Messenger RNA encoding masked proteins and compositions and uses thereof

Abstract

Provided herein are mRNAs encoding masked proteins for use in the treatment of diseases. More specifically, provided herein are mRNAs encoding masking proteins having a therapeutic protein, a masking moiety, at least one cleavable moiety, and, optionally, a half life extending moiety, with the at least one cleavable moiety that is inert in the liver and spleen, and the therapeutic protein is activated upon cleavage of the at least one cleavable moiety in a target tissue, such as a tumor tissue or infection site, aside from the liver or spleen.

Description

Attorney Docket No. 131986-6001MESSENGER RNA ENCODING MASKED PROTEINS AND COMPOSITIONS AND USES THEREOF CROSS-REFERNCE TO RELATED APPLICATION

[0001] This application claims the benefit under 35 U. S. C. § 119(a) of U. S. Provisional Application No. 63 / 724,877, filed November 25, 2024, the entire contents of which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to mRNA-based therapies for the treatment of various diseases. More particularly, this disclosure relates to therapies comprising mRNA-encoded masked proteins that may be expressed in one tissue and selectively activated in a different tissue to treat a disease while preventing on-target side effects of the protein.BACKGROUND

[0003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] Though promising when used in the treatment of various diseases, some therapeutic proteins may have serious negative side effects. For instance, while immunogenic cancer treatments that utilize cytokines to stimulate a subject’s immune system are effective at targeting and eliminating cancer cells, there are drawbacks to these treatments. Cytokines, like many other therapeutic proteins, are capable of stimulating a large number of cell types. However, there use as a therapy can lead to onset of various negative side effects including organ damage and even death. Thus, a continuing need exists for therapeutic proteins that are safe and effective.SUMMARY

[0005] The present disclosure provides a platform technology for mRNAs that encode a masked therapeutic protein comprising a therapeutic protein (e.g., a cytokine, chemokine, or interleukin, such as IL-12), a masking moiety, and a cleavable moiety. The disclosed mRNAs are configured such the masked protein can be transcribed or expressed in one tissue, such as the liver or spleen, and remain stable (i.e., there is no or minimal cleavage of a substrate, such that the mask keeps the therapeutic protein inactive), and after expression of the masked protein from the mRNA, the protein is unmasked (i.e., a substrate is cleaved, thereby14920-5746-9564.2Attorney Docket No. 131986-6001separating the mask and the protein) in a different, distal tissue, such as a tumor or another organ or tissue in need of the therapeutic protein.

[0006] In one aspect, the present disclosure provides mRNA encoding a masked protein, the masked protein comprising:(a) a therapeutic protein;(b) a masking moiety;(c) at least one cleavable moiety; and(d) a half-life extending moiety;wherein the at least one cleavable moiety is inert in mammalian liver and spleen, and wherein the therapeutic protein is activated upon cleavage of the at least one cleavable moiety.

[0007] In some embodiments, the therapeutic protein is a cytokine. In some embodiments, the cytokine is a pro-inflammatory cytokine, optionally selected from IL-ip, IL-6, IL-8, IL-12, IL-17, IL-18, IFN- a, IFN- y, and TNF-a. In some embodiments, the cytokine is an antiinflammatory cytokine, optionally selected from IL-IRA, IL-4, IL-6, IL-10, IL-11, IL-13, IL-35, and TGF-p.

[0008] In some embodiments, the masking moiety is selected from a single-chain antibody, an extracellular domain-based masking moiety, a protein aptamer-based masking moiety, and a steric masking moiety. In some embodiments, the single-chain antibody is an scFv comprising a heavy chain variable sequence and a light chain variable sequence of briakinumab, infliximab, etanercept, adalimumab, certolizumab, golimumab, tocilizumab, siltuximab, sarilumab, olokizumab, sirukumab, ustekinumab, tildrakizumab, guselkumab, BI-655066, LY3074828, secukinumab, ixekizumab, brodalumab, CNTO6785, bimekkizumab, or SCH-900117.

[0009] In some embodiments, the half-life extending moiety comprises an immunoglobulin (IgG) or an Fc domain thereof, a serum albumin, a single-chain antibody that binds to a serum albumin, a single-chain antibody, a hexa-hat glutathione S-transferase (GST), a glutathione affinity moiety, a calmodulin-binding peptide (CGP), a strep-tag, a cellulose binding domain, a maltose binding protein, an s-peptide tag, a chitin binding tag, an immune-reactive epitope, or an epitope tag.

[0010] In some embodiments, the at least one cleavable moiety is cleavable by proteases found in a tumor or in an infection site.24920-5746-9564.2Attorney Docket No. 131986-6001

[0011] In some embodiments, the at least one cleavable moiety can be cleaved by MMP2, MMP9, MP-ST1, uPA, or any combination thereof.

