in vitro using a cell-free protein synthesis system (e.g., in vitro translation, cell-free protein expression, cell-free translation, or cell-free protein synthesis). Following synthesis, in some embodiments, the proteins are purified (e.g., using affinity, ion exchange, hydrophobic interaction, and / or size exclusion) and then administered using techniques known in the art.

[0189] Nucleic Acids Encoding Shuffled Proteins

[0190] In some embodiments, any one of the engineered shuffled proteins described herein may be encoded by nucleic acids. Nucleic acids comprise a polymer of nucleotides (nucleotide monomers). Thus, nucleic acids are also referred to as polynucleotides.

[0191] Nucleic acids may be or may include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras and / or combinations thereof.

[0192] mRNA of the present disclosure comprises an open reading frame (ORF) encoding a shuffled tumor associated antigen. In some embodiments, the mRNA further comprises a 5' untranslated region (UTR), 3' UTR, a poly(A) tail and / or a 5' cap analog. Preferred mRNA molecules are chemically modified.

[0193] Messenger RNA

[0194] Messenger RNA (mRNA) is RNA that encodes a (at least one) protein (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. It isAttorney Docket No. 45817-0180W01 / MTX1503.20

[0195] understood that mRNA is not self-amplifying RNA (saRNA) (see, e.g., Bloom K et al. Gene Therapy 2021; 28: 117-129 for a comparison of mRNA and saRNA). saRNAs include alphavirus replicase sequences that encode an RNA-dependent RNA polymerase. mRNA does not include alphavirus replicase sequences.

[0196] The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application may recite “T”s in a representative DNA sequence but where the sequence represents mRNA, the “T”s would be substituted for “U”s. Thus, any of the DNAs disclosed and identified by a particular sequence identification number herein also disclose the corresponding mRNA sequence complementary to the DNA, where each “T” of the DNA sequence is substituted with “U.”

[0197] Naturally-occurring eukaryotic mRNA molecules can contain stabilizing elements, including, but not limited to, UTRs at their 5'-end (5' UTR) and / or at their 3'-end (3' UTR), in addition to other structural features, such as a 5 '-cap structure or a 3'-poly(A) tail. Both the 5' UTR and the 3' UTR are typically transcribed from the genomic DNA and are elements of the premature mRNA. Characteristic structural features of mature mRNA, such as the 5 '-cap and the 3 '-poly (A) tail are usually added to the transcribed (premature) mRNA during mRNA processing.

[0198] Untranslated Regions (UTRs)

[0199] The mRNAs of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region. A “5’ untranslated region” (UTR) refers to a region of an mRNA that is directly upstream ( / .<?., 5') from the start codon ( / .<?., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide. A “3' untranslated region” (UTR) refers to a region of an mRNA that is directly downstream (i.e., 3') from the stop codon i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide. When RNA transcripts are being generated, the 5’ UTR may comprise a promoter sequence. SuchAttorney Docket No. 45817-0180W01 / MTX1503.20

[0200] promoter sequences are known in the art. It should be understood that such promoter sequences will not be present in a vaccine of the disclosure.

[0201] Where mRNAs are designed to encode a (at least one) protein, the mRNA may comprise a 5’ UTR and / or 3’ UTR. UTRs of an mRNA are transcribed but not translated. In mRNA, the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; the 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is a growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into the polynucleotides of the present disclosure to, among other things, enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down -regulation of the transcript in case they are misdirected to undesired organs sites. A variety of 5’ UTR and 3’ UTR sequences are known.

[0202] It should also be understood that the mRNA of the present disclosure may include any 5’ UTR and / or any 3’ UTR.

[0203] A 5' UTR does not encode a protein (is non-coding). Natural 5' UTRs have features that play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G. 5'UTR also have been known to form secondary structures which are involved in elongation factor binding.

[0204] In some embodiments of the disclosure, a 5’ UTR is a heterologous UTR, z.e., is a UTR found in nature associated with a different ORF. In other embodiments, a 5’ UTR is a synthetic UTR, z.e., does not occur in nature. Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic. Exemplary 5’ UTRs include Xenopus or human derived a-globin or b-globin (8278063; 9012219), human cytochrome b-245 a polypeptide, and hydroxy steroid (17b) dehydrogenase, and Tobacco etch virusAttorney Docket No. 45817-0180W01 / MTX1503.20

[0205] (US8278063, US9012219). CMV immediate-early 1 (IE1) gene (US2014 / 0206753, WO2013 / 185069), the sequence GGGAUCCUACC (SEQ ID NO:21) (WO2014 / 144196) may also be used. In other embodiments, a 5' UTR is a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract) (e.g., W02015 / 101414, W02015 / 101415, WO2015 / 062738, WO2015 / 024667, WO2015 / 024667); 5' UTR element derived from ribosomal protein Large 32 (L32) gene (WO / 2015101414, W02015101415, WO / 2015 / 062738), 5' UTR element derived from the 5' UTR of an hydroxysteroid (17-0) dehydrogenase 4 gene (HSD17B4) (WO2015024667), or a 5' UTR element derived from the 5' UTR of ATP5A1 (WO2015 / 024667) can be used. In some embodiments, an internal ribosome entry site (IRES) is used instead of a 5' UTR.

[0206] A 3' UTR does not encode a protein (is non-coding). Natural or wild type 3' UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif, c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.

[0207] Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of mRNA of the disclosure. When engineering specific nucleic acids, one or more copies of an ARE can be introduced to make nucleic acids of the disclosure less stable and thereby curtail translation and decrease production of theAttorney Docket No. 45817-0180W01 / MTX1503.20

[0208] resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using nucleic acids of the disclosure and protein production can be assayed at various time points posttransfection. For example, cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hours, 12 hours, 1 day, 2 days, and 7 days post-transfection.

[0209] Those of ordinary skill in the art will understand that 5’ UTRs that are heterologous or synthetic may be used with any desired 3’ UTR sequence. For example, a heterologous or synthetic 5’ UTR may be used with a synthetic 3’ UTR or with a heterologous 3’ UTR.

[0210] Non-UTR sequences may also be used as regions or subregions within a nucleic acid. For example, introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. Incorporation of intronic sequences may increase protein production as well as nucleic acid levels.

[0211] Combinations of features may be included in flanking regions and may be contained within other features. For example, the ORF may be flanked by a 5' UTR which may contain a strong Kozak translational initiation signal and / or a 3' UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail. 5' UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes such as the 5' UTRs described in US2010 / 0293625 and WO2015 / 085318A2, each of which is herein incorporated by reference.

[0212] It should be understood that any UTR from any gene may be incorporated into the regions of a nucleic acid. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5' or 3' UTR may be inverted, shortened, lengthened, made with one or more other 5' UTRs or 3' UTRs. As used herein, the termAttorney Docket No. 45817-0180W01 / MTX1503.20

[0213] “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3' UTR or 5' UTR may be altered relative to a wild-type / native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3' or 5') comprise a variant UTR.

[0214] In some embodiments, a double, triple or quadruple UTR such as a 5' UTR or 3' UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. For example, a double beta-globin 3' UTR may be used as described in US2010 / 0129877, which is incorporated herein by reference.

[0215] It is also within the scope of the present disclosure to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as AB AB AB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level.

[0216] In some embodiments, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature, or property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.

[0217] The untranslated region may also include translation enhancer elements (TEE). As a non-limiting example, the TEE may include those described in US 2009 / 0226470, herein incorporated by reference, and those known in the art.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0218] Open Reading Frames

[0219] An open reading frame (ORF) is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA). An ORF typically encodes a protein. It will be understood that the sequences disclosed herein may further comprise additional elements, e.g, 5' and / or 3' UTRs, but that those elements, unlike the ORF, need not necessarily be present in an mRNA of the present disclosure.

[0220] 5’ End Capping

[0221] In some embodiments, an mRNA comprises a 5' terminal cap. 5'-capping of polynucleotides may be completed concomitantly during an in vitro transcription reaction using, for example, the following chemical RNA cap analogs to generate the 5'-guanosine cap structure according to manufacturer protocols: 3'-O-Me-m7G(5')ppp(5') G [the ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). 5 '-capping of modified mRNA may be completed post-transcriptionally using, for example, a Vaccinia Virus Capping Enzyme to generate the “Cap 0” structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2'-0 methyl-transferase to generate: m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-O-methylation of the 5'-antepenultimate nucleotide using a 2'-0 methyl-transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-O-methylation of the 5'-preantepenultimate nucleotide using a 2'-0 methyl-transferase. Enzymes may be derived from a recombinant source. Other cap analogs may be used.

[0222] Polyadenylation Tailing

[0223] A “poly(A) tail” is a region of mRNA that is downstream, e.g., directly downstream (z.e., 3'), from the 3' UTRthat contains multiple, consecutive adenosine monophosphates. A poly(A) tail may contain 10 to 300 adenosine monophosphates. ItAttorney Docket No. 45817-0180W01 / MTX1503.20

[0224] can, in some instances, comprise up to about 400 adenine nucleotides. For example, a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a poly(A) tail contains 50 to 250 adenosine monophosphates (SEQ ID NO:32). In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, and / or export of the mRNA from the nucleus and translation. In some embodiments, the length of the 3'-poly(A) tail may be an essential element with respect to the stability of the individual mRNA. In some embodiments, a poly(A) tail has a length of about 50, about 100, about 150, about 200, about 250, about 300, about 350, or about 400 nucleotides. In some embodiments, a poly(A) tail has a length of 100 nucleotides (SEQ ID NO:33).

[0225] Additional Stabilizing Elements

[0226] mRNA provided herein, in some embodiments, includes an additional stabilizing element. Stabilizing elements may include, for example, a histone stem-loop. A stemloop binding protein (SLBP), a 32 kDa protein has been identified. It is associated with the histone stem-loop at the 3'-end of the histone messages in both the nucleus and the cytoplasm. Its expression level is regulated by the cell cycle; it peaks during the S-phase, when histone mRNA levels are also elevated. The protein has been shown to be essential for efficient 3'-end processing of histone pre-mRNA by the U7 snRNP. SLBP continues to be associated with the stem-loop after processing, and then stimulates the translation of mature histone mRNAs into histone proteins in the cytoplasm. The RNA binding domain of SLBP is conserved through metazoa and protozoa; its binding to the histone stem-loop depends on the structure of the loop. The minimum binding site includes at least three nucleotides 5’ and two nucleotides 3' relative to the stem-loop.

[0227] In some embodiments, an mRNA includes an open reading frame (coding region), a histone stem-loop, and optionally, a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal generally should enhance the expressionAttorney Docket No. 45817-0180W01 / MTX1503.20

[0228] level of the encoded protein. The encoded protein, in some embodiments, is not a histone protein, a reporter protein (e.g., Luciferase, GFP, EGFP, P-Galactosidase, EGFP), or a marker or selection protein (e.g., alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT)).

[0229] In some embodiments, an mRNA includes the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop, even though both represent alternative mechanisms in nature, they act synergistically to increase the protein expression beyond the level observed with either of the individual elements. The synergistic effect of the combination of poly(A) and a histone stem-loop does not depend on the order of the elements or the length of the poly(A) sequence.

[0230] In some embodiments, an mRNA does not include a histone downstream element (HDE). “Histone downstream element” (HDE) includes a purine-rich polynucleotide stretch of approximately 15 to 20 nucleotides 3' of naturally-occurring stem-loops, representing the binding site for the U7 snRNA, which is involved in processing of histone pre-mRNA into mature histone mRNA. In some embodiments, the nucleic acid does not include an intron.

[0231] An mRNA may or may not contain an enhancer and / or promoter sequence, which may be modified or unmodified or which may be activated or inactivated. In some embodiments, the histone stem-loop is generally derived from histone genes and includes an intramolecular base pairing of two neighbored partially or entirely reverse complementary sequences separated by a spacer, consisting of a short sequence, which forms the loop of the structure. The unpaired loop region is typically unable to base pair with either of the stem loop elements. It occurs more often in RNA, as is a key component of many RNA secondary structures but may be present in single-stranded DNA as well. Stability of the stem-loop structure generally depends on the length, number of mismatches or bulges, and base composition of the paired region. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) may result. In some embodiments, the at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0232] In some embodiments, an mRNA has one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES are destabilizing sequences found in the 3 ’UTR. The AURES may be removed from the mRNA. Alternatively, the AURES may remain in the mRNA.

[0233] Sequence Optimization

[0234] In some embodiments, an open reading frame encoding a protein of the disclosure is codon optimized. Codon optimization methods are known in the art. An open reading frame of any one or more of the sequences provided herein may be codon optimized. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art - non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or proprietary methods. In some embodiments, the open reading frame sequence is optimized using optimization algorithms.

[0235] Chemically Unmodified Nucleotides

[0236] In some embodiments, an mRNA is not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g., A, G, C, or U). In some embodiments, nucleotides and nucleosides of the present disclosureAttorney Docket No. 45817-0180W01 / MTX1503.20

[0237] comprise standard deoxyribonucleosides such as those present in DNA (e.g., dA, dG, dC, or dT).

[0238] Chemically Modified Nucleotides

[0239] The compositions of the present disclosure comprise, in some embodiments, a nucleic acid molecule encoding a shuffled cancer / testis (CT) antigen, wherein the nucleic acid comprises nucleotides and / or nucleosides that can be standard (unmodified) or modified as is known in the art. In some embodiments, nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally-occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and / or nucleoside as are recognized in the art.

[0240] In some embodiments, a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally-occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.

[0241] In some embodiments, a non-naturally-occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Nonlimiting examples of such non-naturally-occurring modified nucleotides and nucleosides can be found, inter alia in international publication numbers WO2013052523A1;

[0242] WO2014093924A1; W02015051173A2; WO2015051169A2; W02015089511A2; or WO2017153936A1, each of which is herein incorporated by reference in its entirety.

[0243] Hence, nucleic acids of the disclosure (e.g., DNA and RNA, such as mRNA) can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof.

[0244] Nucleic acids of the disclosure (e.g., DNA and RNA, such as mRNA), in some embodiments, comprise various (more than one) different types of standard and / orAttorney Docket No. 45817-0180W01 / MTX1503.20

[0245] modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and / or modified nucleotides and nucleosides.

[0246] In some embodiments, a modified mRNA introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

[0247] In some embodiments, a modified mRNA introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

[0248] Nucleic acids (e.g., RNA, such as mRNA), in some embodiments, comprise nonnatural modified nucleotides that are introduced during synthesis or post-synthesis of the nucleic acids to achieve desired functions or properties. The modifications may be present on internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a nucleic acid may be chemically modified.

[0249] The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., RNA, such as mRNA). A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0250] Modified nucleotide base pairing encompasses not only the standard adenosinethymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those nucleic acids having at least one chemical modification. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base / sugar or linker may be incorporated into nucleic acids of the present disclosure.

[0251] In some embodiments, modified nucleobases in nucleic acids (e.g., RNA, such as mRNA) comprise 1-methyl-pseudouridine (mly), 1-ethyl-pseudouridine (el \| / ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine (\| / ). In some embodiments, modified nucleobases in nucleic acids (e.g., RNA, such as mRNA) comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1 -methoxymethyl pseudouridine, 5-methyl cytidine, and / or 5-methoxy cytidine. In some embodiments, the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.

[0252] In some embodiments, modified nucleobases in nucleic acids (e.g., RNA, such as mRNA) comprise Nl-methyl-pseudouridine (mly), Nl-ethyl-pseudouridine (el\| / ), 5-methoxy-uridine (mo5U), 5-methyl-uridine (m5U), 5-methyl-cytidine (m5C), and / or pseudouridine (y). In some embodiments, modified nucleobases in nucleic acids (e.g., RNA, such as mRNA) comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and / or 5-methoxy cytidine. In some embodiments, the RNA includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0253] In some embodiments, a mRNA comprises 1 -methyl -pseudouridine (mly) substitutions at one or more or all uridine positions of the mRNA. In some embodiments, the ORF comprises 1-methyl-pseudouridine (ml\| / ) substitutions at one or more or all uridine positions of the ORF. In some embodiments, the mRNA comprises nucleosides consisting of ml\|t, adenosine, guanosine, and cytidine. In some embodiments, the ORF comprises nucleosides consisting of mly, adenosine, guanosine, and cytidine.

[0254] In some embodiments, a mRNA comprises 1-methyl-pseudouridine (ml\| / ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the mRNA. In some embodiments, the ORF comprises 1-methyl-pseudouridine (mly) substitutions at one or more or all uridine positions of the ORF and 5-methyl cytidine substitutions at one or more or all cytidine positions of the ORF. In some embodiments, the mRNA comprises nucleosides consisting of mly, adenosine, guanosine, and 5-methyl cytidine. In some embodiments, the ORF comprises nucleosides consisting of mly, adenosine, guanosine, and 5-methyl cytidine.

[0255] In some embodiments, a mRNA comprises pseudouridine (y) substitutions at one or more or all uridine positions of the mRNA. In some embodiments, the ORF comprises pseudouridine (y) substitutions at one or more or all uridine positions of the ORF. In some embodiments, the mRNA comprises nucleosides consisting of yr, adenosine, guanosine, and cytidine. In some embodiments, the ORF comprises nucleosides consisting of y / , adenosine, guanosine, and cytidine.

[0256] In some embodiments, a mRNA comprises pseudouridine (yr) substitutions at one or more or all uridine positions of the nucleic acid and 5-methylcytidine substitutions at one or more or all cytidine positions of the mRNA. In some embodiments, the ORF comprises pseudouridine (y / ) substitutions at one or more or all uridine positions of the ORF and 5-methyl cytidine substitutions at one or more or all cytidine positions of the ORF. In some embodiments, the mRNA comprises nucleosides consisting of yr, adenosine, guanosine, and 5-methyl cytidine. In some embodiments, the ORF comprises nucleosides consisting of y / , adenosine, guanosine, and 5-methyl cytidine.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0257] In some embodiments, a mRNA comprises uridine at one or more or all uridine positions of the mRNA. In some embodiments, the ORF comprises uridine at one or more or all uridine positions of the ORF. In some embodiments, the mRNA comprises nucleosides consisting of uridine, adenosine, guanosine, and cytidine. In some embodiments, the ORF comprises nucleosides consisting of uridine, adenosine, guanosine, and cytidine.

[0258] In some embodiments, a mRNA comprises 5-methyl-uridine and 5-methyl cytidine at one or more or all uridine and cytidine positions, respectively, of the mRNA. In some embodiments, the ORF comprises 5-methyl-uridine substitutions at one or more or all uridine positions of the ORF and 5-methyl cytidine substitutions at one or more or all cytidine positions of the ORF. In some embodiments, the mRNA comprises nucleosides consisting of 5-methyl-uridine, adenosine, guanosine, and 5-methyl cytidine. In some embodiments, the ORF comprises nucleosides consisting of 5-methyl-uridine, adenosine, guanosine, and 5-methyl cytidine.

[0259] In some embodiments, a mRNA comprises 5-methyl-uridine and 5-methyl cytidine at one or more or all uridine and cytidine positions, respectively, of the mRNA.

[0260] In preferred embodiments, RNAs (e.g., mRNAs) are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a nucleic acid can be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

[0261] The nucleic acids of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a nucleic acid of the disclosure, or in a predetermined sequence region thereof (e.g., in the mRNA including or excluding the poly(A) tail). In some embodiments, all nucleotides X in a nucleic acid of the present disclosure (or in aAttorney Docket No. 45817-0180W01 / MTX1503.20

[0262] sequence region thereof) are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

[0263] The nucleic acid may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

[0264] The mRNA may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the nucleic acids may contain a modified pyrimidine such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid is replaced with a modified uracil (e.g., a 5-substituted uracil). The modified uracil can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the nucleic acid is replaced with a modified cytosineAttorney Docket No. 45817-0180W01 / MTX1503.20

[0265] (e.g., a 5-substituted cytosine). The modified cytosine can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).

[0266] Identification and Ratio Determination (IDR) Sequences

[0267] An Identification and Ratio Determination (IDR) sequence is a sequence of a biological molecule (e.g., nucleic acid or protein) that, when combined with the sequence of a target biological molecule, serves to identify the target biological molecule.

[0268] Typically, an IDR sequence is a heterologous sequence that is incorporated within or appended to a sequence of a target biological molecule and can be used as a reference to identify the target molecule. Thus, in some embodiments, a nucleic acid (e.g., mRNA) comprises (i) a target sequence of interest (e.g., a coding sequence encoding an antigenic peptide or protein); and (ii) a unique IDR sequence.

[0269] An RNA species (e.g., RNA having a given coding sequence) may comprise an IDR sequence that differs from the IDR sequence of other RNA species (e.g., RNA(s) having different coding sequence(s)). Each IDR sequence thus identifies a particular RNA species, and so the abundance of IDR sequences may be measured to determine the abundance of each RNA species in a composition. Use of distinct IDR sequences to identify RNA species allows for analysis of multivalent RNA compositions (e.g., containing multiple RNA species) containing RNA species with similar coding sequences and / or lengths, which could otherwise be difficult to distinguish using PCR- or chromatography-based analysis of full-length RNAs.

[0270] Each RNA species in a multivalent RNA composition may comprise an IDR sequence that is not a sequence isomer of an IDR sequence of another RNA species in a multivalent RNA composition (e.g., the IDR sequence does not have the same number of adenosine nucleotides, the same number of cytosine nucleotides, the same number of guanine nucleotides, and the same number of uracil nucleotides, as another IDR sequence in the composition, even if those sequences have different sequences). Having identical nucleotide compositions causes sequence isomers to have the same mass, presenting aAttorney Docket No. 45817-0180W01 / MTX1503.20

[0271] challenge to distinguishing sequence isomers using mass-based identification methods (e.g., mass spectrometry).

[0272] Each RNA species in a multivalent RNA composition may comprise an IDR sequence having a mass that differs from the mass of IDR sequences of each other RNA species in a multivalent RNA composition. For example, the mass of each IDR sequence may differ from the mass of other IDR sequences by at least 9 Da, at least 25 Da, at least 25 Da, or at least 50 Da. Use of IDR sequences with distinct masses allows RNA fragments comprising different IDR sequences to be distinguished using mass-based analysis methods (e.g., mass spectrometry), which do not require reverse transcription, amplification, or sequencing of RNAs.

[0273] Each RNA species in an RNA composition may comprises an IDR sequence with a different length. For example, each IDR sequence may have a length independently selected from 0 to 25 nucleotides. The length of a nucleic acid influences the rate at which the nucleic acid traverses a chromatography column, and so the use of IDR sequences of different lengths on different RNA species allows RNA fragments having different IDR sequences to be distinguished using chromatography-based methods (e.g., LC-UV).