[0012] In some embodiments, the at least one cleavable moiety comprises 3-25 amino acids, 3-20 amino acids, 3-15 amino acids, 3-12 amino acids, 4-25 amino acids, 4-20 amino acids, 4-15 amino acids, 4-12 amino acids, 5-25 amino acids, 5-20 amino acids, 5-15 amino acids, 5-12 amino acids, 6-25 amino acids, 6-20 amino acids, 6-15 amino acids, 6-12 amino acids, 7-25 amino acids, 7-20 amino acids, 7-15 amino acids, 7-12 amino acids, 8-25 amino acids, 8-20 amino acids, 8-15 amino acids, or 8-12 amino acids.

[0013] In some embodiments, the at least one cleavable moiety comprises a consensus sequence comprising (i) a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid; (ii) a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid; or (ii) both a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid and a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid.

[0014] In some embodiments, the at least one cleavable moiety comprises 3 or fewer negatively charged amino acids; 2 or more special amino acids; 2 or more hydrophobic amino acids; 2 or more polar amino acids; 1 or more positive amino acids; or any combination thereof.

[0015] 1 In some embodiments, the at least one cleavable moiety comprises or consists of an amino acid sequence selected from PWGLSGRS (SEQ ID NO: 7), DHQSRSGPWGLL (SEQ ID NO: 8), and QNQALRSA (SEQ ID NO: 9).

[0016] In another aspect, the present disclosure provides mRNA encoding a first peptide chain of a masked protein and a second peptide chain of the masked protein;the first peptide chain of the masked peptide comprising, in the following order:(a) a therapeutic protein,(b) a first cleavable moiety,(c) a masking moiety,(d) a second cleavable moiety, and(e) a first IgG Fc domain; andthe second peptide chain of the masked peptide comprising a second IgGFc domain.34920-5746-9564.2Attorney Docket No. 131986-6001

[0017] In some embodiments, the therapeutic protein is a cytokine, optionally selected from IL-ip, IL-6, IL-8, IL-12, IL-17, IL-18, IFN- a, IFN- y, TNF-a, IL-IRA, IL-4, IL-10, IL-11, IL-13, IL-35, and TGF-p.

[0018] In some embodiments, the masking moiety is selected from a single-chain antibody, an extracellular domain-based masking moiety, a protein aptamer-based masking moiety, and a steric masking moiety, optionally wherein the single-chain antibody is an scFv comprising a heavy chain variable sequence and a light chain variable sequence of briakinumab, infliximab, etanercept, adalimumab, certolizumab, golimumab, tocilizumab, siltuximab, sarilumab, olokizumab, sirukumab, ustekinumab, tildrakizumab, guselkumab, BI-655066, LY3074828, secukinumab, ixekizumab, brodalumab, CNTO6785, bimekkizumab, or SCH-900117.

[0019] In some embodiments, the first cleavable moiety and the second cleavable moiety each comprise or consist of the same amino acid sequence, or wherein the first cleavable moiety and the second cleavable moiety each independently comprise or consist of a different amino acid sequence.

[0020] In some embodiments, the first cleavable moiety and the second cleavable moiety are each independently cleavable by MMP2, MMP9, MP-ST1, uPA, or any combination thereof.

[0021] In some embodiments, wherein the first cleavable moiety and the second cleavable moiety each independently comprise or consist of an amino acid sequence selected from PWGLSGRS (SEQ ID NO: 7), DHQSRSGPWGLL (SEQ ID NO: 8), and QNQALRSA (SEQ ID NO: 9).

[0022] In some embodiments, the first cleavable moiety and the second cleavable moiety are each independently inert in mammalian liver and spleen.

[0023] In some embodiments, the first IgG Fc domain comprises a knob mutation and the second IgG Fc domain comprises a hole mutation; or wherein the first IgG Fc domain comprises a hole mutation and the second IgG Fc domain comprises a knob mutation.

[0024] In another aspect, the present disclosure provides mRNA comprising a nucleic acid sequence comprising or consisting of any one of SEQ ID NOs: 151-153 or encoding a masked protein comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 135, 136, or 138-148.

[0025] In some embodiments, the mRNA comprises at least one chemical modification.44920-5746-9564.2Attorney Docket No. 131986-6001

[0026] In another aspect, the present disclosure provides compositions comprising the mRNA disclosed herein (e.g., the mRNA of the foregoing aspects or embodiments) and a lipid nanoparticle.

[0027] 25. The composition of claim 24, wherein the lipid nanoparticle comprises an ionizable amino lipid, a neutral lipid, a sterol, and a PEG-modified lipid.

[0028] The present disclosure provides methods of delivering a therapeutic protein to a tissue of interest, comprising administering to a subject an mRNA and compositions disclosed herein (e.g., the mRNA and compositions of the forgoing aspects and embodiments).

[0029] The present disclosure provides methods of stimulating an immune response, comprising administering to a subject an mRNA and compositions disclosed herein (e.g., the mRNA and compositions of the forgoing aspects and embodiments).

[0030] The present disclosure provides methods of treating cancer, comprising administering to a subject with cancer an mRNA and compositions disclosed herein (e.g., the mRNA and compositions of the forgoing aspects and embodiments).

[0031] The present disclosure provides that the mRNA and compositions disclosed herein (e.g., the mRNA and compositions of the forgoing aspects and embodiments) can be used in the manufacture of a medicament for delivering a therapeutic protein to a tissue of interest.

[0032] The present disclosure provides that the mRNA and compositions disclosed herein (e.g., the mRNA and compositions of the forgoing aspects and embodiments) can be used in the manufacture of a medicament for stimulating an immune response in a subject.

[0033] The present disclosure provides that the mRNA and compositions disclosed herein (e.g., the mRNA and compositions of the forgoing aspects and embodiments) can be used in the manufacture of a medicament for treating cancer in a subject.

[0034] The present disclosure provides uses of the mRNA and compositions disclosed herein (e.g., the mRNA and compositions of the forgoing aspects and embodiments) for delivering a therapeutic protein to a tissue of interest.

[0035] The present disclosure provides uses of the mRNA and compositions disclosed herein (e.g., the mRNA and compositions of the forgoing aspects and embodiments) for stimulating an immune response in a subject.54920-5746-9564.2Attorney Docket No. 131986-6001

[0036] The present disclosure provides uses of the mRNA and compositions disclosed herein (e.g., the mRNA and compositions of the forgoing aspects and embodiments) for treating cancer in a subject.

[0037] Additional embodiments are disclosed in Section VII of the Detailed Description.

[0038] The foregoing general description and following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIGs. 1 A-1B shows four illustrative masking strategies to be used in the masked proteins. FIG. 1 A shows different strategies, including the scFv mask, the extracellular domain- (ECD) based mask, the peptide mask, and the steric mask. FIG. IB shows the structures of specific masked proteins disclosed herein

[0040] FIGs. 2A-2D show the structure of and test results of an exemplary ECD-based mask for the IL- 12 masked protein. FIG. 2 A shows the two exemplary IL- 12 masked proteins using two different portions of the IL-12RP1 domain as the masking moiety. FIG. 2B shows a western blot analysis of the masked proteins in the absence or presence of the MMP2 protease to determine whether the masked proteins can be cleaved. FIG. 2C shows the IL- 12 reporter assay results using the MaskProl IL- 12 masked protein construct with and without cleavage. FIG. 2D shows the IL- 12 reporter assay results using the MaskPro2 IL- 12 masked protein construct with and without cleavage.

[0041] FIGs. 3 A-3E show the structure and test results of exemplary peptide masks for the IL-12 masked protein. FIG. 3A shows the configuration of an IL-12 masked protein having the peptide mask as compared to an IL- 12 unmasked protein. FIG. 3B shows the IL- 12 reporter assay results for IL- 12 masked proteins having a first set of first generation peptide masks. FIG. 3C shows the IL- 12 reporter assay results for IL- 12 masked proteins having a second set of first generation peptide masks. FIG. 3D shows the IL- 12 reporter assay results for IL-12 masked proteins having a third set of first generation peptide masks. FIG. 3E shows the IL- 12 reporter assay results for IL- 12 masked proteins having the second generation peptide masks.64920-5746-9564.2Attorney Docket No. 131986-6001

[0042] FIGs. 4A-4C show the strategy for developing a murine IL- 12 masked protein and the test results comparing the human and murine IL- 12 masked proteins. FIG. 4 A shows the murine IL-12 masked proteins and unmasked proteins developed for use in subsequent in vivo studies. FIG. 4B shows the IL-12 reporter assay results for the inactivated and activated forms of both the human IL- 12 masked protein and the murine IL- 12 masked and unmasked protein. FIG. 4C shows the western blot analysis of the proteins analyzed in FIG. 4B in the presence and absence of a protease.