[0274] IDR sequences may be chosen such that no IDR sequence comprises a start codon, ‘AUG’. Lack of a start codon in an IDR sequence prevents undesired translation of nucleotide sequences within and / or downstream from the IDR sequence.

[0275] IDR sequences may be chosen such that no IDR sequence comprises a recognition site for a restriction enzyme. In one example, no IDR sequence comprises a recognition site for Xbal, ‘UCUAG’. Lack of a recognition site for a restriction enzyme (e.g., Xbal recognition site ‘UCUAG’) allows the restriction enzyme to be used in generating and modifying a DNA template for in vitro transcription, without affecting the IDR sequence or sequence of the transcribed RNA or DNA plasmid.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0276] Nucleic Acid Production

[0277] Chemical Synthesis

[0278] Solid-phase chemical synthesis. Nucleic acids the present disclosure may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Solid-phase synthesis is useful in site-specific introduction of chemical modifications in the nucleic acid sequences.

[0279] The synthesis of nucleic acids of the present disclosure by the sequential addition of monomer building blocks may be carried out in a liquid phase.

[0280] The synthetic methods discussed above each has its own advantages and limitations. Attempts have been conducted to combine these methods to overcome the limitations. Such combinations of methods are within the scope of the present disclosure. The use of solid-phase or liquid-phase chemical synthesis in combination with enzymatic ligation provides an efficient way to generate long chain nucleic acids that cannot be obtained by chemical synthesis alone.

[0281] Ligation

[0282] Assembling nucleic acids by a ligase may also be used. DNA or RNA ligases promote intermolecular ligation of the 5’ and 3’ ends of polynucleotide chains through the formation of a phosphodiester bond. Nucleic acids such as chimeric polynucleotides and / or circular nucleic acids may be prepared by ligation of one or more regions or subregions. DNA fragments can be joined by a ligase catalyzed reaction to create recombinant DNA with different functions. Two oligodeoxynucleotides, one with a 5’ phosphoryl group and another with a free 3’ hydroxyl group, serve as substrates for a DNA ligase.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0283] In Vitro Transcription

[0284] cDNA encoding the polynucleotides described herein may be transcribed using an in vitro transcription (IVT) system. In vitro transcription of mRNA is known in the art and is described in International Publication WO 2014 / 152027, which is incorporated by reference herein in its entirety. In some embodiments, the RNA of the present disclosure is prepared in accordance with any one or more of the methods described in WO 2018 / 053209 or WO 2019 / 036682, each of which is incorporated by reference herein.

[0285] In some embodiments, the mRNA transcript is generated using a non-amplified, linearized DNA template in an in vitro transcription reaction to generate the RNA transcript. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of an RNA, for example, but not limited to DENV mRNA. In some embodiments, cells, e.g., bacterial cells, e.g., E. coli, e.g., DH-1 cells are transfected with the plasmid DNA template. In some embodiments, the transfected cells are cultured to replicate the plasmid DNA which is then isolated and purified. In some embodiments, the DNA template includes an RNA polymerase promoter, e.g., a T7 promoter located 5 ' to and operably linked to the gene of interest. Thus, in some embodiments, a DNA plasmid encoding a shuffled cancer / testis (CT) antigen described herein is provided. As used herein, “plasmid DNA” or “pDNA” refers to an extrachromosomal DNA molecule that is physically separated from chromosomal DNA in a cell and can replicate independently. In some embodiments, plasmid DNA is isolated from a cell (e.g., as a plasmid DNA preparation). In some embodiments, plasmid DNA comprises an origin of replication, which may contain one or more heterologous nucleic acids, for example nucleic acids encoding therapeutic proteins that may serve as a template for RNA polymerase. Plasmid DNA may be circularized or linear (e.g., plasmid DNA that has been linearized by a restriction enzyme digest).

[0286] In some embodiments, an in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, and encodes a 3' UTR and a poly(A) tail.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0287] The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template.

[0288] In some embodiments, a nucleic acid (e.g., template DNA and / or RNA) includes 200 to 3,000 nucleotides. For example, a nucleic acid may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.

[0289] An in vitro transcription system typically comprises a transcription buffer (e.g., with magnesium), nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase (e.g., T7 RNA polymerase). In some embodiments, one or more of the NTPs is a chemically modified NTP (e.g., with 1 -methylpseudouridine or other chemical modifications described herein and / or known in the art).

[0290] In some embodiments, the NTPs comprise adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), and guanosine triphosphate (GTP), or an analog of each respective NTP. The ratio of NTPs may vary. In some embodiments, the ratio of GTP: ATP: CTP: UTP is 1: 1: 1: 1. In some embodiments, the amount of the GTP or an analogue thereof is greater than an amount of the UTP or an analogue thereof. In some embodiments, the amount of the GTP is greater than the amount of the UTP. In some embodiments, the amount of ATP is greater than the amount of UTP, and the amount of CTP is greater than the amount of UTP. In some embodiments, the amount of the GTP or an analogue thereof is greater than an amount of the UTP or an analogue thereof. In some embodiments, an IVT system comprises an at least 2: 1 ratio of GTP concentration to ATP concentration, an at least 2: 1 ratio of GTP concentration to CTP concentration, and an at least 4:1 ratio of GTP concentration to UTP concentration. In some embodiments, an IVT system comprises a 2: 1 ratio of GTP concentration to ATP concentration, a 2: 1 ratio of GTP concentration to CTP concentration, and a 4: 1 ratio of GTP concentration to UTP concentration. In some embodiments, an IVT system comprises guanosine diphosphate (GDP). In some embodiments, an IVT system comprises an at least 3:1 ratio of GTP plus GDP concentration to ATP concentration, an at least 6: 1 ratio of GTP plusAttorney Docket No. 45817-0180W01 / MTX1503.20

[0291] GDP concentration to CTP concentration, and an at least 6: 1 ratio of GTP plus GDP concentration to UTP concentration.

[0292] The NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs.

[0293] Any number of RNA polymerases or variants may be used in the method of the present disclosure. The polymerase may be selected from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and / or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids and / or modified nucleotides, including chemically modified nucleic acids and / or nucleotides. Some embodiments exclude the use of DNase.

[0294] An IVT system, in some embodiments, comprises magnesium buffer, dithiothreitol (DTT) spermidine, pyrophosphatase, and / or RNase inhibitor. In some embodiments, an IVT system omits an RNase inhibitor. An IVT system may be incubated at 25 degrees Celsius or at 37 degrees Celsius. Other temperatures may be used, depending in part on the polymerase (e.g., use of a variant polymerase).

[0295] In some embodiments, the RNA transcript is capped via enzymatic capping. In some embodiments, the RNA comprises 5' terminal cap, for example, 7mG(5’)ppp(5’)NlmpNp.

[0296] Purification

[0297] Purification of the nucleic acids described herein may include, but is not limited to, nucleic acid clean-up, quality assurance and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (H1C-HPLC). The term “purified” when used in relation to a nucleic acid such as a “purifiedAttorney Docket No. 45817-0180W01 / MTX1503.20

[0298] nucleic acid” refers to one that is separated from at least one contaminant. A “contaminant” is any substance that makes another unfit, impure or inferior. Thus, a purified nucleic acid (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method.

[0299] A quality assurance and / or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.

[0300] In some embodiments, the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR.

[0301] Lipid Compositions

[0302] In some embodiments, the nucleic acids are formulated as a lipid composition, such as a composition comprising a lipid nanoparticle, a liposome, and / or a lipoplex. In some embodiments, the lipid composition (e.g., lipid nanoparticle, liposome, and / or lipoplex) does not comprise protamine. In some embodiments, the lipid composition does comprise protamine. In some embodiments, nucleic acids are formulated as lipid nanoparticle (LNP) compositions. In some embodiments, nucleic acids are formulated in lipid nanoparticles (LNPs). Lipid nanoparticles typically comprise ionizable lipid (e.g., ionizable amino lipid), non-cationic lipid (e.g., phospholipid, neutral lipid), structural lipid (e.g., sterol), and PEG-modified lipid components along with the nucleic acid cargo of interest. The lipid nanoparticles can be generated using components, compositions, and methods as are generally known in the art. See, e.g., PCT / US2016 / 052352;

[0303] PCT / US2016 / 068300; PCT / US2017 / 037551; PCT / US2015 / 027400;

[0304] PCT / US2016 / 047406; PCT / US2016000129; PCT / US2016 / 014280;

[0305] PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394;

[0306] PCT / US2016 / 52117; PCT / US2012 / 069610; PCT / US2017 / 027492;

[0307] PCT / US2016 / 059575; PCT / US2016 / 069491; PCT / US2016 / 069493; and

[0308] PCT / US2014 / 66242, each of which is incorporated by reference herein to the extent itAttorney Docket No. 45817-0180W01 / MTX1503.20

[0309] describes lipid nanoparticles and components, compositions, and methods for producing lipid nanoparticles.

[0310] In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable lipid, 5-25% non-cationic lipid, 25-55% structural lipid, and 0.5-15%

[0311] PEG-modified lipid.

[0312] In some embodiments, the lipid nanoparticle comprises a molar ratio of

[0313] 20-60% ionizable lipid, 5-30% non-cationic lipid, 10-55% structural lipid, and

[0314] 0.5-15% PEG-modified lipid.

[0315] In some embodiments, the lipid nanoparticle comprises 40-50 mol%

[0316] ionizable lipid, optionally 45-50 mol%, for example, 45-46 mol%, 46-47 mol%,

[0317] 47-48 mol%, 48-49 mol%, or 49-50 mol% for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%.

[0318] In some embodiments, the lipid nanoparticle comprises 20-60 mol%

[0319] ionizable lipid. For example, the lipid nanoparticle may comprise 20-50 mol%,

[0320] 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% ionizable lipid. In some embodiments, the lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% ionizable lipid. In some embodiments, the lipid nanoparticle comprises 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, or 55 mol% ionizable lipid.

[0321] In some embodiments, the lipid nanoparticle comprises 45 - 55 mole percent (mol%) ionizable lipid. For example, lipid nanoparticle may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% ionizable lipid.

[0322] Ionizable lipids

[0323] In some embodiments, the ionizable lipid is a compound of Formula (IL*)Attorney Docket No. 45817-0180W01 / MTX1503.20

[0324] R2

[0325]

[0326] R1H7M'^Rr\3

[0327] (IL*)

[0328] or a salt thereof, wherein:

[0329] R1is -OH, -NRN-C4-IO cycloalkenyl optionally substituted with one or more oxo or -N(RN’RN’ );

[0330] RNis H or Ci-6 alkyl;

[0331] R is H or Ci-6 alkyl;

[0332] RNis H or Ci-6 alkyl;

[0333] o is 1, 2, 3, or 4;

[0334] n is 4, 5, 6, 7, or 8;

[0335] m is 4, 5, 6, 7, or 8;

[0336] M is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R2;

[0337] M’ is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R3;

[0338] R2aR2b

[0339] R2is / L A^R20or -(Ci-6 alkylene)-(C3-8 cycloalkyl)-Ci-6 alkyl;

[0340] R2ais -H or Ci- io alkyl;

[0341] R2bis -H or Ci-io alkyl;

[0342] R2Cis Ci-8 alkyl or C2-8 alkenyl;

[0343]

[0344] R3is R3aR3b;

[0345] R3aisH or Ci-10 alkyl;

[0346] R3bis H or C1-8 alkyl; and

[0347] R3Cis Ci-10 alkyl or C2-8 alkenyl.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0348] In some embodiments, the ionizable lipid is of Formula (IL**-!):

[0349] R2C

[0350] A-N

[0351] R1

[0352]

[0353] R3a

[0354] (IL**-I)

[0355] or a salt thereof, wherein:

[0356] R1is -OH;

[0357] o is 2, 3, or 4;

[0358] n is 4, 5, 6, 7, or 8;

[0359] M is -C(=O)-O-*, wherein * indicates attachment to R2;

[0360] m is 6, 7, or 8;

[0361] M’ is -C(=O)-O-*, wherein * indicates attachment to R3;

[0362] R2Cis C4-8 alkyl;

[0363] R3ais C7-10 alkyl; and

[0364] R3Cis C3-5 alkyl.

[0365] In some embodiments, the ionizable lipid is of Formula (IL**-III):

[0366] R2C

[0367] R1

[0368]

[0369] R3a

[0370] (IL**-III)

[0371] or a salt thereof, wherein:

[0372] R1is NRN-C4-IO cycloalkenyl optionally substituted with one or more oxo or -N(RNRN);

[0373] RNis H;

[0374] RNis C1-2 alkyl;Attorney Docket No. 45817-0180W01 / MTX1503.20

[0375] RN’ is H;

[0376] o is 2, 3, or 4;

[0377] n is 6, 7, or 8;

[0378] M is -C(=O)-O-*, wherein * indicates attachment to R2;

[0379] m is 6, 7, or 8;

[0380] M’ is -C(=O)-O-*, wherein * indicates attachment to R3;

[0381] R2ais C7-10 alkyl;

[0382] R2eis C4-6 alkyl;

[0383] R3ais C1-3 alkyl; and

[0384] R3Cis C4-6 alkyl.

[0385] In some embodiments, the ionizable lipid is of Formula (IL**-IV):

[0386] R2bR2c

[0387]

[0388] R3a

[0389] (IL**-IV)

[0390] or a salt thereof, wherein:

[0391] R1is OH;

[0392] o is 2, 3, or 4;

[0393] n is 6, 7, or 8;

[0394] M is -C(=O)-O-*, wherein * indicates attachment to R2;

[0395] m is 6, 7, or 8;

[0396] M’ is -C(=O)-O-*, wherein * indicates attachment to R3;

[0397] R2bis C3-5 alkyl;

[0398] R2Cis C2-4 alkyl;

[0399] R3ais C7-10 alkyl; and

[0400] R3Cis C4-6 alkyl.

[0401] In some embodiments, the ionizable lipid is of Formula (IL*-I):Attorney Docket No. 45817-0180W01 / MTX1503.20

[0402]

[0403] (IL*-Ia)

[0404] or a salt thereof, wherein:

[0405] R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; and

[0406] R3ais Ci-8 alkyl.

[0407] In some embodiments, ionizable lipid is of Formula (IL*-Ia):

[0408]

[0409] (IL*-Ia)

[0410] or a salt thereof, wherein:

[0411] R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and

[0412] R3ais Ci-8 alkyl.

[0413] In some embodiments, the ionizable lipid is of Formula (IL*-Ia’):

[0414]

[0415] (ILMa’)

[0416] or a salt thereof, wherein:

[0417] o, M, M’, R2cand R3care as defined for variable IL*; and

[0418] R3ais Ci-8 alkyl.

[0419] In some embodiments, the ionizable lipid is of Formula (IL*-Iia):Attorney Docket No. 45817-0180W01 / MTX1503.20

[0420]

[0421] (IL*-Iia)

[0422] or a salt thereof, wherein:

[0423] R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and

[0424] R3ais Ci-8 alkyl.

[0425] In some embodiments, the ionizable lipid is of Formula (IL*-II’):

[0426]

[0427] (iL*-ir)

[0428] or a salt thereof, wherein:

[0429] o, M, M’, R2Cand R3care as defined for variable IL*; and

[0430] R3ais Ci-8 alkyl.

[0431] In some embodiments, the ionizable lipid is of Formula (IL*-III):

[0432]

[0433] R3a

[0434] (IL* -III)

[0435] or a salt thereof, wherein:

[0436] R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;

[0437] R2ais a Ci-8 alkyl; and

[0438] R3ais Ci-8 alkyl.

[0439] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa):Attorney Docket No. 45817-0180W01 / MTX1503.20

[0440]

[0441] (ILMIIa)

[0442] or a salt thereof, wherein:

[0443] R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;

[0444] R2bis a Ci-8 alkyl; and

[0445] R3ais Ct-8 alkyl.

[0446] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa):

[0447]

[0448] (IL*-nia)

[0449] or a salt thereof, wherein:

[0450] R1, o, M, M’, R2C, and R3care as defined for variable IL*;

[0451] R2ais a Ci-8 alkyl; and

[0452] R3ais Ci-8 alkyl.

[0453] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa’):

[0454]

[0455] (IL*-IIIa’)

[0456] or a salt thereof, wherein:

[0457] R1, o, M, M’, R2C, and R3care as defined for variable IL*;

[0458] R2ais a Ci-8 alkyl; and

[0459] R3ais Ci-8 alkyl.

[0460] In some embodiments, the ionizable lipid is of Formula (IL*-IIIb):Attorney Docket No. 45817-0180W01 / MTX1503.20

[0461]

[0462] (IL* -nib)

[0463] or a salt thereof, wherein:

[0464] R1, o, M, M’, R2C, and R3care as defined for variable IL*;

[0465] R2ais a Ci-8 alkyl; and

[0466] R3ais Ci-8 alkyl.

[0467] In some embodiments, the ionizable lipid is of Formula (IL*-IIIb’):

[0468]

[0469] (IL*-IIIb’)

[0470] or a salt thereof, wherein:

[0471] R1, o, M, M’, R2C, and R3care as defined for variable IL*;

[0472] R2ais a Ci-8 alkyl; and

[0473] R3ais Ci-8 alkyl.

[0474] In some embodiments, the ionizable lipid is of Formula (IL*-IV):

[0475] R'

[0476]

[0477] R3a

[0478] (IL*-IV)

[0479] or a salt thereof, wherein:

[0480] R1, o, m, n, M, M’, R2c, and R3eare as defined for variable IL*;Attorney Docket No. 45817-0180W01 / MTX1503.20

[0481] R2bis a Ci-8 alkyl; and

[0482] R3ais Ci-8 alkyl.

[0483] In some embodiments, the ionizable lipid is of Formula (IL*-Iva):

[0484]

[0485] (IL* -Iva)

[0486] or a salt thereof, wherein:

[0487] R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;

[0488] R2bis a Ci-8 alkyl; and

[0489] R3ais Ci-8 alkyl.

[0490] In some embodiments, the ionizable lipid is of Formula (IL*-Iva’):

[0491]

[0492] (IL* -Iva)

[0493] or a salt thereof, wherein:

[0494] o, M, M’, R2e, and R3care as defined for variable IL*;

[0495] R2ais a Ct-8 alkyl; and

[0496] R3ais Ct-8 alkyl.

[0497] Variables o, R1, RN, RN, Rh'' of Ionizable lipid

[0498] In some embodiments of the ionizable lipid, o is 1.

[0499] In some embodiments of the ionizable lipid, o is 2.

[0500] In some embodiments of the ionizable lipid, o is 3.

[0501] In some embodiments of the ionizable lipid, o is 4.

[0502] In some embodiments of the ionizable lipid, R1is -OH.

[0503] In some embodiments of the ionizable lipid, RNis H.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0504] In some embodiments of the ionizable lipid, RNis methyl.

[0505] In some embodiments of the ionizable lipid, RNis ethyl.

[0506] In some embodiments of the ionizable lipid, R1is -NRN-cyclobutenyl, wherein the cyclobutenyl is optionally substituted with one or more oxo or -N(RNRN).

[0507] In some embodiments of the ionizable lipid, RNis H.

[0508] In some embodiments of the ionizable lipid, RNis methyl.

[0509] In some embodiments of the ionizable lipid, RNis ethyl.

[0510] In some embodiments of the ionizable lipid, RNis H.

[0511] In some embodiments of the ionizable lipid, RNis methyl.

[0512] In some embodiments of the ionizable lipid, RNis ethyl.

[0513] In some embodiments of the ionizable lipid, RNis H and RNis methyl.

[0514] In some embodiments of the ionizable lipid,

[0515]

[0516] R1is

[0517] In some embodiments of the ionizable lipid,

[0518]

[0519] R1is

[0520] Variables m and n of the Ionizable lipid

[0521] In some embodiments of the ionizable lipid, m is 4.

[0522] In some embodiments of the ionizable lipid, m is 5.

[0523] In some embodiments of the ionizable lipid, m is 6.

[0524] In some embodiments of the ionizable lipid, m is 7.

[0525] In some embodiments of the ionizable lipid, m is 8.

[0526] In some embodiments of the ionizable lipid, m is 4.

[0527] In some embodiments of the ionizable lipid, n is 5.

[0528] In some embodiments of the ionizable lipid, n is 6.

[0529] In some embodiments of the ionizable lipid, n is 7.

[0530] In some embodiments of the ionizable lipid, n is 8.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0531] In some embodiments of the ionizable lipid, n is 5 and m is 7.

[0532] In some embodiments of the ionizable lipid, n is 7 and m is 7.

[0533] In some embodiments of the ionizable lipid, m is 6 and n is 6.

[0534] Variables R2, R2a, R2b, R2cof Ionizable lipid

[0535] R2aR2b

[0536]

[0537] In some embodiments of the ionizable lipid, R2is 'A

[0538] In some embodiments of the ionizable lipid, R2ais hydrogen.

[0539] In some embodiments of the ionizable lipid, R2ais methyl.

[0540] In some embodiments of the ionizable lipid, R2ais ethyl.

[0541] In some embodiments of the ionizable lipid, R2ais propyl.

[0542] In some embodiments of the ionizable lipid, R2ais butyl.

[0543] In some embodiments of the ionizable lipid, R2ais pentyl.

[0544] In some embodiments of the ionizable lipid, R2ais hexyl.

[0545] In some embodiments of the ionizable lipid, R2ais heptyl.

[0546] In some embodiments of the ionizable lipid, R2ais octyl.

[0547] In some embodiments of the ionizable lipid, R2bis hydrogen.

[0548] In some embodiments of the ionizable lipid, R2bis methyl.

[0549] In some embodiments of the ionizable lipid, R2bis ethyl.

[0550] In some embodiments of the ionizable lipid, R2bis propyl.

[0551] In some embodiments of the ionizable lipid, R2bis butyl.

[0552] In some embodiments of the ionizable lipid, R2bis pentyl.

[0553] In some embodiments of the ionizable lipid, R2bis hexyl.

[0554] In some embodiments of the ionizable lipid, R2bis heptyl.

[0555] In some embodiments of the ionizable lipid, R2bis octyl.

[0556] In some embodiments of the ionizable lipid, R2ais hydrogen and R2bis hydrogen.

[0557] In some embodiments of the ionizable lipid, R2ais hexyl and R2bis

[0558] hydrogen.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0559] In some embodiments of the ionizable lipid, R2ais octyl and R2bis hydrogen. In some embodiments of the ionizable lipid, R2ais hydrogen and R2bis butyl.

[0560] In some embodiments of the ionizable lipid, R2cis methyl.

[0561] In some embodiments of the ionizable lipid, R2cis ethyl.

[0562] In some embodiments of the ionizable lipid, R2cis propyl.

[0563] In some embodiments of the ionizable lipid, R2cis butyl.

[0564] In some embodiments of the ionizable lipid, R2cis pentyl.

[0565] In some embodiments of the ionizable lipid, R2cis hexyl.

[0566] In some embodiments of the ionizable lipid, R2cis heptyl.

[0567] In some embodiments of the ionizable lipid, R2cis octyl.

[0568] In some embodiments of the ionizable lipid, R2is -(C1-6alkylene)-(C3-8cycloalkyl)-C1-6alkyl.

[0569] In some embodiments of the ionizable lipid, R2is -(C1-6alkylene)-(cyclohexyl)-C1-6alkyl.

[0570] In some embodiments of the ionizable lipid, R2is -(C1-6alkylene)-(cyclopentyl)-C1-6alkyl.

[0571] Variables R3, R3a, R3b, and R3cof Ionizable lipid

[0572] In some embodiments of the ionizable lipid,

[0573]

[0574] R3is R3aR3b

[0575] In some embodiments of the ionizable lipid, R3ais hydrogen.

[0576] In some embodiments of the ionizable lipid, R3ais methyl.

[0577] In some embodiments of the ionizable lipid, R3ais ethyl.

[0578] In some embodiments of the ionizable lipid, R3ais propyl.

[0579] In some embodiments of the ionizable lipid, R3ais butyl.

[0580] In some embodiments of the ionizable lipid, R3ais pentyl.

[0581] In some embodiments of the ionizable lipid, R3ais hexyl.

[0582] In some embodiments of the ionizable lipid, R3ais heptyl.

[0583] In some embodiments of the ionizable lipid, R3ais octyl.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0584] In some embodiments of the ionizable lipid, R3bis hydrogen.

[0585] In some embodiments of the ionizable lipid, R3bis methyl.

[0586] In some embodiments of the ionizable lipid, R3bis ethyl.

[0587] In some embodiments of the ionizable lipid, R3bis propyl.

[0588] In some embodiments of the ionizable lipid, R3bis butyl.

[0589] In some embodiments of the ionizable lipid, R3bis pentyl.

[0590] In some embodiments of the ionizable lipid, R3bis hexyl.

[0591] In some embodiments of the ionizable lipid, R3bis heptyl.

[0592] In some embodiments of the ionizable lipid, R3bis octyl.

[0593] In some embodiments of the ionizable lipid, R3ais octyl and R3bis hydrogen.

[0594] In some embodiments of the ionizable lipid, R3ais ethyl and R3bis hydrogen.

[0595] In some embodiments of the ionizable lipid, R3ais hexyl and R3bis hydrogen.

[0596] In some embodiments of the ionizable lipid, R3cis methyl.

[0597] In some embodiments of the ionizable lipid, R3cis ethyl.

[0598] In some embodiments of the ionizable lipid, R3cis propyl.

[0599] In some embodiments of the ionizable lipid, R3cis butyl.

[0600] In some embodiments of the ionizable lipid, R3cis pentyl.

[0601] In some embodiments of the ionizable lipid, R3cis hexyl.

[0602] In some embodiments of the ionizable lipid, R3cis heptyl.

[0603] In some embodiments of the ionizable lipid, R3cis octyl.

[0604] It is understood that, for an ionizable lipid, variables o, R1, RN, RN, RN, m, n, M, M’, R2, R2a, R2b, R2c, R3, R3a, R3b, and R3ccan each be, where applicable, selected from the groups described herein, and any group described herein for any of variables o,. R', RN, RN, RN’, m, n, M, M’, R2, R2a, R2b, R2c, R3, R3a, R3b, and

[0605] R3ccan be combined, where applicable, with any group described herein for oneAttorney Docket No. 45817-0180W01 / MTX1503.20

[0606] or more of the remainder of variables o, R1, RN, RN, R, m, n, M, M’, R2, R2a, R2b, R2c, R3, R3a, R3b, and R3e.

[0607] In some embodiments, the ionizable lipid is a compound selected from:

[0608]

[0609] In some embodiments, the ionizable lipid is

[0610]

[0611] In some embodiments, the ionizable lipid is

[0612]

[0613] In some embodiments, the ionizable lipid is

[0614]

[0615] (1-301).

[0616] In some embodiments, the ionizable lipid is

[0617] (II-6).

[0618]

[0619] Attorney Docket No. 45817-0180W01 / MTX1503.20

[0620] Without wishing to be bound by theory, it is understood that an ionizable lipid may have a positive or partial positive charge at physiological pH. Such

[0621] lipids may be referred to as cationic or ionizable (amino) lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.

[0622] Non-cationic (neutral) lipids

[0623] In certain embodiments, lipid nanoparticles comprise one or more noncationic lipids (neutral lipids). Non-cationic lipids may be phospholipids.

[0624] In some embodiments, the lipid nanoparticle comprises 5-25 mol% noncationic lipid. For example, the lipid nanoparticle may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% non-cationic lipid. In some embodiments, the lipid nanoparticle comprises 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% noncationic lipid.

[0625] In some embodiments, a non-cationic lipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPc), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3 -phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3 -phosphocholine, 1,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-Attorney Docket No. 45817-0180W01 / MTX1503.20

[0626] dilinolenoyl-sn-glycero-3-phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), sphingomyelin, or mixtures thereof.

[0627] In some embodiments, the lipid nanoparticle comprises 5 - 15 mol%, 5 - 10 mol%, or 10 - 15 mol% DSPC. For example, the lipid nanoparticle may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% DSPC.

[0628] In certain embodiments, the lipid composition of the lipid nanoparticle compositions can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[0629] A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.

[0630] A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0631] Particular phospholipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g, LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.

[0632] Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g, an alkenyl group in which one or more double bonds isAttorney Docket No. 45817-0180W01 / MTX1503.20

[0633] replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide.

[0634] Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

[0635] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids.

[0636] Phospholipids also include phosphosphingolipid, such as sphingomyelin.

[0637] In some embodiments, a phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPc), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3 -phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-( 1 -glycerol)

[0638] sodium salt (DOPG), sphingomyelin, or mixtures thereof.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0639] In certain embodiments, a phospholipid is an analog or variant of DSPC. In certain embodiments, a phospholipid is a compound of Formula (HI):

[0640] R1©

[0641] d\© O

[0642] R -N^Os i / Ov. A

[0643]

[0644] R 0(HI),

[0645] or a salt thereof, wherein:

[0646] each R1is independently optionally substituted alkyl; or optionally two R1are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R1are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl;

[0647] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0648] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0649] L2-R2

[0650] xi ( BH-(R2)P

[0651] |_2

[0652] A is of the formula:

[0653]

[0654] ' or ';

[0655] each instance of L2is independently a bond or optionally substituted C1-6alkylene, wherein one methylene unit of the optionally substituted C1-6alkylene is optionally replaced with O, N(RN), S, C(O), C(O)N(RN), NRNC(O), C(O)O, OC(O), OC(O)O, -OC(O)N(RN), NRNC(O)O, or NRNC(O)N(RN);

[0656] each instance of R2is independently optionally substituted C1-30alkyl, optionally substituted C1-30alkenyl, or optionally substituted C1-30alkynyl; optionally wherein one or more methylene units of R2are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclyl ene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), -NRNC(O)N(RN), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), -C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), -NRNC(S), NRNC(S)N(RN), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, -N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O;Attorney Docket No. 45817-0180W01 / MTX1503.20

[0657] each instance of RNis independently hydrogen, optionally substituted

[0658] alkyl, or a nitrogen protecting group;

[0659] Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

[0660] p is 1 or 2.

[0661] In certain embodiments, the compound is not of the formula:

[0662]

[0663] wherein each instance of R2is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0664] In some embodiments, the phospholipids may be one or more of the phospholipids described in PCT Application No. PCT / US2018 / 037922.

[0665] In some embodiments, the lipid nanoparticle comprises a molar ratio of 5-25% non-cationic lipid relative to the other lipid components. For example, the lipid nanoparticle may comprise a molar ratio of 5-30%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, 20-25%, or 25-30% non-cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, 25%, or 30% non-cationic lipid.

[0666] In some embodiments, the lipid nanoparticle comprises a molar ratio of 5-25% phospholipid relative to the other lipid components. For example, the lipid nanoparticle may comprise a molar ratio of 5-30%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, 20-25%, or 25-30% phospholipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, 25%, or 30% phospholipid lipid.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0667] Structural lipids

[0668] The lipid composition of a pharmaceutical composition can comprise one or more structural lipids. As used herein, the term “structural lipid” includes

[0669] sterols and also to lipids containing sterol moieties.

[0670] Incorporation of structural lipids in the lipid nanoparticle may help

[0671] mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alphatocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol.

[0672] In some embodiments, the structural lipids may be one or more of the structural lipids described in U. S. Patent No. 11,969,506, which is incorporated herein by reference to the extent it describes structural lipids.

[0673] In some embodiments, the lipid nanoparticle comprises a molar ratio of 25-55% structural lipid relative to the other lipid components. For example, the lipid nanoparticle may comprise a molar ratio of 10-55%, 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55% structural lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% structural lipid.

[0674] In some embodiments, the lipid nanoparticle comprises 30-45 mol% sterol, optionally 35-40 mol%, for example, 30-31 mol%, 31-32 mol%, 32-33 mol%, 33-34 mol%, 35-35 mol%, 35-36 mol%, 36-37 mol%, 38-38 mol%, 38-39 mol%, or 39-40 mol%. In some embodiments, the lipid nanoparticle comprises 25-55 mol% sterol. For example, the lipid nanoparticle may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%,Attorney Docket No. 45817-0180W01 / MTX1503.20

[0675] 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% sterol. In some embodiments, the lipid nanoparticle comprises 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% sterol.

[0676] In some embodiments, the lipid nanoparticle comprises 35 -40 mol% cholesterol. For example, the lipid nanoparticle may comprise 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, or 40 mol% cholesterol.

[0677] Polyethylene glycol (PEG)-modified lipids

[0678] The lipid composition, such as a lipid nanoparticle composition, can comprise one or more polyethylene glycol (PEG)-modified lipids.

[0679] As used herein, the term “PEG-lipid” or “PEG-modified lipid” refers to polyethylene glycol (PEG)-modifted lipids. Non-limiting examples of PEG-modified lipids include PEG-modified phosphatidylethanolamine and

[0680] phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. For example, a

[0681] PEG-modified lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE,

[0682] PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0683] In some embodiments, the PEG-modified lipid includes, but is not limited to 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)]

[0684] (PEG-DSPE), PEG-distearyl glycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA).

[0685] In some embodiments, the PEG-modified lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modifiedAttorney Docket No. 45817-0180W01 / MTX1503.20

[0686] phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, and / or PEG-DPG

[0687] In some embodiments, the lipid moiety of the PEG-modified lipids includes those having lengths of from about Cwto about C22, preferably from about Ci4to about Ci6. In some embodiments, a PEG moiety, for example an mPEG-NFE, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In some embodiments, the PEG-modified lipid is PEG2k-DMG.

[0688] In some embodiments, lipid nanoparticles can comprise a PEG-modified lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE.

[0689] PEG-modified lipids are known in the art, such as those described in U. S. Patent No. 8,158,601 and PCT Publication. No. WO 2015 / 130584 A2, which are incorporated herein by reference to the extent they describe PEG-modified lipids.

[0690] In general, some of the other lipid components (e.g., PEG-modified lipids) of various formulae may be synthesized as described PCT Application No.

[0691] PCT / US2016 / 000129, which is incorporated by reference to the extent it discloses lipid components and production.

[0692] The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG-modified lipid is a lipid modified with polyethylene glycol. A PEG-modified lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG-modified lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0693] In some embodiments the PEG-modified lipids are a modified form of

[0694] PEG DMG. PEG-DMG has the following structure:

[0695]

[0696] O

[0697] In some embodiments, PEG-modified lipids can be PEGylated lipids described in PCT Publication No. WO 2012 / 099755, which is herein incorporated by reference to the extent it discloses PEG-modified lipids. Any of these

[0698] exemplary PEG-modified lipids may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG-modified lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an -OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment.

[0699] In certain embodiments, a PEG-modified lipid is a compound of Formula (PI):

[0700]

[0701] or salts thereof, wherein:

[0702] R3is -OR°;

[0703] R° is hydrogen, optionally substituted alkyl, or an oxygen protecting

[0704] group;

[0705] r is an integer between 1 and 100, inclusive;

[0706] L1is optionally substituted Ci-io alkylene, wherein at least one methylene of the optionally substituted Ci-io alkylene is independently replaced with

[0707] optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(RN), S,Attorney Docket No. 45817-0180W01 / MTX1503.20

[0708] C(O), C(O)N(RN), NRNC(O), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, or -NRNC(O)N(RN);

[0709] D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions;

[0710] m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0711] L2— R2,

[0712] vI. ( B 4-(R2)P

[0713] \ / ^L2-R2

[0714] A is of the formula:

[0715]

[0716] ' or ';

[0717] each instance of L2is independently a bond or optionally substituted Ci-6 alkylene, wherein one methylene unit of the optionally substituted Ci-6 alkylene is optionally replaced with O, N(RN), S, C(O), C(O)N(RN), NRNC(O), C(O)O, OC(O), -OC(O)O, OC(O)N(RN), NRNC(O)O, or NRNC(O)N(RN);

[0718] each instance of R2is independently optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30 alkynyl; optionally wherein one or more methylene units of R2are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), -NRNC(O)N(RN), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), -C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), -NRNC(S), NRNC(S)N(RN), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, -N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O;

[0719] each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;

[0720] Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

[0721] p is 1 or 2.

[0722] In certain embodiments, the compound of Formula (PI) is a PEG-OH lipid (z.e., R3is -OR°, and R° is hydrogen). In certain embodiments, the compound of Formula (PI) is of Formula (PI-OH):Attorney Docket No. 45817-0180W01 / MTX1503.20

[0723]

[0724] or a salt thereof.

[0725] In certain embodiments, a PEG-modified lipid is a PEGylated fatty acid.

[0726] In certain embodiments, a PEG-modified lipid is a compound of Formula (PII). In some embodiments, compounds of Formula (PII) have the following formula:

[0727] O

[0728]

[0729] or a salts thereof, wherein:

[0730] R3is-OR°;

[0731] R° is hydrogen, optionally substituted alkyl or an oxygen protecting

[0732] group;

[0733] r is an integer between 1 and 100, inclusive;

[0734] R3is optionally substituted C10-40 alkyl, optionally substituted C10-40 alkenyl, or optionally substituted C10-40 alkynyl; and optionally one or more methylene groups of R5are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), -NRNC(O)N(RN), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, -SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), -C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), -N(RN)S(O)O, S(O)2, n(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O; and

[0735] each instance of RNis independently hydrogen, optionally substituted

[0736] alkyl, or a nitrogen protecting group.

[0737] In certain embodiments, the compound of Formula (PII) is of Formula

[0738] (PII-OH):Attorney Docket No. 45817-0180W01 / MTX1503.20

[0739] O

[0740]

[0741] (pn-OH),

[0742] or a salt thereof.

[0743] In some embodiments, r is 40-50.

[0744] In yet other embodiments the compound of Formula (PII) is:

[0745] o

[0746]

[0747] or a salt thereof.

[0748] In some embodiments, the compound of Formula (PII) is

[0749] o

[0750]

[0751] In some embodiments, the lipid composition does not comprise a PEG-modified lipid.

[0752] In some embodiments, the PEG-modified lipids may be one or more of the PEG-modified lipids described in U. S. Patent No. 10,207,010, which is incorporated by reference herein to the extent it discloses PEG-modified lipids.

[0753] In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5-15% PEG-modified lipid relative to the other lipid components. For example, the lipid nanoparticle may comprise a molar ratio of 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% PEG-modified lipid.

[0754] In some embodiments, the lipid nanoparticle comprises 1-5% PEG-modified lipid, optionally 1-3 mol%, for example 1.5 to 2.5 mol%, 1-2 mol%, 2-3 mol%, 3-4 mol%, or 4-5 mol%. In some embodiments, the lipid nanoparticle comprises 0.5-15 mol% PEG-modified lipid. For example, the lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol%. In some embodiments, the lipid nanoparticleAttorney Docket No. 45817-0180W01 / MTX1503.20

[0755] comprises 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG-modified lipid.

[0756] Some embodiments comprise adding PEG to a composition comprising an LNP in which a nucleic acid is formulated (e.., which already includes PEG in the amounts listed above). In embodiments comprise adding about 0.5mo% or more PEG to an LNP composition, such as about lmol%, about 1.5mol%, about 2mol%, about 2.5mol%, about 3mol%, about 3.5mol%, about 4mol%, about 5mol%, or more after formation of an LNP composition e.g., which already contains PEG in amount listed elsewhere herein).

[0757] In some embodiments, a lipid nanoparticle comprises a first PEG-modified lipid in a core of the LNP, and a second PEG-modified lipid outside of the core of the LNP. The first and second PEG-modified lipids of the core and outside the core may the same PEG-modified lipids (z.e., have the same structure), or be different PEG-modified lipids (z.e., have different structures). In some embodiments, both PEG-modified lipids are 134-hydroxy-3, 6; 9,12,15,18,: 21, 24,27,30,33,36,39, 42, 45, 48, 51, 54,57, 60, 63, 66,69,72,75,78, 81, 8 4,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate. In some embodiments, both PEG-modified lipids are PEG-DMG. In some embodiments, the first PEG-modified lipid is PEG-DMG and the second PEG-modified lipid is 134-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,8 4,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate. In some embodiments, the first

[0758] PEG-modified lipid is ^-hydroxySy, 9, 12, 15, 18, 21,24, 27, 30, 33, 36, 39, 42, 45, 48, 51,54, 57, 60, 63, 66, 69, 72, 75, 78, 81,8 4,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate and the second PEG-modified lipid is PEG-DMG. In some embodiments, 0.25 mol% to 1.0 mol% (as a percentage ofAttorney Docket No. 45817-0180W01 / MTX1503.20

[0759] lipids in the LNP) of the first PEG-modified lipid is in the core of the lipid nanoparticle. In some embodiments, 0.25 mol% to 0.50 mol% of the first PEG-modified lipid is in the core of the lipid nanoparticle. In some embodiments, 0.25 mol%, 0.50 mol%, 0.75 mol%, or 1.0 mol% of the first PEG-modified lipid is in the core of the LNP. In some embodiments, 2.0 mol% to 2.75 mol% of the second PEG-modified lipid is outside the core of the LNP. In some embodiments, 2.0 mol%, 2.25 mol%, 2.5 mol%, or 2.75 mol% of the second PEG-modified lipid is outside the core of the LNP. In some embodiments, the LNP comprises 3.0 mol% PEG-modified lipids.

[0760] LNPs having certain amounts of a PEG-modified lipid in the core and certain amounts of a PEG-modified lipid outside of the core, and methods of producing the same, are disclosed in PCT Publication No. WO 2023 / 018773, which is incorporated by reference herein to the extent it discloses lipid nanoparticles and methods of producing lipid nanoparticles.

[0761] In some embodiments, the lipid nanoparticle comprises 20-60 mol% ionizable lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid.

[0762] In some embodiments, a LNP comprises an ionizable lipid of Compound (1-18), wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG-modified lipid is DMG-PEG. In some embodiments, a LNP comprises 20-60 mol% ionizable lipid of Compound (1-18), 5-25 mol% DSPC, 25-55 mol% cholesterol, and 2-4 mol% DMG-PEG.

[0763] In some embodiments, a LNP comprises an ionizable lipid of Compound (1-25), wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG-modified lipid is DMG-PEG. In some embodiments, a LNP comprises 20-60 mol% ionizable lipid of Compound (1-25), 5-25 mol% DSPC, 25-55 mol% cholesterol, and 2-4 mol% DMG-PEG.

[0764] In some embodiments, a LNP comprises an ionizable lipid of Compound (1-301), wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG-modified lipid is DMG-PEG. In some embodiments, a LNP comprises 20-60 mol%Attorney Docket No. 45817-0180W01 / MTX1503.20

[0765] ionizable lipid of Compound (1-301), 5-25 mol% DSPC, 25-55 mol% cholesterol, and 2-4 mol% DMG-PEG.

[0766] In some embodiments, a LNP comprises an ionizable lipid of Compound (II-6), wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG-modified lipid is DMG-PEG. In some embodiments, a LNP comprises

[0767] 20-60 mol% ionizable lipid of Compound (11-6), 5-25 mol% DSPC, 25-55 mol% cholesterol, and 2-4 mol% DMG-PEG.

[0768] In some embodiments, a LNP comprises an ionizable lipid of Compound (IL**), wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG-modified lipid is DMG-PEG. In some embodiments, a LNP comprises 20-60 mol% ionizable lipid of Compound (IL**), 5-25 mol% DSPC, 25-55 mol% cholesterol, and 2-4 mol% DMG-PEG.

[0769] In some embodiments, a LNP comprises an ionizable lipid of any of Formula (IL*), a phospholipid comprising DSPC, a structural lipid, and a PEG-modified lipid comprising PEG-DMG. In some embodiments, a LNP comprises

[0770] 20-60 mol% ionizable lipid of Formula (IL*), 5-25 mol% phospholipid

[0771] comprising DSPC, 25-55 mol% structural lipid, and 2-4 mol% PEG-modified

[0772] lipid comprising DMG-PEG.

[0773] In some embodiments, a LNP comprises an ionizable lipid of any of Formula (IL*), a phospholipid comprising DSPC, a structural lipid, and a PEG-modified lipid comprising a compound having Formula (PII). In some embodiments, a LNP comprises 20-60 mol% ionizable lipid of Formula (IL*), 5-25 mol% phospholipid comprising DSPC, 25-55 mol% structural lipid, and 2-4 mol% PEG-modified lipid comprising a compound having Formula (PII).

[0774] In some embodiments, a LNP comprises an ionizable lipid of Formula (IL*), a phospholipid comprising a compound having Formula (HI), a structural lipid, and the PEG-modified lipid comprising a compound having Formula (PI) or (Pll). In some embodiments, a LNP comprises 20-60 mol% ionizable lipid of Formula (IL*), 5-25 mol% phospholipid comprising a compound having FormulaAttorney Docket No. 45817-0180W01 / MTX1503.20

[0775] (HI), 25-55 mol% structural lipid, and 2-4 mol% PEG-modified lipid comprising a compound having Formula (PI) or (PII).

[0776] In some embodiments, a LNP comprises an ionizable lipid of Formula (IL*), a phospholipid comprising a compound having Formula (HI), a structural lipid, and the PEG-modified lipid comprising a compound having Formula (PI) or (PII). In some embodiments, a LNP comprises 20-60 mol% ionizable lipid of Formula (IL*), 5-25 mol% phospholipid comprising a compound having Formula (HI), 25-55 mol% structural lipid, and 2-4 mol% PEG-modified lipid modified lipid comprising a compound having Formula (PI) or (PII).