[0043] FIGs. 5A-5C show the structure and analysis of an exemplary IL-12 masked protein having an anti-HSA antibody as the half-life extending moiety. FIG. 5A shows the configuration of the IL- 12 masked protein with anti-HSA antibody as the half-life extending moiety. FIG. 5B shows a western blot analysis of the IL-12 masked protein in the absence or presence of various concentrations of the MMP2 protease. FIG. 5C shows the IL-12 reporter assay results of IL- 12 masked proteins with or without the anti-HSA antibody half-life extending moiety.

[0044] FIGs. 6A-6D show the relative cleavage of various cleavable moieties in human spleen or human liver. FIG. 6A shows the percent of cleavable moiety cleaved in human spleen for Substrate-3, Substrate-1, and Substrate-2, which have been identified as potentially resistant to spleen / liver cleavage. FIG. 6B shows the percent of cleavable moiety cleaved in human liver for Substrate-3, Substrate-1, and Substrate-2, which have been identified as potentially resistant to spleen / liver cleavage. FIG. 6C shows the percent of cleavable moiety cleaved in human spleen for Substrate-4, which was not selected by the selection method described herein. FIG. 6D shows the percent of cleavable moiety cleaved in human liver for Substrate-4, which was not selected by the selection method described herein.

[0045] FIG. 7A-7D shows exemplary configurations of the IL- 12 masked protein used in subsequent experiments. FIG. 7A shows an unmasked configuration, FIG. 7B shows a non-cleavable configuration, FIG. 7C shows a dual substrate configuration, and FIG. 7D shows a single substrate configuration.

[0046] FIG. 8 show the mean tumor volume to determine tumor growth in the MC38 mouse model when various IL-12 masked and unmasked proteins are administered.

[0047] FIGs. 9A-9F show the tumor volume to determine tumor growth in the MC38 mouse model when various IL-12 masked and unmasked proteins are administered. FIG. 9A shows the mean tumor volume at each time point for the various treatments. FIG. 9B shows the74920-5746-9564.2Attorney Docket No. 131986-6001tumor volume measured over time for each individual member of the vehicle only treatment group. FIG. 9C shows the tumor volume measured over time for each individual member of the MaskPro3 treatment group. FIG. 9D shows the tumor volume measured over time for each individual member of the MaskPro22 treatment group. FIG. 9E shows the tumor volume measured over time for each individual member of the MaskPro23 treatment group. FIG. 9F shows the tumor volume measured over time for each individual member of the MaskPro4 treatment group.

[0048] FIG. 10 shows the mean body weight measured over time for animals administered various IL- 12 masked and unmasked proteins.

[0049] FIGs. 11 A and 1 IB show the AST / ALT results measured at day 12 of the treatment time course for animals administered various IL-12 masked and unmasked proteins. FIG. 11 A shows the AST levels measured. FIG. 1 IB shows the ALT levels measured.

[0050] FIGs. 12A-12E show the concentration of various cytokines measured in plasma samples taken from animals at 24 or 72 hours following administration the first dose of various IL-12 masked and unmasked proteins. FIG. 12A shows the levels of IFN-y measured 24 hours post-first dose for various IL- 12 masked and unmasked proteins treatments. FIG.12B shows the level of IFN-y measured 72 hours post-first dose for various IL- 12 masked and unmasked proteins when administered in low dosages. FIG. 12C shows the level of IFN-y measured 72 hours post-first dose for various IL- 12 masked and unmasked proteins when administered in high dosages. FIG. 12D shows the level of IL-10 measured 72 hours post-first dose for various IL-12 masked and unmasked proteins when administered in low and high dosages. FIG. 12E shows the level of TNF-a measured 72 hours post-first dose for various IL-12 masked and unmasked proteins when administered in low and high dosages.

[0051] FIGs. 13A and 13B show the structures and test results of the exemplary IL-12 proteins encoded by the mRNA constructs. FIG. 13 A shows the structures and corresponding names of the various IL- 12 proteins encoded by the mRNA constructs tested. FIG. 13B shows the IL-12 reporter assays following administration of the various IL-12 masked proteins and corresponding mRNA constructs.

[0052] FIG. 14 shows murine body weight following administration of various mRNA constructs encoding the different IL- 12 masked or unmasked proteins.

[0053] FIGs. 15A-15D show results of various tests used to determine toxicity of the mRNA constructs encoding the IL- 12 masked and unmasked proteins. FIG. 15A shows the ALT84920-5746-9564.2Attorney Docket No. 131986-6001levels measured for the various treatment groups. FIG. 15B shows the AST levels measured for the various treatment groups. FIG. 15C shows the spleen weight measured for the various treatment groups. FIG. 15D shows the liver weight measured for the various treatment groups.

[0054] FIG. 16 shows the mean tumor volume to determine tumor growth in the MC38 mouse model when the mRNA encoding various IL- 12 masked and unmasked proteins are administered.