[0777] In some embodiments, a LNP comprises an ionizable lipid of Formula (IL*), a phospholipid having Formula (HI), a structural lipid, and a PEG-modified lipid comprising a compound having Formula (PIT). In some embodiments, a LNP comprises 20-60 mol% ionizable lipid of Formula (IL*), 5-25 mol% phospholipid having Formula (HI), 25-55 mol% structural lipid, and 2-4 mol% PEG-modified lipid comprising a compound having Formula (PII).

[0778] In some embodiments, the lipid nanoparticle comprises 49 mol% ionizable lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG.

[0779] In some embodiments, the lipid nanoparticle comprises 49 mol% ionizable lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.

[0780] In some embodiments, the lipid nanoparticle comprises 48 mol% ionizable lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG.

[0781] In some embodiments, a LNP comprises an N: P ratio of from about 2: 1 to about 30:1.

[0782] In some embodiments, a LNP comprises an N: P ratio of about 6: 1.

[0783] In some embodiments, aLNP comprises anN: P ratio of about 3:1, 4:1, or 5:1. In some embodiments, a LNP comprises a wt / wt ratio of the ionizable lipid component to the RNA of from about 10:1 to about 100: 1.

[0784] In some embodiments, a LNP comprises a wt / wt ratio of the ionizable lipid component to the RNA of about 20: 1.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0785] In some embodiments, a LNP comprises a wt / wt ratio of the ionizable

[0786] lipid component to the RNA of about 10:1.

[0787] Some embodiments comprise a composition having one or more LNPs having a diameter of about 150 nm or less, such as about 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm or less. Some embodiments comprise a composition having a mean LNP diameter of about 150 nm or less, such as about 140 nm, 130 nm, 120 nm, 110 nm, 100 nm,

[0788] 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm or less. In some embodiments, the composition has a mean LNP diameter from about 30nm to

[0789] about 150nm, or a mean diameter from about 60nm to about 120nm.

[0790] In some embodiments, an LNP further comprises one or more cargo molecules, including but not limited to nucleic acids (e.g, mRNA, plasmid DNA, DNA or RNA oligonucleotides, siRNA, shRNA, snRNA, snoRNA, IncRNA,

[0791] etc.), small molecules, proteins, and peptides.

[0792] Effective in vivo delivery of nucleic acids represents a continuing medical challenge. Exogenous nucleic acids (z.e., originating from outside of a cell or organism) are readily degraded in the body, e.g, by the immune system.

[0793] Accordingly, effective delivery of nucleic acids to cells often requires the use of a particulate carrier (e.g., lipid nanoparticles). The particulate carrier should be formulated to have minimal particle aggregation, be relatively stable prior to intracellular delivery, effectively deliver nucleic acids intracellularly, and illicit no or minimal immune response. To achieve minimal particle aggregation and pre-delivery stability, many conventional particulate carriers have relied on the presence and / or concentration of certain components (e.g., PEG-modified lipid).

[0794] However, it has been discovered that certain components may decrease the

[0795] stability of formulated nucleic acids (e.g., mRNA molecules). The reduced

[0796] stability may limit the broad applicability of the particulate carriers. As such,

[0797] there remains a need for methods by which to improve the stability of nucleic acid (e.g, mRNA) formulated in lipid nanoparticles.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0798] In some embodiments, a LNP comprises one or more ionizable molecules (e.g., amino lipids or ionizable lipids). The ionizable molecule may comprise a charged group and may have a certain pKa. In certain embodiments, the pKa of the ionizable molecule may be greater than or equal to about 6, greater than or equal to about 6.2, greater than or equal to about 6.5, greater than or equal to about 6.8, greater than or equal to about 7, greater than or equal to about 7.2, greater than or equal to about 7.5, greater than or equal to about 7.8, greater than or equal to about 8. In some embodiments, the pKa of the ionizable molecule may be less than or equal to about 10, less than or equal to about 9.8, less than or equal to about 9.5, less than or equal to about 9.2, less than or equal to about 9.0, less than or equal to about 8.8, or less than or equal to about 8.5. Combinations of the above referenced ranges are also possible (e.g., greater than or equal to 6 and less than or equal to about 8.5). Other ranges are also possible. In embodiments in which more than one type of ionizable molecule are present in a particle, each type of ionizable molecule may independently have a pKa in one or more of the ranges described above.

[0799] In general, an ionizable molecule comprises one or more charged groups. In some embodiments, an ionizable molecule may be positively charged or negatively charged. For instance, an ionizable molecule may be positively charged. For example, an ionizable molecule may comprise an amine group. As used herein, the term “ionizable molecule” has its ordinary meaning in the art and may refer to a molecule or matrix comprising one or more charged moiety. As used herein, a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc. The charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively-charged moi eties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively-charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example,Attorney Docket No. 45817-0180W01 / MTX1503.20

[0800] changes in pH may alter the charge of the moiety, and / or cause the moiety to become charged or uncharged. In general, the charge density of the molecule

[0801] and / or matrix may be selected as desired.

[0802] In some embodiments, an ionizable molecule (e.g., an amino lipid or ionizable lipid) may include one or more precursor moieties that can be converted to charged moieties. For instance, the ionizable molecule may include a neutral moiety that can be hydrolyzed to form a charged moiety, such as those described above. As a non-limiting specific example, the molecule or matrix may include an amide, which can be hydrolyzed to form an amine, respectively. Those of

[0803] ordinary skill in the art will be able to determine whether a given chemical moiety carries a formal electronic charge (for example, by inspection, pH titration, ionic conductivity measurements, etc.), and / or whether a given chemical moiety can be reacted (e.g., hydrolyzed) to form a chemical moiety that carries a formal

[0804] electronic charge.

[0805] The ionizable molecule e.g., amino lipid or ionizable lipid) may have any suitable molecular weight. In certain embodiments, the molecular weight of an ionizable molecule is less than or equal to about 2,500 g / mol, less than or equal to about 2,000 g / mol, less than or equal to about 1,500 g / mol, less than or equal to about 1,250 g / mol, less than or equal to about 1,000 g / mol, less than or equal to about 900 g / mol, less than or equal to about 800 g / mol, less than or equal to about 700 g / mol, less than or equal to about 600 g / mol, less than or equal to about 500 g / mol, less than or equal to about 400 g / mol, less than or equal to about 300

[0806] g / mol, less than or equal to about 200 g / mol, or less than or equal to about 100 g / mol. In some instances, the molecular weight of an ionizable molecule is greater than or equal to about 100 g / mol, greater than or equal to about 200 g / mol, greater than or equal to about 300 g / mol, greater than or equal to about 400 g / mol, greater than or equal to about 500 g / mol, greater than or equal to about 600 g / mol, greater than or equal to about 700 g / mol, greater than or equal to about 1000 g / mol,

[0807] greater than or equal to about 1,250 g / mol, greater than or equal to about 1,500Attorney Docket No. 45817-0180W01 / MTX1503.20

[0808] g / mol, greater than or equal to about 1,750 g / mol, greater than or equal to about 2,000 g / mol, or greater than or equal to about 2,250 g / mol. Combinations of the above ranges (e.g., at least about 200 g / mol and less than or equal to about 2,500 g / mol) are also possible. In embodiments in which more than one type of ionizable molecules are present in a particle, each type of ionizable molecule may independently have a molecular weight in one or more of the ranges described above.

[0809] In some embodiments, the percentage (e.g., by weight, or by mole) of a single type of ionizable molecule (e.g., amino lipid or ionizable lipid) and / or of all the ionizable molecules within a particle may be greater than or equal to about 15%, greater than or equal to about 16%, greater than or equal to about 17%, greater than or equal to about 18%, greater than or equal to about 19%, greater than or equal to about 20%, greater than or equal to about 21%, greater than or equal to about 22%, greater than or equal to about 23%, greater than or equal to about 24%, greater than or equal to about 25%, greater than or equal to about 30%, greater than or equal to about 35%, greater than or equal to about 40%, greater than or equal to about 42%, greater than or equal to about 45%, greater than or equal to about 48%, greater than or equal to about 50%, greater than or equal to about 52%, greater than or equal to about 55%, greater than or equal to about 58%, greater than or equal to about 60%, greater than or equal to about 62%, greater than or equal to about 65%, or greater than or equal to about 68%. In some instances, the percentage (e.g, by weight, or by mole) may be less than or equal to about 70%, less than or equal to about 68%, less than or equal to about 65%, less than or equal to about 62%, less than or equal to about 60%, less than or equal to about 58%, less than or equal to about 55%, less than or equal to about 52%, less than or equal to about 50%, or less than or equal to about 48%. Combinations of the above referenced ranges are also possible (e.g., greater than or equal to 20% and less than or equal to about 60%, greater than or equal to 40% and less than or equal to about 55%, etc.). In embodiments in which more than one type of ionizable molecule is present in a particle, each type of ionizable molecule may independently have a percentage (e.g., by weight, or by mole) in one or more of the ranges described above. The percentage (e.g, by weight, or by mole) may be determinedAttorney Docket No. 45817-0180W01 / MTX1503.20

[0810] by extracting the ionizable molecule(s) from the dried particles using, e.g.,

[0811] organic solvents, and measuring the quantity of the agent using high pressure

[0812] liquid chromatography (i.e., HPLC), liquid chromatography -mass spectrometry

[0813] (LC-MS), nuclear magnetic resonance (NMR), or mass spectrometry (MS). Those of ordinary skill in the art would be knowledgeable of techniques to determine the quantity of a component using the above-referenced techniques. For example, HPLC may be used to quantify the amount of a component, by, e.g., comparing the area under the curve of a HPLC chromatogram to a standard curve.

[0814] It should be understood that the terms “charged” or “charged moiety” does not refer to a “partial negative charge" or “partial positive charge" on a molecule.

[0815] The terms “partial negative charge" and “partial positive charge" are given their ordinary meaning in the art. A “partial negative charge" may result when a functional group comprises a bond that becomes polarized such that electron

[0816] density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way. In some embodiments, the composition comprises a liposome. A liposome is a lipid particle comprising lipids arranged into one or more concentric lipid bilayers around a central region. The central region of a liposome may comprise an aqueous solution, suspension, or other aqueous composition.

[0817] In some embodiments, the composition comprises a lipoplex. A lipoplex is a lipid particle comprising a cationic liposome and a nucleic acid e.g., mRNA).

[0818] Lipoplexes may be formed by contacting a liposome comprising a cationic lipid with a nucleic acid. A lipoplex may comprise multiple concentric lipid bilayers, each concentric bilayer separated by one or more nucleic acids. The central region of the lipoplex may comprise an aqueous solution, suspension, or other aqueous composition.

[0819] In some embodiments, the composition comprises a lipopolyplex. A lipopolyplex is a lipid particle comprising a lipid bilayer surrounding a complexAttorney Docket No. 45817-0180W01 / MTX1503.20

[0820] of a cationic polymer and a nucleic acid (e.g., mRNA). See Midoux & Pi chon, Expert Rev Vaccines. 2015. 14(2):221-234. A lipopolyplex may be formed by contacting a cationic liposome (e.g., liposome comprising a cationic lipid) with the complex of nucleic acid and cationic polymer. The central region of the lipopolyplex may comprise an aqueous solution, suspension, or other aqueous composition.

[0821] In some embodiments, the composition comprises a cationic nanoemulsion. A cationic nanoemulsion comprises a cationic lipid, hydrophilic surfactant, and hydrophobic surfactant.

[0822] A liposome, lipoplex, lipopolyplex, or cationic nanoemulsion may comprise a sterol. A liposome, lipoplex, lipopolyplex, or cationic nanoemulsion may comprise a neutral lipid. A liposome, lipoplex, lipopolyplex, or cationic nanoemulsion may comprise a PEG-modified lipid.

[0823] Other Lipid Composition Components

[0824] The lipid composition of a pharmaceutical composition disclosed herein can include one or more components in addition to those described above. For example, the lipid composition can include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents (e.g., surfactants), or other components. For example, a permeability enhancer molecule can be a molecule described by U. S. Patent Application Publication No. 2005 / 0222064. Carbohydrates can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).

[0825] A polymer can be included in and / or used to encapsulate or partially encapsulate a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition in lipid nanoparticle form). A polymer can be biodegradable and / or biocompatible. A polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0826] The ratio between the lipid composition and the polynucleotide range can be from about 10:1 to about 60:1 (wt / wt).

[0827] In some embodiments, the ratio between the lipid composition and the polynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt / wt). In some embodiments, the wt / wt ratio of the lipid composition to the polynucleotide encoding a therapeutic agent is about 20:1 or about 15:1.

[0828] In some embodiments, the pharmaceutical composition disclosed herein can contain more than one polypeptides. For example, a pharmaceutical composition disclosed herein can contain two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) polynucleotides (e.g., RNA, e.g., mRNA).

[0829] In one embodiment, the lipid nanoparticles described herein can comprise polynucleotides (e.g., mRNA) in a lipid:polynucleotide weight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or 70:1, or a range or any of these ratios such as, but not limited to, 5: 1 to about 10:1, from about 5: 1 to about 15:1, from about 5:1 to about 20:1, from about 5:1 to about 25:1, from about 5:1 to about 30:1, from about 5:1 to about 35:1, from about 5:1 to about 40:1, from about 5:1 to about 45:1, from about 5:1 to about 50:1, from about 5:1 to about 55:1, from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about 10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 to about 25:1, from about 10:1 to about 30:1, from about 10:1 to about 35:1, from about 10:1 to about 40:1, from about 10:1 to about 45:1, from about 10:1 to about 50:1, from about 10:1 to about 55:1, from about 10:1 to about 60:1, from about 10:1 to about 70:1, from about 15:1 to about 20:1, from about 15:1 to about 25:1, from about 15:1 to about 30:1, from about 15:1 to about 35:1, from about 15:1 to about 40:1, from about 15:1 to about 45:1, from about 15:1 to about 50:1, from about 15:1 to about 55:1, from about 15:1 to about 60:1 or from about 15:1 to about 70:1.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0830] In one embodiment, the lipid nanoparticles described herein can comprise the polynucleotide in a concentration from approximately 0.1 mg / ml to 2 mg / ml such as, but not limited to, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, 2.0 mg / ml or greater than 2.0 mg / ml.

[0831] Nanoparticle Compositions

[0832] In some embodiments, the pharmaceutical compositions disclosed herein are formulated as lipid nanoparticles (LNPs). Accordingly, the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent such as compound as described herein, and (ii) polynucleotides encoding each of the polypeptides described herein (e.g., a shuffled MAGEA4 polypeptide, a shuffled MAGEA6 polypeptide, a shuffled MAGEC2 polypeptide, a shuffled NY-ESO-1 (CTAG1B) polypeptide, a shuffled PRAME polypeptide, a shuffled SSX1 polypeptide, and a shuffled XAGE1B polypeptide). In such nanoparticle composition, the lipid composition disclosed herein can encapsulate the polynucleotide encoding any of the polypeptides described herein (e.g., polynucleotides encoding each of the polypeptides described herein (e.g., a shuffled MAGEA4 polypeptide, a shuffled MAGEA6 polypeptide, a shuffled MAGEC2 polypeptide, a shuffled NY-ESO-1 (CTAG1B) polypeptide, a shuffled PRAME polypeptide, a shuffled SSX1 polypeptide, and a shuffled XAGE1B polypeptide).

[0833] Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0834] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes. In some embodiments, nanoparticle compositions are vesiclesAttorney Docket No. 45817-0180W01 / MTX1503.20

[0835] including one or more lipid bilayers. In certain embodiments, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers can be functionalized and / or crosslinked to one another. Lipid bilayers can include one or more ligands, proteins, or channels.

[0836] In one embodiment, a lipid nanoparticle comprises an ionizable amino lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNP comprises an ionizable amino lipid, a PEG-modified lipid, a sterol and a structural lipid. In some embodiments, the LNP has a molar ratio of about 40-50% ionizable amino lipid; about 5-15% structural lipid; about 30-45% sterol; and about 1-5% PEG-modified lipid.

[0837] In some embodiments, the LNP has a polydispersity value of less than 0.4. In some embodiments, the LNP has a net neutral charge at a neutral pH. In some embodiments, the LNP has a mean diameter of 50-150 nm. In some embodiments, the LNP has a mean diameter of 80-100 nm.

[0838] As generally defined herein, the term “lipid” refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic. Examples of classes of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids. In some instances, the amphiphilic properties of some lipids leads them to form liposomes, vesicles, or membranes in aqueous media.

[0839] In some embodiments, a lipid nanoparticle (LNP) may comprise an ionizable amino lipid. As used herein, the term “ionizable amino lipid” has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties. In some embodiments, an ionizable amino lipid may be positively charged or negatively charged. An ionizable amino lipid may be positively charged, in which case it can be referred to as “cationic lipid”. In certain embodiments, an ionizable amino lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipid. As used herein, a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc. The chargedAttorney Docket No. 45817-0180W01 / MTX1503.20

[0840] moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively- charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and / or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired.

[0841] It should be understood that the terms “charged” or “charged moiety” does not refer to a “partial negative charge" or “partial positive charge" on a molecule. The terms “partial negative charge" and “partial positive charge" are given its ordinary meaning in the art. A “partial negative charge" may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way.

[0842] The ionizable amino lipid is sometimes referred to in the art as an “ionizable cationic lipid”. In one embodiment, the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail that are connected via a linker structure.

[0843] In addition to these, an ionizable amino lipid may also be a lipid including a cyclic amine group.

[0844] In one embodiment, the ionizable amino lipid may be selected from, but not limited to, an ionizable amino lipid described in International Publication Nos.

[0845] WO2013086354 and WO2013116126; the contents of each of which are herein incorporated by reference in their entirety.

[0846] In yet another embodiment, the ionizable amino lipid may be selected from, but not limited to, Formula CL1-CLXXXX11 of US Patent No. 7,404,969; each of which is herein incorporated by reference in their entirety.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0847] In one embodiment, the lipid may be a cleavable lipid such as those described in International Publication No. WO2012170889, herein incorporated by reference in its entirety. In one embodiment, the lipid may be synthesized by methods known in the art and / or as described in International Publication Nos. WO2013086354; the contents of each of which are herein incorporated by reference in their entirety.

[0848] Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) can be used to measure zeta potentials. Dynamic light scattering can also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

[0849] The size of the nanoparticles can help counter biological reactions such as, but not limited to, inflammation, or can increase the biological effect of the polynucleotide.

[0850] As used herein, “size” or “mean size” in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition.

[0851] In one embodiment, the polynucleotide encoding any of the polypeptides described herein (e.g., polynucleotides encoding each of the polypeptides described herein (e.g., a shuffled MAGEA4 polypeptide, a shuffled MAGEA6 polypeptide, a shuffled MAGEC2 polypeptide, a shuffled NY-ESO-1 (CTAG1B) polypeptide, a shuffled PRAME polypeptide, a shuffled SSX1 polypeptide, and a shuffled XAGE1B polypeptide) are Formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100Attorney Docket No. 45817-0180W01 / MTX1503.20

[0852] nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and / or about 90 to about 100 nm.

[0853] In one embodiment, the nanoparticles have a diameter from about 10 to 500 nm. In one embodiment, the nanoparticle has a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.

[0854] In some embodiments, the largest dimension of a nanoparticle composition is 1 pm or shorter (e.g., 1 pm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter).

[0855] A nanoparticle composition can be relatively homogenous. A polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3)

[0856] polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition can have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a nanoparticle composition disclosed herein can be from about 0.10 to about 0.20.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0857] The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species can interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a nanoparticle composition disclosed herein can be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about 10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0858] In some embodiments, the zeta potential of the lipid nanoparticles can be from about 0 mV to about 100 mV, from about 0 mV to about 90 mV, from about 0 mV to about 80 mV, from about 0 mV to about 70 mV, from about 0 mV to about 60 mV, from about 0 mV to about 50 mV, from about 0 mV to about 40 mV, from about 0 mV to about 30 mV, from about 0 mV to about 20 mV, from about 0 mV to about 10 mV, from about 10 mV to about 100 mV, from about 10 mV to about 90 mV, from about 10 mV to about 80 mV, from about 10 mV to about 70 mV, from about 10 mV to about 60 mV, from about 10 mV to about 50 mV, from about 10 mV to about 40 mV, from about 10 mV to about 30 mV, from about 10 mV to about 20 mV, from about 20 mV to about 100 mV, from about 20 mV to about 90 mV, from about 20 mV to about 80 mV, from about 20 mV to about 70 mV, from about 20 mV to about 60 mV, from about 20 mV to about 50 mV, from about 20 mV to about 40 mV, from about 20 mV to about 30 mV, from about 30 mV to about 100 mV, from about 30 mV to about 90 mV, from about 30 mV to about 80 mV, from about 30 mV to about 70 mV, from about 30 mV to about 60 mV, from about 30 mV to about 50 mV, from about 30 mV to about 40 mV, from about 40Attorney Docket No. 45817-0180W01 / MTX1503.20

[0859] mV to about 100 mV, from about 40 mV to about 90 mV, from about 40 mV to about 80 mV, from about 40 mV to about 70 mV, from about 40 mV to about 60 mV, and from about 40 mV to about 50 mV. In some embodiments, the zeta potential of the lipid nanoparticles can be from about 10 mV to about 50 mV, from about 15 mV to about 45 mV, from about 20 mV to about 40 mV, and from about 25 mV to about 35 mV. In some embodiments, the zeta potential of the lipid nanoparticles can be about 10 mV, about 20 mV, about 30 mV, about 40 mV, about 50 mV, about 60 mV, about 70 mV, about 80 mV, about 90 mV, and about 100 mV.

[0860] The term “encapsulation efficiency” of a polynucleotide describes the amount of the polynucleotide that is encapsulated by or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. As used herein, “encapsulation” can refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement.

[0861] Encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of the polynucleotide in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents.

[0862] Fluorescence can be used to measure the amount of free polynucleotide in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a polynucleotide can be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0863] The amount of a polynucleotide present in a pharmaceutical composition disclosed herein can depend on multiple factors such as the size of the polynucleotide, desired target and / or application, or other properties of the nanoparticle composition as well as on the properties of the polynucleotide.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0864] For example, the amount of an mRNA useful in a nanoparticle composition can depend on the size (expressed as length, or molecular mass), sequence, and other characteristics of the mRNA. The relative amounts of a polynucleotide in a nanoparticle composition can also vary.

[0865] The relative amounts of the lipid composition and the polynucleotide present in a lipid nanoparticle composition of the present disclosure can be optimized according to considerations of efficacy and tolerability. For compositions including an mRNA as a polynucleotide, the N: P ratio can serve as a useful metric.

[0866] As the N: P ratio of a nanoparticle composition controls both expression and tolerability, nanoparticle compositions with lowN: P ratios and strong expression are desirable. N: P ratios vary according to the ratio of lipids to RNA in a nanoparticle composition.

[0867] In general, a lower N: P ratio is preferred. The one or more RNA, lipids, and amounts thereof can be selected to provide an N: P ratio from about 2: 1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, theN: P ratio can be from about 2:1 to about 8:1. In other embodiments, theN: P ratio is from about 5:1 to about 8:1. In certain embodiments, the N: P ratio is between 5:1 and 6:1. In one specific aspect, the N: P ratio is about is about 5.67:1.

[0868] In addition to providing nanoparticle compositions, the present disclosure also provides methods of producing lipid nanoparticles comprising encapsulating a polynucleotide. Such method comprises using any of the pharmaceutical compositions disclosed herein and producing lipid nanoparticles in accordance with methods of production of lipid nanoparticles known in the art. See, e.g., Wang et al. (2015) “Delivery of oligonucleotides with lipid nanoparticles” Adv.

[0869] Drug Deliv. Rev. 87:68-80; Silva et al. (2015) “Delivery Systems for Biopharmaceuticals. Part I: Nanoparticles and Microparticles” Curr. Pharm.

[0870] Technol. 16: 940-954; Naseri et al. (2015) “Solid Lipid Nanoparticles and Nanostructured Lipid Carriers: Structure, Preparation and Application” Adv.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0871] Pharm. Bull. 5:305-13; Silva et al. (2015) “Lipid nanoparticles for the delivery of biopharmaceuticals” Curr. Pharm. Biotechnol. 16:291-302, and references cited therein.

[0872] Stabilizing Compositions

[0873] Some embodiments of compositions are stabilized pharmaceutical compositions. Various non-viral delivery systems, including nanoparticle formulations, present attractive opportunities to overcome many challenges associated with RNA delivery. Lipid nanoparticles (LNPs) have drawn particular attention in recent years as various LNP formulations have shown promise in a variety of pharmaceutical applications.

[0874] However, lipids have been shown to degrade nucleic acids, including mRNA, and lipid nanoparticle formulations undergo rapid loss of purity when stored as refrigerated liquids. Moreover, the storage stability of mRNA formulated in LNPs is lower than that of unformulated mRNA.

[0875] A class of compounds has been found to stabilize nucleic acids within a lipid carrier such as an LNP, an unexpected and unprecedented discovery which enables applications including extended refrigerated liquid shelf-life, extended in-use periods at room temperature, and extended in-use stability at physiological temperatures up to higher temperatures such as 40°C. Such stabilizing compounds solve a critical problem, as current manufacturing processes and formulations experience a 5-10% purity loss during LNP formation and processing that is typical with current large-scale LNP production.

[0876] In some embodiments, the stabilized pharmaceutical composition comprises a nucleic acid formulation comprising an mRNA and a stabilizing compound (e.g., a compound of Formula (I), of Formula (II), or a tautomer or solvate thereof). In some embodiments, the stabilized pharmaceutical composition comprises a nucleic acid formulation comprising an mRNA and a lipid, and a compound of Formula (I):Attorney Docket No. 45817-0180W01 / MTX1503.20

[0877]

[0878] * (I),

[0879] or a tautomer or solvate thereof, wherein:

[0880] = is a single bond or a double bond;

[0881] R1is H; R2is OCH3, or together with R3is OCH2O; R3is OCH3, or

[0882] together with R2is OCH2O; R4is H; R5is H or OCH3; R6is OCH3; R7is H or OCH3; R8is H; R9is H or CH3; and X is a pharmaceutically acceptable anion, e.g., a halide such as chloride.

[0883] In some embodiments, the compound of Formula (I) has the structure of:

[0884]

[0885] Formula (la) Formula (lb) Formula (Ic) or a tautomer or solvate thereof.

[0886] In some embodiments, the stabilized pharmaceutical composition

[0887] comprises a nucleic acid formulation comprising an mRNA and a lipid, and a compound of Formula (II):Attorney Docket No. 45817-0180W01 / MTX1503.20

[0888]

[0889] or a tautomer or solvate thereof, wherein:

[0890] R10is H; R11is H; R12together with R13is OCH2O; R14is H; R15together with R16is OCH2O; R17is H; and X is a pharmaceutically acceptable anion, e.g., a halide such as chloride.

[0891] In some embodiments, the compound of Formula (II) has the structure of:

[0892] 0

[0893]

[0894] Formula (Ila),

[0895] or a tautomer or solvate thereof.

[0896] Stabilizing compounds of Formulas (I), (la), (lb), (Ic), (II), and (lia) are described in PCT Application No. PCT / US2022 / 025967, which is incorporated by reference herein to the extent it discloses compounds of such Formulas (I), (la), (lb), (Ic), (II), and (lia).

[0897] In some embodiments, the nucleic acid formulation comprises lipid nanoparticles. In some embodiments, the stabilizing compound (“the compound”) has a purity of at least 70%, 80%, 90%, 95%, or 99%. In some embodiments, the compound contains fewer than lOOppm of elemental metals. In some embodiments, the stabilized pharmaceutical composition (“the composition”) comprises a pharmaceutically acceptable metal chelator, e.g., EDTA (ethylenediaminetetraacetic acid) or DTPA (di ethyl enetri aminepentaacetic acid).

[0898] In some embodiments, the composition is an aqueous solution. In some embodiments, the compound is present at a concentration between about 0.1 mM and about lOmM in the aqueous solution. In some embodiments, the aqueous solution has a pH of or about 5 to 8, including pH of about 5, 5.5, 6, 6.5, 7, 7.5, or 8. In some embodiments, the aqueous solution does not comprise NaCl. In some embodiments, theAttorney Docket No. 45817-0180W01 / MTX1503.20

[0899] aqueous solution comprises NaCl in a concentration of or about 150mM. In some embodiments, the aqueous solution comprises a phosphate buffer, a Tris buffer, an acetate buffer, a histidine buffer, or a citrate buffer.

[0900] In some embodiments, microbial growth in the composition is inhibited by the compound.

[0901] In some embodiments, the composition is characterized as having an

[0902] mRNA purity level of greater than 60%, greater than 70%, greater than 80%, or greater than 90% main peak mRNA purity after at least thirty days of storage. In some embodiments, the composition comprises an mRNA purity level of greater than 50% main peak mRNA purity after at least six months of storage. In some embodiments, the storage is at room temperature.

[0903] In some embodiments, the composition comprises an mRNA formulated in a lipid nanoparticle, and the composition comprises less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% RNA

[0904] fragments after at least thirty days of storage. In some embodiments, the storage temperature is greater than room temperature. In some embodiments, the storage temperature is about 4 °C.

[0905] In some embodiments, the compound interacts with the mRNA comprised within a lipid nanostructure (e.g., a lipid nanoparticle, liposome, or lipoplex), e.g., via pi-pi stacking and / or by changing backbone helicity of the mRNA. In some embodiments, the compound intercalates with a mRNA. In some embodiments, the compound binds with a mRNA, e.g., reversible binding, and / or binding to the stranded regions of the mRNA. In some embodiments, the compound selfassociates, binds to mRNA ribose contacts, and / or binds to mRNA base contacts.

[0906] In some embodiments, the compound does not substantially bind to mRNA phosphate contacts. In some embodiments, the positive charge of the compound contributes to mRNA binding. In some embodiments, the interacts with the

[0907] mRNA with a binding affinity defined by an equilibrium dissociation constant ofAttorney Docket No. 45817-0180W01 / MTX1503.20

[0908] less than 10'3M (e.g., less than 10’4M, less than 10‘5M, less than IO’5M, less than 10‘7M, less than 10'8M, or less than 10'9M).

[0909] In some embodiments, the compound interacts with a mRNA and provides shielding from solvent, e.g., water. In some embodiments, the compound shields ribose from solvent more than the compound shields the phosphate groups of the mRNA. In some embodiments, the solvent exposure is measured by the solvent accessible surface area (SASA). In some embodiments, a stabilizing compound decreases the solvent accessible area of ribose to about 5-10 nm2. In some embodiments, a stabilizing compound decreases the solvent accessible area of ribose to about 6-8 nm2. In some embodiments, a stabilizing compound decreases the solvent accessible area of phosphate to about 9-12 nm2. In some embodiments, a stabilizing compound decreases the solvent accessible area of phosphate to about 10-11 nm2.

[0910] In some embodiments, a mRNA that is conformationally stabilized by the compound exhibits thermal unfolding temperatures (measured by circular dichroism or DSC, for example) that are higher than in the absence of the compound. In some embodiments, the compound confers increased stability, e.g., thermal stability, to the mRNA in a folded structure, e.g., relative to its unfolded or less folded or more linear form. In some embodiments, the compound causes compaction of the mRNA upon interaction with the mRNA. In some embodiments, the compound causes a decrease in the hydrodynamic radius of the mRNA molecule upon interaction with the mRNA. In some embodiments, a stabilizing compound causes compaction or a decrease in the hydrodynamic radius of a mRNA molecule by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more. In some embodiments, a stabilizing compound causes compaction or a decrease in the hydrodynamic radius of a mRNA molecule when the compound is in a concentration of 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, or 100 pM.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0911] Compositions and Formulations for Use

[0912] Certain aspects of the invention are directed to compositions or formulations comprising any of the polynucleotides disclosed above.

[0913] In some embodiments, the composition or formulation comprises:

[0914] (i) polynucleotides encoding each of the polypeptides described herein (e.g., a shuffled MAGEA4 polypeptide (SEQ ID NO: 1), a shuffled MAGEA6 polypeptide (SEQ ID NO:2), a shuffled MAGEC2 polypeptide (SEQ ID NO:3), a shuffled NY-ESO-1 (CTAG1B) polypeptide (SEQ ID NO:4), a shuffled PRAME polypeptide (SEQ ID NO:5), a shuffled SSX1 polypeptide (SEQ ID NO:6), and a shuffled XAGE1B polypeptide (SEQ ID NO:7)); and

[0915] (ii) a delivery agent comprising, e.g., an ionizable lipid (e.g., Compound 1-25), a phospholipid (e g., DSPC or DOPE), a structural lipid (e.g., Cholesterol), and a PEG lipid (e.g., PEG-DMG), e.g., with a mole ratio in the range of about (i) 40-50 mol% ionizable lipid (e.g., Compound 1-25), optionally 45-50 mol% ionizable lipid, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol% for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%; (ii) 30-45 mol% structural lipid (e.g., cholesterol), optionally 35-42 mol% structural lipid, for example, 30-31 mol%, 31-32 mol%, 32-33 mol%, 33-34 mol%, 35-35 mol%, 35-36 mol%, 36-37 mol%, 37-38 mol%, 38-39 mol%, or 39-40 mol%, or 40-42 mol% structural lipid; (iii) 5-15 mol% phospholipid (e.g., DSPC), optionally 10-15 mol% phospholipid, for example, 5-6 mol%, 6-7 mol%, 7-8 mol%, 8-9 mol%, 9-10 mol%, 10-11 mol%, 11-12 mol%, 12-13 mol%, 13-14 mol%, or 14-15 mol% phospholipid; and (iv) 1-5% PEG lipid (e.g., PEG-DMG), optionally 1-5 mol% PEG lipid, for example 1.5 to 2.5 mol%, 1-2 mol%, 2-3 mol%, 3-4 mol%, or 4-5 mol% PEG lipid.

[0916] Pharmaceutical Compositions

[0917] Provided are pharmaceutical compositions comprising mRNAs, optionally mRNAs with lipid delivery vehicles, and pharmaceutically acceptable excipients.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0918] Pharmaceutical compositions may not comprise proteins per se, but rather comprise nucleic acids, in particular mRNA(s), that encode antigens or therapeutic proteins that, once delivered to a cell, tissue or subject, can be translated and ultimately produce an immune response or exert a therapeutic effect. Delivery of nucleic acids, in particular mRNA(s), can be achieved by inclusion of nucleic acids in appropriate carriers or delivery vehicles (e.g, lipid nanoparticles) such that upon administration to cells, tissues or subjects, nucleic acid is taken up by cells which, in turn, express protein(s) encoded by the nucleic acids, e.g, mRNAs. Upon delivery and uptake by cells of the body, the mRNAs are translated in the cytosol and protein antigens are generated by the host cell machinery. The encoded protein(s) are presented and elicit an adaptive humoral and cellular immune response, or exert a therapeutic effect, such as protein replacement of a mutant endogenous form of the encoded protein.

[0919] The term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for therapeutic use in vivo or ex vivo. A “pharmaceutically acceptable carrier”, after being administered to or upon a subject, does not cause undesirable physiological effects.

[0920] The carrier in the pharmaceutical composition must be “acceptable” also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences. In some embodiments, the pharmaceutical composition does not comprise protamine.

[0921] Pharmaceutical compositions may be in a form for administration intramuscularly, intranasally, subcutaneously, or intradermally. Preferably, the pharmaceutical compositions are in a form for administration intramuscularly.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0922] Methods of Use

[0923] Provided are methods of administering a composition to a subject (e.g., a mammalian subject, including human subjects) in an effective amount to induce an antigen-specific immune response.

[0924] An “effective amount,” “pharmaceutically effective amount,” or “immunogenically effective amount” of a composition refers to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be induction of an antibody response and / or T cell response to an antigen. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. Typically, an effective amount of a composition provides an induced or boosted immune response as a function of antigen production in the cells of the subject.

[0925] In another embodiment, the polynucleotides, pharmaceutical compositions, or formulations of the present disclosure can be repeatedly administered such that protein is expressed at a therapeutic level for a period of time sufficient to have a beneficial biological effect as described herein.

[0926] The skilled artisan will appreciate that the therapeutic effectiveness of a drug or a treatment of the instant invention can be characterized or determined by measuring the level of expression of an encoded protein (e.g., enzyme) in a sample or in samples taken from a subject (e.g., from a preclinical test subject (rodent, primate, etc.) or from a clinical subject (human). Likewise, the therapeutic effectiveness of a drug or a treatment of the instant invention can be characterized or determined by measuring the level of activity of an encoded protein (e.g., enzyme) in a sample or in samples taken from a subject (e.g., from a preclinical test subject (rodent, primate, etc.) or from a clinical subject (human). Furthermore, the therapeutic effectiveness of a drug or a treatment of the instant invention can be characterized or determined by measuring the level of an appropriate biomarker in sample(s) taken from a subject. Levels of protein and / or biomarkers can be determined post-administration with a single dose of an mRNAAttorney Docket No. 45817-0180W01 / MTX1503.20

[0927] therapeutic of the invention or can be determined and / or monitored at several time points following administration with a single dose or can be determined and / or monitored throughout a course of treatment, e.g., a multi-dose treatment.

[0928] In some embodiments, a nucleic acid vaccine described herein (e.g., an mRNA cancer vaccine which includes multiple mRNAs encoding shuffled full-length versions of MAGEA4, MAGEA6, MAGEC2, NYESO1, PRAME, SSX1, and XAGE-1B), and compositions and formulations comprising the nucleic acid vaccine, are used to treat and / or prevent advanced solid tumor malignancies. Specifically, in some instances, provided herein are methods for treating advanced solid tumor malignancies, such as, melanoma, non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), urinary bladder cancer (UBC), bladder cancer, head and neck squamous cell carcinoma (HNSCC), esophageal carcinoma, breast cancer, colon / rectal adenocarcinoma, gastric carcinoma, ovarian carcinoma, cervical carcinoma, endometrial carcinoma, and renal cell carcinoma, wherein the method comprises administering an “effective amount,” “pharmaceutically effective amount,” or “immunogenically effective amount” of a nucleic acid vaccine described herein (or compositions and formulations comprising the vaccine) to a subject in need thereof.

[0929] In other aspects the disclosure provides anti-cancer immunotherapies, such as checkpoint inhibitors, for use in combination with the cancer vaccines that are described herein. Immune checkpoint modulators include both stimulatory checkpoint molecules and inhibitory checkpoint molecules (e.g., an anti-CTLA4 and / or an anti-PDl antibody).

[0930] Stimulatory checkpoint inhibitors function by promoting the checkpoint process. Several stimulatory checkpoint molecules are members of the tumor necrosis factor (TNF) receptor superfamily (e.g., CD27, CD40, 0X40, GITR, or CD137), while others belong to the B7-CD28 superfamily (e.g., CD28 or ICOSO. 0X40 (CD134), is involved in the expansion of effector and memory T cells. Anti-OX40 monoclonal antibodies have been shown to be effective in treating advanced cancer. MEDI0562 is a humanized 0X40 agonist. GITR, Glucocorticoid-Induced TNFR family Related gene, is involved in T cell expansion. Several antibodies to GITR have been shown to promote an anti-tumorAttorney Docket No. 45817-0180W01 / MTX1503.20

[0931] responses. ICOS, Inducible T-cell costimulator, is important in T cell effector function. CD27 supports antigen-specific expansion of naive T cells and is involved in the generation of T and B cell memory. Several agonistic anti-CD27 antibodies are in development. CD 122 is the Interleukin-2 receptor beta sub-unit. NKTR-214 is a CD 122-biased immune-stimulatory cytokine.

[0932] Inhibitory checkpoint molecules include, but are not limited to: PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3. CTLA-4, PD-1, and ligands thereof are members of the CD28-B7 family of co-signaling molecules that play important roles throughout all stages of T-cell function and other cell functions. CTLA-4, Cytotoxic T- Lymphocyte-Associated protein 4 (CD152), is involved in controlling T cell proliferation.

[0933] The PD-1 receptor is expressed on the surface of activated T cells (and B cells) and, under normal circumstances, binds to its ligands (PD-L1 and PD-L2) that are expressed on the surface of antigen-presenting cells, such as dendritic cells or macrophages. This interaction sends a signal into the T cell and inhibits it. Cancer cells take advantage of this system by driving high levels of expression of PD-L1 on their surface. This allows them to gain control of the PD-1 pathway and switch off T cells expressing PD-1 that may enter the tumor microenvironment, thus suppressing the anticancer immune response. Pembrolizumab (formerly MK-3475 and lambrolizumab, trade name KETRUDA) is a human antibody used in cancer immunotherapy and targets the PD- 1 receptor.

[0934] The checkpoint inhibitor is a molecule such as a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein or a combination thereof or a small molecule. For instance, the checkpoint inhibitor inhibits a checkpoint protein which may be CTLA-4, PD-L1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands or a combination thereof.

[0935] Ligands of checkpoint proteins include but are not limited to CTLA-4, PD-L1, PDL2, PD1, B7- H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4,Attorney Docket No. 45817-0180W01 / MTX1503.20

[0936] CD 160, CGEN- 15049, CHK 1, CHK2, A2aR, and B-7 family ligands. In some embodiments the anti-PD-1 antibody is BMS-936558 (nivolumab). In some embodiments the anti-LAG3 antibody is relatlimab. In other embodiments the anti-CTLA-4 antibody is ipilimumab (trade name Yervoy, formerly known as MDX-010 and MDX-101). In yet other embodiments the checkpoint inhibitor is pembrolizumab. In some embodiments, a combination of checkpoint inhibitors (e.g., nivolumab and relatlimab) is administered in addition to the cancer vaccine described herein.

[0937] Pembrolizumab is a potent humanized immunoglobulin G4 monoclonal antibody with high specificity of binding to the PD-1 receptor, thus inhibiting its interaction with PD- LI and programmed cell death 1 ligand 2. Based on preclinical in vitro data, pembrolizumab has high affinity and potent receptor blocking activity for PD-1.

[0938] Pembrolizumab has an acceptable preclinical safety profile and is in clinical development as an IV immunotherapy for advanced malignancies. KEYTRUDA™ (pembrolizumab) is approved for the treatment of patients across a number of indications. Pembrolizumab is approved for use in several cancer types, and is under investigation in several phases of clinical development for many more. Despite much progress in the field of immune-oncology therapeutics, not all subjects respond to pembrolizumab therapy, most responses are not complete, and it is only approved for use in limited tumor types.

[0939] Combining pembrolizumab with mRNA cancer vaccine may allow more subjects to derive greater clinical benefit than with pembrolizumab monotherapy.

[0940] The dose of pembrolizumab in some embodiments is 200 mg administered every 3 weeks. The dose recently approved in the United States for treatment of cutaneous melanoma subjects is 2 mg / kg every 3 weeks. It has been concluded that a dose of 200 mg consistently across multiple tumor types is similar to 2 mg / kg.

[0941] In some embodiments the cancer therapeutic agents, including the checkpoint inhibitors, are delivered in the form of mRNA encoding the cancer therapeutic agents. In other embodiments the checkpoint inhibitors are delivered in the form of peptides.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0942] Definitions

[0943] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0944] The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0945] In this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein. In certain aspects, the term "a" or "an" means "single." In other aspects, the term "a" or "an" includes "two or more" or "multiple."

[0946] Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as " A and / or B" herein is intended to include " A and B," " A or B," " A" (alone), and " B" (alone). Likewise, the term "and / or" as used in a phrase such as " A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0947] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0948] Wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially of are also provided.

[0949] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the invention. Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the invention. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the invention. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of an invention is disclosed as having a plurality of alternatives, examples of that invention in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of an invention can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0950] Nucleotides are referred to by their commonly accepted single-letter codes.

[0951] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Nucleobases are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, U represents uracil.

[0952] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB BiochemicalAttorney Docket No. 45817-0180W01 / MTX1503.20

[0953] Nomenclature Commission. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation.

[0954] About: The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art, such interval of accuracy is ± 10 %.

[0955] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0956] Approximately: As used herein, the term "approximately," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0957] Dosing regime. As used herein, a "dosing regimen" or a "dosing regimen" is a schedule of administration or physician determined regimen of treatment, prophylaxis, or palliative care.

[0958] Effective Amount: As used herein, the term "effective amount" of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an "effective amount" depends upon the context in which it is being applied. The term "effective amount" can be used interchangeably with "effective dose," "therapeutically effective amount," or "therapeutically effective dose."

[0959] Methods of Administration'. As used herein, “methods of administration” can include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. A method of administration can be selected to target delivery (e.g., to specifically deliver) to a specific region or system of a body.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0960] Nanoparticle Composition'. As used herein, a “nanoparticle composition” is a composition comprising one or more lipids. Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0961] The phrase "nucleotide sequence encoding" refers to the nucleic acid (e.g., an mRNA or DNA molecule) coding sequence which encodes a polypeptide. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence can further include sequences that encode signal peptides.

[0962] Pseudouridine: As used herein, pseudouridine (\| / ) refers to the C-glycoside isomer of the nucleoside uridine. A "pseudouridine analog" is any modification, variant, isoform or derivative of pseudouridine. For example, pseudouridine analogs include but are not limited to 1-carboxymethyl-pseudouridine, 1-propynyl -pseudouridine, 1-taurinomethyl-pseudouridine, 1 -taurinomethyl-4-thio-pseudouridine, 1 -methylpseudouridine (m1!] / ) (also known as Nl-methyl-pseudouridine), l-methyl-4-thio-pseudouridine (m's ), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m3\| / ), 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouri dine, 2-thio-l -methyl- 1 -deazapseudouridine, dihydropseudouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3y), and 2'-O-methyl-pseudouridine (\| / m).

[0963] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder,Attorney Docket No. 45817-0180W01 / MTX1503.20

[0964] and / or condition, to treat, improve symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0965] Uracil'. Uracil is one of the four nucleobases in the nucleic acid of RNA, and it is represented by the letter U. Uracil can be attached to a ribose ring, or more specifically, a ribofuranose via a P-Ni-glycosidic bond to yield the nucleoside uridine. The nucleoside uridine is also commonly abbreviated according to the one letter code of its nucleobase, i.e., U. Thus, in the context of the present disclosure, when a monomer in a polynucleotide sequence is U, such U is designated interchangeably as a "uracil" or a "uridine."

[0966] Uridine Content. The terms "uridine content" or "uracil content" are interchangeable and refer to the amount of uracil or uridine present in a certain nucleic acid sequence. Uridine content or uracil content can be expressed as an absolute value (total number of uridine or uracil in the sequence) or relative (uridine or uracil percentage respect to the total number of nucleobases in the nucleic acid sequence).

[0967] Uridine -Modified Sequence'. The terms "uridine-modified sequence" refers to a sequence optimized nucleic acid (e.g., a synthetic mRNA sequence) with a different overall or local uridine content (higher or lower uridine content) or with different uridine patterns (e.g., gradient distribution or clustering) with respect to the uridine content and / or uridine patterns of a candidate nucleic acid sequence. In the content of the present disclosure, the terms "uridine-modified sequence" and "uracil-modified sequence" are considered equivalent and interchangeable.

[0968] Nucleobase'. As used herein, the term “nucleobase” (alternatively “nucleotide base” or “nitrogenous base”) refers to a purine or pyrimidine heterocyclic compound found in nucleic acids, including any derivatives or analogs of the naturally occurring purines and pyrimidines that confer improved properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof. Adenine, cytosine, guanine, thymine, and uracil are the nucleobases predominately found in natural nucleic acids. Other natural, non-natural, and / or synthetic nucleobases, as known in the art and / or described herein, can be incorporated into nucleic acids. Unless otherwiseAttorney Docket No. 45817-0180W01 / MTX1503.20

[0969] specified, the nucleobase sequence of a SEQ ID NO described herein encompasses both natural nucleobases and chemically modified nucleobases (e.g., a “U” designation in a SEQ ID NO encompasses both uracil and chemically modified uracil).

[0970] Nucleoside / Nucleotide'. As used herein, the term “nucleoside” refers to a compound containing a sugar molecule (e g., a ribose in RNA or a deoxyribose in DNA), or derivative or analog thereof, covalently linked to a nucleobase (e.g., a purine or pyrimidine), or a derivative or analog thereof (also referred to herein as “nucleobase”), but lacking an internucleoside linking group (e.g., a phosphate group). As used herein, the term “nucleotide” refers to a nucleoside covalently bonded to an intemucleoside linking group (e.g., a phosphate group), or any derivative, analog, or modification thereof that confers improved chemical and / or functional properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof.

[0971] Nucleic acid: As used herein, the term “nucleic acid” is used in its broadest sense and encompasses any compound and / or substance that includes a polymer of nucleotides, or derivatives or analogs thereof. These polymers are often referred to as “polynucleotides”. Accordingly, as used herein the terms “nucleic acid” and “polynucleotide” are equivalent and are used interchangeably. Exemplary nucleic acids or polynucleotides of the disclosure include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, mRNAs, modified mRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization) or hybrids thereof.

[0972] Open Reading Frame: As used herein, the term “open reading frame”, abbreviated as “ORF”, refers to a segment or region of an mRNA molecule that encodes a polypeptide. The ORF comprises a continuous stretch of non-overlapping, in-frameAttorney Docket No. 45817-0180W01 / MTX1503.20

[0973] codons, beginning with the initiation codon and ending with a stop codon, and is translated by the ribosome.

[0974] Equivalents and Scope

[0975] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[0976] In the claims, articles such as "a," "an," and "the" can mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0977] It is also noted that the term "comprising" is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of is thus also encompassed and disclosed.

[0978] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0979] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art can be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they can be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any nucleic acid or protein encoded thereby; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0980] All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

[0981] Section and table headings are not intended to be limiting.

[0982] EXAMPLES

[0983] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0984] Example 1 — Methods for Making a Shuffled Polypeptide Vaccine

[0985] As shown in FIGs. 1A and 1B, provided herein are methods for generating a shuffled polypeptide vaccine for use in eliciting an immune response against a target polypeptide. The steps of the method include identifying a target polypeptide (e.g., MAGEA4, MAGEA6, MAGEC2, NYES01, PRAME, SSX1, and XAGE-1B) and identifying at least 3 fragments of the target polypeptide that are generated by introducing breaks in the target polypeptide at break points. The break points are chosen at least in part based on the identification and preservation of one or more T cell epitopes of the target polypeptide (so as to elicit an effective immune response against the target polypeptide), the identification and disruption of one or more regions in the target polypeptide that contribute to function of the target polypeptide (so as to reduce theAttorney Docket No. 45817-0180W01 / MTX1503.20

[0986] likelihood that the vaccine produces a functional protein), and / or the identification and disruption of one or more regions in the target polypeptide that share sequence identity with a portion of a naturally occurring polypeptide other than the target polypeptide (so as to avoid an off-target immune response). In some cases, the fragments are truncated at one or both ends to remove one or more amino acid residues that contribute to function of the target polypeptide. Additionally, in some cases, a fragment at one or both ends is truncated to remove one or more amino acid residues that are part of a sequence of amino acids that shares sequence identity with a portion of a naturally occurring polypeptide other than the target polypeptide (as outlined in FIG. IB). The fragments can also be modified to add 1-14 amino acids at the C-terminal end and / or the N-terminal end of the one or more fragments, wherein the 1-14 amino acids are identical to the 1-14 amino acids that were present on either side of the one or more break points prior to introducing breaks in the target polypeptide. These modifications are intended to ensure that T cell epitopes present in the target polypeptide are maintained in the shuffled polypeptide.

[0987] Once the fragments are identified, the fragments are rearranged to generate a plurality of different predicted shuffled polypeptides. Each of the plurality of predicted shuffled polypeptides retains in shuffled form at least 95% of the amino acid sequence from the target polypeptide. Each of the plurality of predicted shuffled polypeptides is analyzed to identify any pseudo-epitopes (i.e., epitopes that are not present in the target polypeptide but are created by rearranging the fragments) present in the predicted shuffled polypeptides. In some cases, the plurality of different predicted shuffled polypeptides generated are limited to exclude any permutations that contain one or more pseudo-epitopes that are nine amino acids in length and are identical with a portion of a naturally occurring polypeptide other than the target polypeptide (as shown in FIG. IB). Additionally, when more than 10 fragments are identified, the method further comprises implementing a genetic algorithm to select 10-20 predicted shuffled polypeptides, wherein the genetic algorithm selects predicted shuffled polypeptides with relatively fewer pseudo-epitopes.Attorney Docket No. 45817-0180W01 / MTX1503.20

[0988] The predicted 3-dimensional (3D) structures of each of the plurality of predicted shuffled polypeptides is then compared a 3D structure of the target polypeptide. A shuffled polypeptide is selected based on it having the greatest structural deviation from the target polypeptide. Once the polypeptide is selected, a nucleic acid that encodes the shuffled polypeptide is created.

[0989] mRNAs encoding shuffled versions MAGEA4, MAGEA6, MAGEC2, NYES01, PRAME, SSX1, and XAGE-1B were generated by the above-described method. The shuffled polypeptides generated for each of the target polypeptides are as follows:

[0990] MAGEA4:

[0991] MLGVMGVYDGREHTVYGEPRKLLTQDWVQENYLEYRQVPGSNPARYEFLWGP RALAETSYVKVLEHVVRVNARVRIAYPSLREAALLEEEEGVMSSEQKSQHCKPE EGVEAQEE ALGL VGAQ APTTEEQEAAVS S S SPL VPGTLEEVP AAES AGPPQ SPQG ANKVDELAHFLLRKYRAKELVTKAEMLERVLGTIAMEGDSASEEEIWEEGNNQI FPKTGLLIIVLGTIAMEGDSASESALPTTISFTCWRQPNEGSSSQEEEGPSTSPDAES LFREALSNKVDELAHFLLRKYAAESAGPPQSPQGASALPTTISFTCWRQRAKELV TKAEMLERVIKNYKRCFPVIFGKASESLKMIFGIDVKEVDPASNTYTLVTCLGLS YDGLLGNNQIFPKTGLLIIMEGDSASEEEIWEELGVMGVYDGREHTV (SEQ ID NO:1).

[0992] MAGEA6:

[0993] MRK VAKL VHFLLLK YRAREP VTKAEMLGS VVGNWQ YFFP VIF SKASD SLQLVF GIELMPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLM EVDPIGHVYIFATCLGLSYDGLLGDNQIMPKTGFLIIILAIIAKEGDCAPEEKIWEEL SVLEVFEGREDSIFGDPKKLLTFPDLESEFQAALSRKVAKLVHFLLLKYKASDSL QLVFGIELMEVDPIGHVYIFATGEVPAAESPDPPQSPQGASSLPTTMNYPLWSQSY EDSSNQEEEGPSTFPDLESEFQAALSGREDSIFGDPKKLLTQYFVQENYLEYRQEA ASSSSTLVEVTLGEVPAAESPDPPQSTQYFVQENYLEYRQVPGSDPACYEFLWGP RALIETSYVKVLHHM VKISGGPRISYPLLHEWALREGEE (SEQ ID NO:2).Attorney Docket No. 45817-0180W01 / MTX1503.20

[0994] MAGEC2:

[0995] MLTKVWVQGHYLEYREVPHSSPPYYEFLWIEVGPDHFCVFANTVGLTDEGSDD EGMPENSLLIIILSVIFIKGNCASEEVIWEVLNAVGVYAGREHFVYGEPRELLTKV WVQGHYLEYREEAS SAS STL YLVF SP S SF ST S S SLILGEVPHS SPPYYEFLWGPRA HSESIKKKVLEFL AKLNNT VP S SFP S WYKD ALKD VEERVQ ATIDT ADD AT VM AS E SL S VMS SNVSF SEGLPD SES SFTYTLDEKVAEL VEFLLLKYRAREFMELLFGL AL

[0996] lEVGPDHFCVFANTEKVAELVEFLLLKYEAEEPVTEAEMLMIVIKYKDYFPVILK RAREFMELLFGLALSPSSFSTSSSLILGGPEEEEVPSGVIPNLTESIPSSPPQGPPQGP SQSPLSSCCSSFSWSSFSEESSSQKGEDTGTCQGLPDSESSFTYTLDMPPVPGVPFR NVDNDSPTSVELEDWVDAQHPTDEEEEEASSASSTLYLVF (SEQ ID NO:3).

[0997] NY-ESO-1 (CTAG1B):

[0998] MYLAMPFATPMEAELARRSLAQDAPPLPVDHRQLQLSISSCLQQLSLLMWITQC FLPVFLAQPPSGQRRSLLMWITQVTVSGNILTIRLTAADHRQLQLSISSCLQARRS LAQDAPPLPVPGVLLKEFTVSGNILTIRLTAAMQAEGRGTGGSTGDADGPGGPGI PDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGC CRCGARGPESRLLEFYLAMPFATPMEAEL (SEQ ID NO:4).

[0999] PRAME:

[1000] MLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKA VLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAA QPMTCLQ AL YVD SLFFLRGRLDQLLRHVMNPLETL SITNCRL SEGD VMHLSQ SP S VSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLS HCSQLTTLSFYGNSISISALQSLLQHLIMERRRLWGSIQSRYISMSVWTSPRRLVEL AGQ SLLKDE AL AIA ALELISIS ALQ SLLQHLIGL SNLTHVLYP VPLCELGRP SMVW LSANPCPHCGDRTFYDPEPILCPCFMPNLLKDEALAIAALELLPRELFPPLFMAAF LRLCCKKLKIFAMPMQDIKMI LKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMI NLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQDGLSTEAEQPFIPVEVLVDLFLAttorney Docket No. 45817-0180W01 / MTX1503.20

[1001] KEGACDELFSYLIEQYIAQFTSQFLSLQCLQALYVDSLFFLRGLSNLTHVLYPVPL ESYEDIHGTLHLERLAYLHAR (SEQ ID NO: 5).

[1002] SSX1:

[1003] MERKQLVIYEEISDPEEDDEMNGDDTFAKRPRDDAKASEKRSKAFDDIATYFSK KEGRLHRIIPKIMPKKPAEDENDSKGVSEASGPQNDGKQLHPPGKANISEKINKRS GPKRGKHAWTHRLRHNRRIQVEHPQMTFGRLHRIIPKIMPKKGPKRGKHAWTH RLRERKQLVIYEEISDPWKKMKYSEKISYVYMKRNYKAMTKLGFKVTLPPFMC NKQATDFQGNDFDNDHNRRIQVEHPQMTFKAFDDIATYFSKKEWKKMKYSEKI SYVY (SEQ ID NO:6).

[1004] XAGE1B:

[1005] MVKVKIIPKEEHCKMPEAGEEQPQVMESPKKKNQQLKVGILHLGSISQTPGINLD LGSGVKVKIIPKEEHCKMRQKKIRIQLRSQCATWKVICKSCISQTPGINLDLGSGK NQQLKVGILHLGSRQKKIRIQLRSQCA (SEQ ID NO:7).

[1006] A composition (“Composition A”) comprising seven mRNAs encoding each of the seven shuffled polypeptides above (targeting each of MAGEA4, MAGEA6, MAGEC2, NYESO1, PRAME, SSX1, and XAGE-1B) was generated. The mRNAs were encapsulated in lipid nanoparticles containing a mixture of four lipids: Compound 1-25 (ionizable lipid), PEG2000-DMG, DSPC and cholesterol in a buffer containing tromethamine (Tris), sucrose, and acetate at pH 7.5.

[1007] Example 2 - Composition A target antigen expression and protein detection in human normal and tumor tissues

[1008] All Composition A targets are unmutated, germline cancer testis antigens (CTAs) that are expressed in the testis and placenta and could potentially be detected in other normal human tissues. Therefore, primary human tissues were evaluated for targetAttorney Docket No. 45817-0180W01 / MTX1503.20

[1009] detection using RNA-seq, in situ hybridization (ISH), and immunohistochemistry (IHC) methods.

[1010] Primary human normal tissue sections were sourced and screened for Composition A target detection using in situ hybridization and immunohistochemistry methods. The targets of Composition A were all detected in the testis at the mRNA and protein level. mRNA and protein were co-expressed in non-testis tissue for PRAME (adrenal gland, ovary, and uterus), MAGEA4 (placenta), SSX (thyroid; based on the internal RNASeq for mRNA expression), and MAGEA3 / 6 (placenta; based on the literature for mRNA expression). mRNA in the absence of protein signal was identified in the uterus (MAGEA3 / 6, MAGEA4, MAGEC2, and NY-ESO-1), tonsil (MAGEA3 / 6, MAGEA4, MAGEC2, NY-ESO-1, and PRAME), kidney (PRAME), and oviduct (PRAME). mRNA expression without detectable protein might indicate differential regulation of RNA versus protein expression or might indicate a limitation of the IHC assay. In addition, XAGE1, for which an IHC assay is unavailable, was expressed at the RNA level in the testis, lung, tonsil, and uterus. See FIG. 2.

[1011] The Composition A targets were also evaluated by in situ hybridization (ISH), immunohistochemistry (IHC), and / or RNA sequencing (RNA-Seq) in 5 cancer indications: head and neck squamous cell carcinoma (HNSC), hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and melanoma (SKCM). At least 1 target of Composition A was expressed at > 1 transcript per million (TPM) in 57 - 96% of the tumors evaluated for each of the indications based on RNA-Seq; and at a H score > 3 for IHC and / or an ISH grade > 1 in 60 - 94% of the tumors based on IHC / ISH data. There was concordance between the RNA-Seq data and the IHC / ISH data at the indication level, with SKCM and LUSC being the indications with the highest prevalence of expression for the targets of Composition A. See FIG. 3.Attorney Docket No. 45817-0180W01 / MTX1503.20

[1012] Example 3: Analysis of cellular processing and HLA class I presentation of Composition A mRNAs by immunopeptidomics

[1013] To assess the protein translation and HLA class I presentation of peptides derived from Composition A, monoallelic (HLA-A*02:01, HLA-A*ll:01, or HLA-B*07:02) A549 cells were transfected individually with the Composition A mRNAs and subjected to immunoprecipitation and mass spectrometry (IP-MS) analysis. These three HLA alleles were selected due to their high prevalence across the global population, including genetic variations. High frequency of these alleles and their genetic variants could potentially increase probability of greater target expression across a polymorphic population. HLA allele frequency data covering 16 diverse populations was acquired from the National Marrow Donor Program (NMDP) data published in Gragert, Loren et al. “Six-locus high resolution HLA haplotype frequencies derived from mixed-resolution DNA typing for the entire US donor registry.” Human immunology vol. 74,10 (2013): 1313-20. For each HLA locus, HLA-A, HLA-B, and HLA-C, alleles with frequency higher than 1 / 2000 (0.05%) in all the 16 populations and worldwide were selected and their frequencies were visualized as a heatmap.

[1014] Based on the worldwide frequency of the 3 HLA alleles (HLA-A*02:01, HLA-A*11:01, HLA-B*07:02), it is estimated that 55.81% of the population would have at least one of the 3 alleles. To estimate the global population coverage achieved by the 3 HLA alleles, the cumulative phenotypic frequency (CPF) was first computed for each HLA locus (HLA-A, and HLA-B) using C

[1015]

[1016] PFL = 1 - (1 - EieL(P'))2, where pi is the population frequency of the ithalleles within locus L, assuming Hardy-Weinberg proportions for the genotypes (Zhang et al., 2010, Dawson et al., 2001). Next, the CPF estimates were combined for the different HLA loci to compute a global population coverage according to: CPFtotai = 1 - fl LEH (1- CPFL), where H is the set of HLA loci (Bui et al., 2006).

[1017] HLA-I molecules were immunoprecipitated using a pan-HLA-I antibody, and bound peptides were eluted and analyzed by mass spectrometry. All mRNAs in Composition A encoded proteins that were expressed and detected. Specific peptidesAttorney Docket No. 45817-0180W01 / MTX1503.20

[1018] were presented for all constructs of Composition A across all three HLA alleles evaluated (FIG. 4). No peptides were detected from a non-translated protein control. No XAGE-1B peptide was detected on the HLA-A*02:01 cell line, which aligns with the in silico predicted absence of an HLA-A*02: 01 -restricted XAGE-1B epitope.

[1019] The mRNA target sequences of Composition A were modified from their original wildtype form to break native protein function. This modification process introduced novel junctional sequences that do not align to any other sequence in the human proteome and are predicted to bind poorly to human HLA class I alleles. Despite low predicted binding affinities, some peptide sequences derived from these novel junctions, or “pseudo epitopes,” were detected for 5 / 7 drug product sequences. However, pseudo epitope detection was generally fractionally very small relative to wildtype peptide detection and given the lack of homology with any known human protein, not expected to increase on or off target effects.

[1020] Example 4 — mRNAs of Composition A target-specific human T cell functional activity The cancer testis antigens targeted by Composition A are all germline sequences that could be subject to immune tolerization. The presence of Composition A targetspecific T cells from both the naive and memory primary human T cell compartments supports that TCRs that recognize these targets are not entirely removed during central tolerization, and that the T cells can functionally respond when encountering cognate antigen.

[1021] To confirm that human TCRs functionally respond to polypeptides encoded by Composition A mRNAs, TCR sequences specific for epitopes derived from 5 / 7 targets (PRAME, NY-ESO-1, MAGE-A4, MAGE-A6, and MAGE-C2) were cloned into Jurkat T cells that express luciferase under the control of a nuclear factor of activated T cells (NF AT) reporter. TCR-engineered T cells were co-cultured with monoallelic HLA class I-expressing A549 target cells that were transfected with the corresponding Composition A target sequences across a dose titration. All TCRs were robustly stimulated in an antigen-specific manner, as measured by luciferase activity (top of FIG. 5 (panel A)).Attorney Docket No. 45817-0180W01 / MTX1503.20

[1022] Furthermore, Jurkat reporter T cell viability remained >80% for all targets at high doses, except for MAGEA4 (bottom of FIG. 5 (panel B)). These data confirm that Composition A target sequences specifically activate cognate human TCRs in a dose titratable manner. The data also indicate that T-cell lines expressing target-specific T-cell receptors functionally respond to polypeptides encoded by Composition A mRNAs.

[1023] Next, human T-cells derived from patients were expanded in vitro, with functional expansion and restimulation tested in response to Composition A to assess T-cell responses. T-cell responses in this particular assay were assessed by interferon gamma (IFNy) and tumor necrosis factor (TNF) production in primary human T-cells from melanoma or NSCLC patient-derived PBMCs. Antigen-specific T-cell responses were observed (FIG. 6).

[1024] Finally, naive T cells from healthy donor PBMC were expanded with Composition A mRNAs for ~30 days. Target-specific T cell responses were induced across multiple HLA alleles (FIG. 7). This further cemented the T-cell reactivity findings, and also further confirmed with the IFNy ELISpot analysis.

[1025] Example 5 — In Vivo Composition A mRNA immunogenicity assessments in transgenic HLA-A*02: 1 mice

[1026] To evaluate whether polypeptides encoded by Composition A mRNAs are translated, processed, and presented to the immune system in vivo, a transgenic mouse strain expressing the human HLA-A*02:01 allele was used in immunogenicity assessments. HLA-A*02:01 transgenic mice received 3 weekly IM administrations of either the individual LNP -formulated (i.e., Compound 1-25, PEG2000-DMG, DSPC and cholesterol) mRNA sequences of Composition A or LNP -formulated (i.e., Compound I-25, PEG2000-DMG, DSPC and cholesterol) a non-translating Factor IX (NTFIX; control mRNA that does not translate and is not functional) and spleens were collected 1 week post-last dose mouse T cell responses to target epitopes presented on HLA-A*02:01 was assessed. All mice immunized with Composition A mRNAs produced robust, antigenspecific T cell responses, as measured by IFNg ELISpot (FIG. 8).Attorney Docket No. 45817-0180W01 / MTX1503.20

[1027] Example 6 — WT vs. Shuffled HPV16 constructs as measurement of Shuffled format -antigen potency

[1028] To evaluate the feasibility of using the presently described methods to develop a construct encoding a shuffled polypeptide for viral oncogenic proteins, a shuffled HPV16 E6-E7 concatenated construct was generated by identifying five fragments between the E6 and E7 proteins, adding back 28 amino acids at four breakpoint junctions (14 amino acids on each side of the breakpoints), and shuffling the fragments to generate a shuffled polypeptide that was selected according to criteria described in Example 1. The wild type and shuffled polypeptides are shown below:

[1029] WT: HPV16 E6 E7 tandem MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFR DLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRC INCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLMHGDTP TLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKC DSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP (SEQ ID NO:8).

[1030] Shuffled: HPV E6 - E7 concatenated Shuffled MSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTG RCMSCCRSSRTRRETQLMHGDTPTLHEYMLMHQKRTAMFQDPQERPRKLPQLC TELQTTIHDIILECVYCKQQLMHGDTPTLHEYMLDLQPETTDLYCYEQFCCKCDS TLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPKFYSKISEYRHYCYSLYGTTL EQQYNKPIHDIILECVYCKQQLLRREVYDFAFRDLGQAEPDRAHYNIVTFCCKCD STLRLCVQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYD LQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVT (SEQ ID NO: 9).

[1031] Immunogenicity of the two polypeptides were evaluated in normal mice. As shown in FIG. 9, mice were injected intramuscularly with mRNAs encoding either WTAttorney Docket No. 45817-0180W01 / MTX1503.20

[1032] E6-E7 or Shuffled HPV16 E6-E7 (formulated in lipid nanoparticles containing Compound 1-25). The table under the schematic shows the 4 groups of mice, the mRNA construct they received, and what concentration. Mice received 3 doses of the indicated construct and 7 days after the final dose mice were killed, and spleens were collected.

[1033] As shown in FIG. 10, the mean body weight of the mice stayed constant through the study period (mean body weight change on left; mean body weight on right). Spleen weight at the time of harvest were also roughly consistent among the groups (bottom right).

[1034] As shown in FIG. 11, bulk phenotyping showed comparable T cell proportions across groups; generally, with a trend more towards an effector like response in the groups treated with the shuffled mRNA. Additionally, it was confirmed that the constructs could generate HPV16 E6-E7 epitope specific CD8 T cell responses (see FIGs.

[1035] 12 and 13).

[1036] The constructs were then tested to determine if they could be used to treat a mouse model of a TCI tumor prophylactically or therapeutically. C57BL / 6 mice transformed with oncogenic human papillomavirus (HPV) E6 and E7 genes are a model for lung cancer and HPV-associated tumors. C57BL / 6 mice were injected with 250,000 cells of the TCI cell line. These mice develop subcutaneous tumors over time.

[1037] As shown on the top of FIG. 14, to evaluate prophylactic use of the constructs, mice that do not yet have any tumor growth were injected intramuscularly with a single dose of mRNA (formulated in lipid nanoparticles containing Compound 1-25) encoding either WT or Shuffled HPV 16 E6-E7 (formulated in lipid nanoparticles containing Compound 1-25). Mice were then monitored for tumor growth and survival.

[1038] As shown on the bottom of FIG. 14, to evaluate the therapeutic use of the mRNA constructs, mice were first transformed with TC 1 cells and monitored for tumor growth. Once tumor size is greater than 40mm3, mice were injected intramuscularly with mRNA (formulated in lipid nanoparticles containing Compound 1-25) encoding either WT or Shuffled HPV 16 E6-E7 (formulated in lipid nanoparticles containing Compound 1-25).Attorney Docket No. 45817-0180W01 / MTX1503.20

[1039] Mice were given two more doses (once every two weeks) and then monitored for survival.

[1040] The shuffled HPV16 E6-E7 proteoform vaccine was more effective in delaying tumor growth (see FIG. 15) and also increased overall survival (see FIG. 16) as compared to the WT vaccine.

[1041] Prior to treatment, tumor volume was assessed. Tumor volume was roughly the same among each group. 16 days after tumor implant, tumor volume was assessed.

[1042] Tumor volume in mice treated with shuffled HPV16 E6-E7 proteoform vaccine was lower than the non-treated group and mice treated with the WT vaccine (see FIG. 17).

[1043] 24 hours post 2nddose, mice were evaluated to determine presence of HPV16 E7 epitope specific T cells. HPV16 E7 epitope specific T cells were present in tumors of WT and shuffled vaccine treated mice (see FIG. 18). Additionally, elevated KLRG1 expression was observed on E7 multimer+ CD8 T cells across tissues, and lower PD1 expression was seen in tumor-draining lymph node (TDLN) and spleen of mice vaccinated with shuffled HPV16 E6-7 vs. the WT proteoform (see FIG. 19).

[1044] Overall, the data show that treatment with shuffled HPV16 E6-7 resulted in a therapeutic effect.

[1045] Example 7: WT vs. Shuffled PD-L1 constructs as measurement of Shuffled format To evaluate the feasibility of using the presently described methods to develop a construct encoding a shuffled polypeptide, a shuffled PD-L1 construct was generated according to criteria described in Example 1. A differential tag approach was employed by tagging the antigen at the N-terminus with a FLAG sequence and at the C-terminus with an HA sequence (see FIG. 20A). FIG. 20B shows the expected flow cytometry results based on the detection of one or more tags. Cells that stain positively for both FLAG and HA tags express the full-length polypeptide. In FIG. 20C, A549 cells were transfected with either WT PD-L1, a tagged version of WT PD-L1, or a tagged version of Shuffled PD-L1 (SH PD-L1 Tag). The top panel of FIG. 20C shows that WT PD-L1 is localized at the surface based on staining with an anti-PD-Ll antibody. However, theAttorney Docket No. 45817-0180W01 / MTX1503.20

[1046] bottom panel of FIG. 20C shows that Shuffled PD-L1 is localized intracellularly based on staining with anti-FLAG and anti-HA antibodies.

[1047] Sequences for the wild type, shuffled PD-L1, and tagged shuffled PD-L1 polypeptides are shown below:

[1048] WT PD-L1 MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQF VHGEEDLK VQHS S YRQRARLLKDQL SLGNAALQITD VKLQD AGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPK AE VIWTS SDHQ VL SGKTTTTNSKREEKLFNVT STLRINTTTNEIF YC TFRRLDPEE NHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGI QDTNSKKQSDTHLEET (SEQ ID NO 22).

[1049] WT Tagged PD-L1 MDYKDDDDKMRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPV EKQLDL AALIVYWEMEDKNIIQF VHGEEDLKVQHS S YRQRARLLKDQL SLGNAA LQ1TDVI< LQDAGVYRCMISYGGADYI< RITVI< VNAPYNI< INQRILVVDPVTSEHE LTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFY CTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGR MMDVKKCGIQDTNSKKQSDTHLEETYPYDVPDYA (SEQ ID NO:23).

[1050] Shuffled PD-L1 MFPVEKQLDLAALIVYWEMEDKNIIQFVHMRIFAVFIFMTYWHLLNAFTVTVPK DLYVVEYGSNMTIECKFPVEKQLDLAALIVRITVKVNAPYNKINQRILVVDPVTS EHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTN EIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAYWEMEDKNIIQFVH GEEDLKVQHS S YRQRARLLKDQL SLGNAALQITD VKLQD AGVYRCMISYGGAD YK1LLCLGVALTF1FRLRKGRMMDVKKCG1QDTNSKKQSDTHLEET (SEQ ID NO:24).Attorney Docket No. 45817-0180W01 / MTX1503.20

[1051] Shuffled Tagged PD-L1 MDYKDDDDKMFPVEKQLDLAALIVYWEMEDKNIIQFVHMRIFAVFIFMTYWHL LNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVRITVKVNAPYNKINQ RILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVT STLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAYWEME DKNIIQF VHGEEDLK VQHS S YRQRARLLKDQL SLGNAALQITD VKLQD AGVYRC MISYGGADYKILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET YPYDVPDYA (SEQ ID NO: 25).

[1052] Example 8: Shuffled Antigens Lose Biological Functionality

[1053] In this Example, the functionality of two shuffled polypeptides were assessed. WT PD-L1 is a surface protein that binds PD1. Accordingly, the functionality of Shuffled PD-L1 was assessed by its ability to bind a fluorophore conjugated version of PD1 (FIG. 21A). As shown in FIG. 21B, WT PD-L1 binding to PD1 was exhibited by fluorescence detection by a shifting of the curve to the right. However, the same shift was not observed for Shuffled PD-L1, indicating that Shuffled PD-L1 does not bind PD1 and has lost its wild-type functionality. The sequences from Example 7 were used for this part of this Example.

[1054] WT IDO1 catalyzes the conversion of L-tryptophan to N-formyl-L-kynurenine (see FIG. 21C). To test the functionality of Shuffled IDO-1, the ability of the shuffled polypeptide to catalyze the conversion of L-tryptophan to N-formyl-L-kynurenine was addressed. As shown in FIG. 21D, the shuffled polypeptide (unlike WT IDO1) was not able to catalyze the conversion of L-tryptophan to N-formyl-L-kynurenine. The sequences for WT IDO1 and Shuffled IDO1 are below:

[1055] WT IDO1 MAHAMENSWT1SKEYH1DEEVGFALPNPQENLPDFYNDWMF1AKHLPDL1ESGQ LRERVEKLNMLSIDHLTDHKSQRLARLVLGCITMAYVWGKGHGDVRKVLPRNIAttorney Docket No. 45817-0180W01 / MTX1503.20

[1056] AVPYCQLSKKLELPPILVYADCVLANWKKKDPNKPLTYENMDVLFSFRDGDCS KGFFLVSLLVEIAAASAIKVIPTVFKAMQMQERDTLLKALLEIASCLEKALQVFH QIHDHVNPKAFFSVLRIYLSGWKGNPQLSDGLVYEGFWEDPKEFAGGSAGQSSV FQCFDVLLGIQQTAGGGHAAQFLQDMRRYMPPAHRNFLCSLESNPSVREFVLSK GDAGLREAYDACVKALVSLRSYHLQIVTKYILIPASQQPKENKTSEDPSKLEAKG TGGTDLMNFLKTVRSTTEKSLLKEG (SEQ ID NO:26).

[1057] Shuffled IDO1 MSKKLELPPILVYADCVLANWKKKDPNKPLTYENMDVLFSFRDGDCSKGFFLVS LLVEIAAASAIKVIPTVFKAMQMQERDTLLKALLEIASCLEKALQVFHQIHDHVN PKAFFSVLESNPSVREFVLSKGDAGLREAYDACVKALVSLRSYHLQIVTKYILIPA SQQPKENKTSEDPSKLEAKGTGGTDLMNFLKTVRSTTEKSLLKEGMAHAMENS WTISKEYHIDEEVGFALPNPQENLPDFYNDWMFIAKHLPDLIESGQLRERVEKLN MLSIDHLTDHKSQRLARLVLGCITMAYVWGKGHGDVRKVLPRNIAVPYCQLRIY LSGWKGNPQLSDGLVYEGFWEDPKEFAGGSAGQSSVFQCFDVLLGIQQTAGGG HAAQFLQDMRRYMPP AHRNFLC SLKVLPRNIAVP YCQL SKKLELPPILVYADRY MPPAHRNFLCSLESNPSVREFVLSKG (SEQ ID NO:27).

[1058] Example 9: Analysis of cellular processing and HLA class I presentation of Shuffled PD-L1 or Shuffled I DOI mRNAs by immunopeptidomics

[1059] To assess the protein translation and HLA class I presentation of peptides derived from Shuffled PD-L1 or Shuffled IDO1, monoallelic HLA-A*02:01 A549 cells were transfected with mRNAs encoding either WT or Shuffled proteins and subjected to immunoprecipitation and mass spectrometry (IP -MS) analysis. HLA-I molecules were immunoprecipitated using a HLA-A*02:01 antibody, and bound peptides were eluted and analyzed by mass spectrometry.

[1060] FIGs. 22A and B are lollipop plots displaying epitope intensities detected by mass spectrometry immunopeptidomics. FIGs. 22A and B plot peptides by their position along the construct sequence (x-axis). Vertical bars with filled circles represent individualAttorney Docket No. 45817-0180W01 / MTX1503.20

[1061] construct-derived peptides detected to bind HLA-A*02:01. The height of each bar corresponds to the intensity of the detected peptide (y-axis). The ticks below each x-axis mark the positions of all predicted 8-12mer epitopes within the construct. Top of FIG.

[1062] 22A is WT PD-L1 and bottom is Shuffled PD-L1. Top of FIG. 22B is WT IDO1 and bottom is Shuffled IDO1.

[1063] A table summarizing the number of unique and pseudoepitope peptides detected to bind HLA-A*02:01 is shown below.

[1064] Na. of Detected Detected / Predicted No. of Detected Target HLA Allele Length Unique Epitopes Epitopes Pseudoepitopes WTPOll A*02.01 290 15.15< % 0 Shuffled POLI A*C2: CH 319 6 18.7S% 0 WT1DO1 A*C2;01 403 2 4.65 % 0

[1065]

[1066] Shuffled IDOL A*C2:01 460 3 <5.98 %

[1067] Tables summarizing the epitopes identified by mass spectrometry immunopeptidomics are shown below.

[1068] PD-L1 Peptide Detected in WT Detected in SH ALQITDVKL (SEQ ID NO:34) X X

[1069] HLLNAFTVTV (SEQ ID NO:35) X

[1070] KLFNVTSTL (SEQ ID NO: 36) X X

[1071] KLQDAGVYRC (SEQ ID NO:37) X

[1072] MTYWHLLNA (SEQ ID NO: 38) X X

[1073] VIWTSSDHQV (SEQ ID NO:39) X X

[1074] VTVPKDLYV (SEQ ID NO:4Q) X

[1075]

[1076] Attorney Docket No. 45817-0180W01 / MTX1503.20

[1077] IDO1 Peptide Detected in WT Detected in SH KVIPTVFKA (SEQ ID NO:41) X X

[1078] QLRERVEKL (SEQ ID NO:42) X X

[1079] SAIKVIPTV (SEQ ID NO:43) X

[1080] SLLKEGMAHA* (SEQ ID NO:44) X

[1081] SVLESNPSV* (SEQ ID NO:45) X

[1082]

[1083] * = pseudoepitope.

[1084] To evaluate whether human TCRs functionally respond to the polypeptide encoded by Shuffled PD-L1 mRNA, mono-allelic HLA-A*02: 01 -expressing Expi293F cells were transfected with mRNA encoding Shuffled PD-L1 for 18-22 hrs. Jurkat reporter cells expressing a TCR specific for the LLNFATVTV epitope (SEQ ID NO:46) were then added to the culture and incubated for 3 hrs. Data shown in FIG. 23 demonstrates that Shuffled PD-L1 target sequences specifically activated cognate human TCRs in a dose titratable manner. Data points in FIG. 23 are mean of n=3 technical replicates; error bars are SD.

[1085] Example 10: Tumor Efficacy, Survival, and Immunogenicity Study using WT v.v.

[1086] Shuffled Constructs

[1087] Tumor growth and survival were assessed in a melanoma mouse model. As shown in FIG. 24A, C57BL / 6 mice were implanted subcutaneously (SC) with 5 * 104B16. F10 melanoma cells on Day 0. B16. F10 melanoma cells express the tumor-associated antigens gplOO and Trp2. When tumors reached approximately 100 mm3(Day 8), mice received three therapeutic doses of either wild-type (WT) tumor-associated antigen (TAA)-encoding mRNA, Shuffled TAA-encoding mRNA, or control mRNA at 7-day intervals (Q7D). The TAA vaccine targets included gplOO and Trp2 (sequences are shown below). Each WT or Shuffled target (gplOO, Trp2) were singly encoded on an mRNA. The mRNAs were LNP -formulated (i.e., Compound 1-25, PEG2000-DMG, DSPC and cholesterol) and then co-delivered to mice. Tumor volume and survival were monitored throughout the study.Attorney Docket No. 45817-0180W01 / MTX1503.20

[1088] WT gplOO MGVQRRSFLPVLVLSALLAVGALEGSRNQDWLGVPRQLVTKTWNRQLYPEWTE VQGSNCWRGGQVSLRVINDGPTLVGANASFSIALHFPGSQKVLPDGQVIWANNT IINGSQVWGGQPVYPQEPDDACVFPDGGPCPSGPKPPKRSFVYVWKTWGKYWQ VLGGPVSRLSIATGHAKLGTHTMEVTVYHRRGSQSYVPLAHASSTFTITDQVPFS VSVSQLQALDGETKHFLRNHPLIFALQLHDPSGYLAEADLSYTWDFGDGTGTLIS RALDVTHTYLESGSVTAQVVLQAAIPLVSCGSSPVPGTTDGYMPTAEAPGTTSRQ GTTTKVVGTTPGQMPTTQPSGTTVVQMPTTEVTATTSEQMLTSAVIDTTLAEVST TEGTGTTPTRPSGTTVAQATTTEGPDASPLLPTQSSTGSISPLLDDTDTIMLVKRQ VPLDCVLYRYGSFSLALDIVQGIESAEILQAVPFSEGDAFELTVSCQGGLPKEACM DISSPGCQPPAQRLCQSVPPSPDCQLVLHQVLKGGSGTYCLNVSLADANSLAVAS TQLVVPGQDGGLGQAPLLVGILLVLVAVVLASLIHRHRLKKQGSVSQMPHGSTH WLRLPPVFRARGLGENSPLLSGQQV (SEQ ID NO:28).

[1089] Shuffled gplOO MSSTFTITDQVPFSVSVSQLQALDGETKHFLRNHPLIFALQLHDPSGYLAEADLSY TWDFGDGTGTLISRALDVTHTYLESGMGVQRRSFLPVLVLSALLAVGALEGSRN QDWLGVPRQLVTKTWNRQLYPEWTEVQGSNCWRGGQVSLRVINDGPTLVGAN ASFSIALHFPGSQKVLPDGQVIWANNTIINGSQVWGGQPVYPQEPDDACVFPDGG PCPSGPKPPKRSFVYVWKTWGKYWQVLGGPVSRLSIATGHAKLGTHTMEVTVY HRRGSQSYVPLAHASRALDVTHTYLESGSVTAQVVLQAAIPLLVLHQVLKGGSG TYCLNVSLADANSLAVPTTEVTATTSEQMLTSAVIDTTLAEVSTCLNVSLADANS LAVASTQLVVPGQDGGLGQAPLLVGILLVLVAVVLASLIHRHRLKKQGSVSQMP HGSTHWLRLPPVFRARGLGENSPLLSGQQVSVTAQVVLQAAIPLVSCGSSPVPGT TDGYMPTAEAPGTTSRQGTTTKVVGTTPGQMPTTQPSGTTVVQMPTTEVTATTS EQMLHRRGSQSYVPLAHASSTFTITDQVPFSVTSAVIDTTLAEVSTTEGTGTTPTR PSGTTVAQATTTEGPDASPLLPTQSSTGS1SPLLDDTDT1MLVKRQVPLDCVLYRYAttorney Docket No. 45817-0180W01 / MTX1503.20

[1090] GSFSLALDIVQGIESAEILQAVPFSEGDAFELTVSCQGGLPKEACMDISSPGCQPPA QRLCQSVPPSPDCQLVLHQVLKGGSGTY (SEQ ID NO:29).

[1091] WT Trp2 MGLVGWGLLLGCLGCGILLRARAQFPRVCMTLDGVLNKECCPPLGPEATNICGF LEGRGQCAEVQTDTRPWSGPYILRNQDDREQWPRKFFNRTCKCTGNFAGYNCG GCKFGWTGPDCNRKKPAILRRNIHSLTAQEREQFLGALDLAKKSIHPDYVITTQH WLGLLGPNGTQPQIANC S VYDFF VWLHYYS VRDTLLGPGRP YKAIDF SHQGPAF VTWHRYHLLWLERELQRLTGNESFALPYWNFATGKNECDVCTDELLGAARQD DPTLISRNSRFSTWEIVCDSLDDYNRRVTLCNGTYEGLLRRNKVGRNNEKLPTLK NVQDCLSLQKFDSPPFFQNSTFSFRNALEGFDKADGTLDSQVMNLHNLAHSFLN GTNALPHSAANDPVFVVLHSFTDAIFDEWLKRNNPSTDAWPQELAPIGHNRMYN MVPFFPPVTNEELFLTAEQLGYNYAVDLSEEEAPVWSTTLSVVIGILGAFVLLLGL LAFLQYRRLRKGYAPLMETGLSSKRYTEEA (SEQ ID NO: 30).

[1092] Shuffled Trp2 MVIGILGAFVLLLGLLAFLQYRRLRKGYAPLMETGLSSKRYTEEAHYYSVRDTLL GPGRPYKAIDFSHQGPAFVTWHRYHLLWLERELQRLTGNESFALPYWNFATGK NECDVCTDELLGAARQDDPTLISRNSRF STWEIVCD SLDDYNRRVTLCNGTYEGL LRRNKVGRNNEKLPTLKNVQDCLSLQKFDSPPFFQNSTFSFRNALEGFDKADGTL DSQVMNLHNLAHSFLNGTNALMGLVGWGLLLGCLGCGILLRARAQFPRVCMTL DGVLNKECCPPLGPEATNICGFLEGRGQCAEVQTDTRPWSGPYILRNQDDREQW PRKFFNRTCKCTGNFAGYNCGGCKFGWTGPDCNRKKPAILRRNIHSLTAQEREQ FLGALDLAKKSIHPDYVITTQHWLGLLGPNGTQPQIANCSVYDFFVWLPSTDAW PQELAPIGHNRMYNMVPFFPPVPHSAANDPVFVVLHSFTDAIFDEWLKRNNPSTD AWPQELAPIGQIANCSVYDFFVWLHYYSVRDTLLGPGRHNLAHSFLNGTNALPH SAANDPVFVVLHSEEEAPVWSTTLSVVIGILGAFVLLLGLHNRMYNMVPFFPPVT NEELFLTAEQLGYNYAVDLSEEEAPVWSTTLSV (SEQ ID NO:31).Attorney Docket No. 45817-0180W01 / MTX1503.20

[1093] Tumor growth kinetics following treatment are shown in FIG. 24B. Mean tumor volume (± SEM) was plotted over time (Days Post Implant, DPI) for control mRNA (black triangles), WT TAA mRNA (gray squares), and Shuffled TAA mRNA (black circles). Tumor volumes were assessed until 50% survival in each group. Statistical comparisons were performed using multiple unpaired t-tests; values with p < 0.05 were considered statistically significant. The data show that the WT and shuffled mRNAs reduced tumor growth rate.

[1094] Kaplan-Meier survival analysis of mice treated with control mRNA (dotted line), WT TAA mRNA (dashed line), or Shuffled TAA mRNA (solid line) is shown in FIG. 24C. Statistical comparisons were performed using the log-rank (Mantel-Cox) test; values with p < 0.05 were considered statistically significant. The data show that the WT and shuffled mRNAs increase survival outcomes.

[1095] Immunogenicity of WT and TAA mRNA vaccines in mice was also assessed. As shown in FIG. 25A, C57BL / 6 mice were immunized with either wild-type (WT) or Shuffled tumor-associated antigen (TAA)-encoding mRNA encapsulated in lipid nanoparticles (LNP) at 7-day intervals for a total of three doses (Q7D x 3). Seven days after the final immunization, splenocytes were harvested and stimulated ex vivo with Trp2 or gplOO minimal peptides for 5 hours, followed by surface and intracellular cytokine staining. FIG. 25B shows the flow cytometric analysis of CD8+T-cell responses. Frequencies of IFNy CD44 CD8+T cells were quantified after 5-hour peptide stimulation with Trp2 or gplOO. FIG. 25C is a IFNy ELISpot assay. Splenocytes were stimulated with Trp2 or gplOO peptides for 24 hours, and IFNy spot-forming units (SFU) per 2 x 105cells were enumerated after DMSO background subtraction. Statistical comparisons were performed using Mann-Whitney (ICS) or Kruskal -Wallis (ELISpot) test and corrected for multiple comparisons; values with p < 0.05 were considered statistically significant.

[1096] Example 11: A Study of Composition A Administered Alone and in Combination with Immune Checkpoint Blockade in Participants with Solid TumorsAttorney Docket No. 45817-0180W01 / MTX1503.20

[1097] This Example describes a Phase 1, first-in-human, multicenter, open-label, doseescalation study to investigate the safety, tolerability, pharmacokinetic (PK), pharmacodynamics, and preliminary clinical efficacy of Composition A administered alone and in combination with immune checkpoint blockade in participants with solid tumors.

[1098] Arm 1 (Dose Escalation): Composition A alone; participants will receive Composition A at an applicable dose as monotherapy.

[1099] Arm 2 (Dose Confirmation): Composition A in combination with nivolumab / relatlimab; participants will receive mRNA-4106 in combination with nivolumab / relatlimab at an applicable dose.

[1100] Composition A is administered by intramuscular injection. Nivolumab / relatlimab are administered by intravenous injection.

[1101] Inclusion criteria include the following:

[1102] - All participants must be a minimum age of 18 years old.

[1103] - For Arm 1, Monotherapy Arm: Histologically confirmed advanced or metastatic cancer (melanoma, non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), esophageal carcinoma, head and neck squamous cell carcinoma (HNSCC), urinary bladder cancer (UBC), colon / rectal adenocarcinomas, gastric, ovarian, cervical, and endometrial carcinomas) with measurable disease as determined by RECIST vl.l and have completed or refused all standard therapies (no limit to prior lines of therapy). Participants must have a tumor lesion amenable to biopsy, or alternatively archival tumor tissue is acceptable as long as the collection date is within one year of the enrollment date.

[1104] - Arm 2, Combination with Nivolumab / Relatlimab Arm: Histologically confirmed unresectable or metastatic melanoma, with measurable disease as determined by RECIST vl.l and have not had any prior therapy for this cancer in this setting (that is, first-line therapy). Note that prior adjuvant, neoadjuvant, or perioperative melanoma therapy (that is, anti-CTLA-4, anti-PDl / Ll, BRAF / MEK inhibitors, or interferon) is permitted if disease recurrence did not occur within 3 months from the last treatment date.Attorney Docket No. 45817-0180W01 / MTX1503.20

[1105] - Participant has an Eastern Cooperative Oncology Group (ECOG) performance status of <1.

[1106] - Participant has adequate hematological and biological function.

[1107] - Participants who could become pregnant: negative pregnancy test within 24 hours before the first dose of study treatment.

[1108] Exclusion criteria include the following:

[1109] - Participant has active central nervous system tumors or metastases.

[1110] - Participant has received treatment with prohibited medications / treatments (i.e., concurrent anticancer therapy including other chemotherapy, hormonal anticancer therapy, biologic therapy, or immunotherapy) or investigational agents within 5 half-lives or 14 days prior to the first day of study treatment (Cycle 1 Day 1), whichever is shorter.

[1111] - Participant has required the use of immunosuppressive doses of systemic steroids or absorbed topical steroids (doses >10 mg prednisone daily equivalent) within 2 weeks before study treatment administration or currently requiring maintenance doses of >10 mg prednisone or equivalent per day.

[1112] - Participant has any plan to receive a live attenuated vaccine during study treatment or has received a live vaccine within 30 days before the first dose of study treatment.

[1113] - Participant has reversible toxicities from prior cancer therapy that have not recovered to Grade 1 or baseline. Any unresolved toxicity National Cancer Institute (NCI) Common Terminology Criteria for AEs (CTCAE) Grade >2 from previous anticancer therapy with the exception of alopecia, vitiligo, and prespecified laboratory values.

[1114] - Participant has any unstable or clinically significant concurrent medical / psychiatric illness or social situation that would limit compliance with study requirements or compromise the ability of the participant to provide written informed consent, per the discretion of the Investigator.

[1115] - Participant has concurrent enrollment in another clinical study (unless it is an observational noninterventional clinical study).Attorney Docket No. 45817-0180W01 / MTX1503.20

[1116] Outcomes measured by this study are the number of participants with dose limiting toxicities. For Arm 1, this will be assessed on days 1-21 and for Arm 2 on days 1-28. The number of participants with treatment emergent adverse events, serious adverse events and adverse events of special interest will be measured for up to 4 years after the study.

[1117] OTHER EMBODIMENTS

[1118] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 45817-0180W01 / MTX1503.20WHAT IS CLAIMED IS:

1. A method of generating a nucleic acid, the method comprising:(a) identifying at least 3 fragments of a target polypeptide generated by introducing breaks in the target polypeptide at break points, optionally wherein additionally one or more of the fragments are truncated to remove one or more amino acid residues that contribute to function of the target polypeptide, optionally wherein additionally one or more of the fragments are modified with one, two, or three amino acid residues to enhance immune recognition and response, optionally wherein additionally one or more of the fragments are truncated to remove one or more amino acid residues that are part of a sequence of amino acids that shares sequence identity to 9 or more consecutive amino acids of a portion of a naturally occurring polypeptide other than the target polypeptide, and optionally wherein additionally one or more of the fragments are modified to add 1-14 amino acids at the C-terminal end and / or the N-terminal end of the one or more fragments, wherein the 1-14 amino acids are identical to the 1-14 amino acids that were present on either side of the one or more break points prior to introducing breaks in the target polypeptide;(b) rearranging the fragments identified and optionally truncated and / or modified in step (a) in different permutations, thereby generating a plurality of different predicted shuffled polypeptides, wherein each of the plurality of predicted shuffled polypeptides retains in shuffled form at least 95% of the amino acid sequence from the target polypeptide;(c) comparing predicted 3-dimensial (3D) structures of each of the plurality of predicted shuffled polypeptides to a 3D structure of the target polypeptide;(d) selecting a shuffled polypeptide that is determined in step (c) to have the greatest structural deviation from the target polypeptide; and(e) creating a nucleic acid that encodes the shuffled polypeptide.

2. The method of claim 1, wherein the method comprises identifying the break points used in step (a) at least in part based on one or more of: (i) the identification andAttorney Docket No. 45817-0180W01 / MTX1503.20preservation and / or amplification of one or more T cell epitopes of the target polypeptide, (ii) the identification and disruption of one or more regions in the target polypeptide that contribute to function of the target polypeptide, and (iii) the identification and disruption of one or more regions in the target polypeptide that share sequence identity with a portion of a naturally occurring polypeptide other than the target polypeptide.

3. The method of claim 1 or 2, wherein at least 4, 5, 6, 7, 8, 9, or 10 fragments are identified in step (a).

4. The method of any one of claims 1-3, wherein each of the fragments is between 9 amino acids and 300 amino acids in length.

5. The method of any one of claims 1-4, further comprising eliminating or minimizing one or more pseudo-epitopes present in the predicted shuffled polypeptides, wherein a pseudo-epitope is an epitope not present in the target polypeptide and created by rearrangement of the fragments.

6. The method of any one of claims 1-5, wherein the plurality of different predicted shuffled polypeptides generated in step (b) exclude any permutations that contain one or more pseudo-epitopes that comprise nine or more consecutive amino acids in length and are identical with a portion of a naturally occurring polypeptide other than the target polypeptide.

7. The method of any one of claims 1-6, wherein one or more of the fragments are truncated to remove one or more amino acid residues that associated with a biological function of the target polypeptide.

8. The method of any one of claims 1-7, wherein one or more of the fragments are truncated to remove one or more amino acid residues that are part of a sequence of amino acids comprising 9 or more consecutive amino acids identical to a portion of a naturally occurring polypeptide other than the target polypeptide.Attorney Docket No. 45817-0180W01 / MTX1503.

209. The method of any one of claims 1-7, wherein one or more of the fragments are modified by adding 1-14 amino acids to the C-terminal end and / or the N-terminal end of the one or more fragments, wherein the added 1-14 amino acids are identical to the 1-14 amino acids that were present adjacent to the one or more break points prior to introducing breaks in the target polypeptide.

10. The method of claim 9, wherein one or more of the fragments are modified by adding 14 amino acids to the C-terminal end and / or the N-terminal end of the one or more fragments, wherein the added 14 amino acids are identical to the 14 amino acids that were adjacent to the one or more break points prior to introducing breaks in the target polypeptide.

11. The method of claim 9, wherein all of the fragments are modified by adding 1-14 amino acids to the C-terminal end and / or the N-terminal end of all of the fragments, wherein the added 1-14 amino acids are identical to the 1-14 amino acids that were adjacent to each respective break point prior to introducing breaks in the target polypeptide.

12. The method of claim 9, wherein all of the fragments are modified by adding 14 amino acids to the C-terminal end and / or the N-terminal end of all of the fragments, wherein the added 14 amino acids are identical to the 14 amino acids that were adjacent to each respective break point prior to introducing breaks in the target polypeptide.

13. The method of any one of claims 1-12, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more predicted shuffled polypeptides are generated in step (b).

14. The method any one of claims 1-13, wherein, when more than 10 fragments are identified in step (a), the method further comprises, prior to step (c), implementing a computational optimization method, such as a genetic algorithm, to select 10-20 predicted shuffled polypeptides that are compared in step (c), wherein the geneticAttorney Docket No. 45817-0180W01 / MTX1503.20algorithm selects predicted shuffled polypeptides with fewer pseudo-epitopes than predicted shuffled polypeptides that are not selected by the genetic algorithm.

15. The method of any one of claims 1-14, wherein the shuffled polypeptide retains in shuffled form at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid residues from the target polypeptide.

16. The method of any one of claims 1-15, wherein the target polypeptide is a naturally occurring human polypeptide.

17. The method of claim 16, wherein the naturally occurring human polypeptide is a tumor associated antigen.

18. The method of claim 17, wherein the tumor associated antigen is MAGEA4, MAGEA6, MAGEC2, NY-ESO-1 (CTAG1B), PRAME, SSX1, or XAGE1B.

19. The method of any one of claims 1-18, wherein the nucleic acid is a mRNA.

20. A nucleic acid generated by the method of any one of claims 1-19.

21. A nucleic acid encoding a shuffled polypeptide, wherein the shuffled polypeptide comprises at least three fragments derived from a target polypeptide, wherein the at least three fragments are a in the shuffled polypeptide in an order different from the order of the fragments in the target polypeptide, wherein the shuffled polypeptide retains in shuffled form at least 95% of the amino acid sequence from the target polypeptide, and wherein one or more of the fragments are optionally:(i) modified with one, two, or three residues to enhance immune recognition and response;(ii) truncated to remove one or more amino acid residues that contribute to function of the target polypeptide;Attorney Docket No. 45817-0180W01 / MTX1503.20(iii) truncated to remove one or more amino acid residues that are part of a sequence of amino acids that shares sequence identity to 9 or more consecutive amino acids of a portion of a naturally occurring polypeptide other than the target polypeptide; and(iv) modified by adding 1-14 amino acids to the C-terminal end and / or the N-terminal end of the one or more fragments, wherein the added 1-14 amino acids are identical to the 1-14 amino acids that were adjacent to the one or more break points prior to introducing breaks in the target polypeptide.

22. The nucleic acid of claim 21, wherein the shuffled polypeptide comprises at least 4, 5, 6, 7, 8, 9, or 10 fragments derived from the target polypeptide.

23. The nucleic acid of claim 21 or 22, wherein each of the fragments is between 9 amino acids and 300 amino acids in length.

24. The nucleic acid of any one of claims 21-23, wherein one or more of the fragments are truncated to remove one or more amino acid residues associated with a biological function of the target polypeptide.

25. The nucleic acid of any one of claims 21-24, wherein one or more of the fragments are truncated to remove one or more amino acid residues that are part of a sequence of amino acids comprising 9 or more consecutive amino acids identical to a portion of a naturally occurring polypeptide other than the target polypeptide.

26. The nucleic acid of any one of claims 21-25, wherein one or more of the fragments are modified by adding 1-14 amino acids to the C-terminal end and / or the N-terminal end of the one or more fragments, wherein the added 1-14 amino acids are identical to the 1-14 amino acids adjacent to the one or more break points prior to introducing breaks in the target polypeptide.Attorney Docket No. 45817-0180W01 / MTX1503.2027. The nucleic acid of claim 26, wherein one or more of the fragments are modified by adding 14 amino acids to the C-terminal end and / or the N-terminal end of the one or more fragments, wherein the added 14 amino acids are identical to the 14 amino acids adjacent to the one or more break points prior to introducing breaks in the target polypeptide.

28. The nucleic acid of claim 26, wherein each of the fragments are modified by adding 1-14 amino acids to the C-terminal end and / or the N-terminal end of each of the fragments, wherein the added 1-14 amino acids are identical to the 1-14 amino acids adjacent to each respective break point prior to introducing breaks in the target polypeptide.

29. The nucleic acid of claim 26, wherein each of the fragments are modified by adding 14 amino acids to the C-terminal end and / or the N-terminal end of each of the fragments, wherein the added 14 amino acids are identical to the 14 amino acids adjacent to each respective break point prior to introducing breaks in the target polypeptide.

30. The nucleic acid of any one of claims 21-29, wherein the shuffled polypeptide retains in shuffled form at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the amino acid residues from the target polypeptide.

31. The nucleic acid of any one of claims 21-30, wherein the target polypeptide is a naturally occurring human polypeptide.

32. The nucleic acid of claim 31, wherein the naturally occurring human polypeptide is a tumor associated antigen.

33. The nucleic acid of claim 32, wherein the tumor associated antigen is MAGEA4, MAGEA6, MAGEC2, NY-ESO-1 (CTAG1B), PRAME, SSX1, or XAGE1B.Attorney Docket No. 45817-0180W01 / MTX1503.2034. The nucleic acid of claim 21, wherein the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1.

35. The nucleic acid of claim 21, wherein the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

36. The nucleic acid of claim 21, wherein the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3.

37. The nucleic acid of claim 21, wherein the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4.

38. The nucleic acid of claim 21, wherein the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:5.

39. The nucleic acid of claim 21, wherein the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:6.

40. The nucleic acid of claim 21, wherein the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:7.

41. The nucleic acid of claim 32, wherein the tumor associated antigen is PD-Ll.

42. The nucleic acid of claim 41, wherein the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:24.

43. The nucleic acid of claim 32, wherein the tumor associated antigen is a viral oncogenic protein.Attorney Docket No. 45817-0180W01 / MTX1503.2044. The nucleic acid of claim 43, wherein the viral oncogenic protein is human papillomavirus (HPV) protein.

45. The nucleic acid of claim 44, wherein the HPV protein is HPV16 E6 and / or HPV16E7.

46. The nucleic acid of claim 45, wherein the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:8.

47. The nucleic acid of claim 31, wherein the naturally occurring human polypeptide is a tumor associated immune regulator.

48. The nucleic acid of claim 47, wherein the tumor associated immune regulator is IDO 1.

49. The nucleic acid of claim 21, wherein the shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:27.

50. The nucleic acid of any one of claims 21-49, wherein the nucleic acid is a mRNA.

51. A pharmaceutical composition comprising one or more of the nucleic acids of any one of claims 21-50.

52. The pharmaceutical composition of claim 51, comprising at least 2, 3, 4, 5, or 6 different nucleic acids selected from the group consisting of:a first nucleic acid encoding a first shuffled polypeptide comprising at least 3 fragments derived from MAGEA4;a second nucleic acid encoding a second shuffled polypeptide comprising at least 3 fragments derived from MAGEA6;Attorney Docket No. 45817-0180W01 / MTX1503.20a third nucleic acid encoding a third shuffled polypeptide comprising at least 3 fragments derived from MAGEC2;a fourth nucleic acid encoding a fourth shuffled polypeptide comprising at least 3 fragments derived from NY-ESO-1 (CTAG1B);a fifth nucleic acid encoding a fifth shuffled polypeptide comprising at least 3 fragments derived from PRAME;a sixth nucleic acid encoding a sixth shuffled polypeptide comprising at least 3 fragments derived from SSX1; anda seventh nucleic acid encoding a seventh shuffled polypeptide comprising at least 3 fragments derived from XAGE1B.

53. The pharmaceutical composition of claim 51, comprising:a first nucleic acid encoding a first shuffled polypeptide comprising at least 3 fragments derived from MAGEA4;a second nucleic acid encoding a second shuffled polypeptide comprising at least 3 fragments derived from MAGEA6;a third nucleic acid encoding a third shuffled polypeptide comprising at least 3 fragments derived from MAGEC2;a fourth nucleic acid encoding a fourth shuffled polypeptide comprising at least 3 fragments derived from NY-ESO-1 (CTAG1B);a fifth nucleic acid encoding a fifth shuffled polypeptide comprising at least 3 fragments derived from PRAME;a sixth nucleic acid encoding a sixth shuffled polypeptide comprising at least 3 fragments derived from SSX1; anda seventh nucleic acid encoding a seventh shuffled polypeptide comprising at least 3 fragments derived from XAGE1B.

54. The pharmaceutical composition of claim 53, wherein:the first shuffled polypeptide comprises the amino acid sequence set forth in SEQ IDNO:1;Attorney Docket No. 45817-0180W01 / MTX1503.20the second shuffled polypeptide comprises the amino acid sequence set forth in SEQ IDN0:2;the third shuffled polypeptide comprises the amino acid sequence set forth in SEQ IDN0:3;the fourth shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4;the fifth shuffled polypeptide comprises the amino acid sequence set forth in SEQ IDN0:5;the sixth shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6; andthe seventh shuffled polypeptide comprises the amino acid sequence set forth in SEQ ID NO:7.

55. The pharmaceutical composition of claim 52, comprising at least 2, 3, 4, 5, or 6 of the nucleic acids of claims 34-40.

56. The pharmaceutical composition of any one of claims 51-55, wherein each of the nucleic acids are mRNAs.

57. The pharmaceutical composition of any one of claims 51-56, wherein the pharmaceutical composition comprises a lipid nanoparticle.

58. The pharmaceutical composition of claim 57, wherein the lipid nanoparticle comprises:(i) an ionizable lipid,(ii) a phospholipid,(iii) a structural lipid, and(iv) a PEG-lipid.

59. The pharmaceutical composition of claim 57 or 58, wherein the lipid nanoparticle comprises a compound of Formula (IL*):Attorney Docket No. 45817-0180W01 / MTX1503.20or a salt thereof, wherein:R1is -OH, -NRN-C4-IO cycloalkenyl optionally substituted with one ormore oxo or -N(RNRN);RNis H or Ci-6 alkyl;RNis H or Ci-6 alkyl;RNis H or Ci-6 alkyl;o is 1, 2, 3, or 4;n is 4, 5, 6, 7, or 8;m is 4, 5, 6, 7, or 8;M is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R2;M’ is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R3;R2aR2bor -(C1-6 alkylene)-(C3-8 cycloalkyl)-Ci-6 alkyl;R2ais -H or Ci-io alkyl;R2bis -H or Ci-io alkyl;R2Cis Ci-8 alkyl or C2-8 alkenyl;R3isR3aR3b.R3ais H or Ci-10 alkyl;R3bis H or C1-8 alkyl; andR3eis Ci-10 alkyl or C2-8 alkenyl.Attorney Docket No. 45817-0180W01 / MTX1503.2060. The pharmaceutical composition of any one of claims 57 to 59, wherein the lipid nanoparticle comprises (i) Compound 1-25, (ii) DSPC or DOPE, (iii) cholesterol, and (iv) PEG2000-DMG or the compound of Formula (PII).

61. The pharmaceutical composition of any one of claims 57 to 59, wherein the lipid nanoparticle comprises (i) Compound 1-25, (ii) DSPC, (iii) cholesterol, and (iv) PEG2000-DMG.

62. The pharmaceutical composition of any one of claims 58 to 61, wherein the lipid nanoparticle comprises a molar ratio of about 20-60% ionizable lipid: 5-25% phospholipid: 25-55% cholesterol: and 0.5-15% PEG lipid.

63. The pharmaceutical composition of any one of claims 51 to 62, wherein the pharmaceutical composition is formulated for intravenous, subcutaneous, intramuscular, intranasal, intraocular, rectal, pulmonary or oral delivery.

64. A method of generating an immune response in a human subject in need thereof, comprising administering to the human subject an effective amount of one or more of the nucleic acids of any one of claims 21-50 or the pharmaceutical composition of any one of claims 51-63.

65. A method of treating a cancer in a human subject in need thereof, comprising administering to the human subject an effective amount of one or more of the nucleic acids of any one of claims 21-50 or the pharmaceutical composition of any one of claims 51-63.

66. The method of claim 65, wherein the cancer is an advanced solid tumor malignancy.

67. The method of claim 65 or 66, wherein the cancer is melanoma, non-small cell lung cancer, hepatocellular carcinoma, urinary bladder cancer, bladder cancer, headAttorney Docket No. 45817-0180W01 / MTX1503.20and neck squamous cell carcinoma, esophageal carcinoma, breast cancer, colon / rectal adenocarcinoma, gastric carcinoma, ovarian carcinoma, cervical carcinoma, endometrial carcinoma, or renal cell carcinoma.

68. The method of any one of claims 65 to 67, wherein the method comprises further administering an immune checkpoint inhibitor.

69. The method of claim 68, wherein the immune checkpoint inhibitor is nivolumab, relatlimab, or a combination thereof.

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