[0055] FIG. 17 shows the tumor volume measured for each member of a treatment group in a MC38 mouse model when mRNA encoding various IL- 12 masked and unmasked proteins was administered.

[0056] FIG. 18 shows the IFN-y levels measured at day 2, day 4, and day 5 when mRNA encoding various IL-12 masked and unmasked proteins was administered.

[0057] FIG. 19 shows the GM-CSF levels measured at day 2, day 4, and day 5 when mRNA encoding various IL-12 masked and unmasked proteins was administered.

[0058] FIG. 20 shows the IL- la levels measured at day 2, day 4, and day 5 when mRNA encoding various IL-12 masked and unmasked proteins was administered.

[0059] FIG. 21 shows the IL-6 levels measured at day 2, day 4, and day 5 when mRNA encoding various IL-12 masked and unmasked proteins was administered.

[0060] FIG. 22 shows the IL- 10 levels measured at day 2, day 4, and day 5 when mRNA encoding various IL-12 masked and unmasked proteins was administered.

[0061] FIG. 23 shows the TNFa levels measured at day 4 and day 5 when mRNA encoding various IL-12 masked and unmasked proteins was administered.

[0062] FIG. 24 shows the IL-12 levels measured at day 2, day 4, and day 5 when mRNA encoding various IL-12 masked and unmasked proteins was administered.DETAILED DESCRIPTION

[0063] Provided herein are mRNA molecules encoding masked proteins for the treatment of a disease or disorder. In general terms, the masked proteins comprise a therapeutic protein, a masking moiety, and at least one cleavable moiety. The masked protein may further comprise a half-life extending moiety. The disclosed masked proteins are suitable for systemic delivery, such as intravenous administration. Upon translation and production of the masked94920-5746-9564.2Attorney Docket No. 131986-6001proteins from the mRNA molecules in the liver or spleen, the masked proteins may be inert or inactivated. However, the masked protein may be activated at a distal target tissue (e.g., a tumor) via cleavage of the substrate. To modulate the therapeutic effect of the masked protein, the cleavable moiety may be cleaved by proteases present in the target cell, cell population, or tissue in need of treatment.

[0064] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology. It is to be understood that the present disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein for the purpose of describing particular embodiments only and is not intended to be limiting.I. Definitions

[0065] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to a “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0066] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0067] It is understood that aspects and variation of the invention described herein include “consisting” and / or “consisting essentially of’ aspects and variations. Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the disclosed components, and that104920-5746-9564.2Attorney Docket No. 131986-6001there are processes and methods according to the present disclosure that consist essentially of, or consist of, the disclosed processing steps.

[0068] Numeric ranges are inclusive of the numbers defining the range.

[0069] As used herein, the terms “activatable” and “inducible” when used together with the term “therapeutic protein” refer to a masked therapeutic protein that exhibits attenuated activity (e.g., attenuated or nonexistent ligand / receptor binding, agonist, or antagonist activity) compared to the activity of the therapeutic protein itself, wherein exposure of the activatable or inducible therapeutic protein to an activation condition yields an active (or “activated”) therapeutic protein.

[0070] The terms “antibody” and “immunoglobulin” are used interchangeably herein. An antibody or immunoglobulin, as used herein, is intended to refer to immunoglobulin molecules comprised of two heavy (H) chains. Typically, antibodies in mammals (e.g., humans, rodents, and monkeys) comprise four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2, and CH3. Each light chain is comprised of a light chain variable region (LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0071] Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multi-specific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, or tetrameric antibodies comprising two heavy chain and two light chain molecules. One of skill in the art would recognize that other forms of antibodies exist.

[0072] The term “Fc domain” refers to the portion of an IgG molecule that is associated with a crystalline fragment obtained by papain digestion of an IgG molecule. For the purposes of the present disclosure, the term “Fc domain” includes variants of Fc domains, such as “knob and hole” variants discussed herein. An Fc domain generally consists of the C-terminal114920-5746-9564.2Attorney Docket No. 131986-6001halves of two IgG molecular heavy chains linked via disulfide bonds. Fc domains do not have antigen binding activity, but contain binding sites and carbohydrate moieties for Fc receptors, including FcRn receptors. The Fc domain generally contains the entire second constant domain CH2 and the third constant domain CH3. The second constant domain CH2 and the third constant domain CH3 may be derived from IgGl, IgG2, IgG3, or IgG4. The second constant domain CH2 and the third constant domain CH3 may be domains found in nature or mutant domains.

[0073] The term “attenuated” as used herein means reduced activity or binding capacity relative to a reference protein (i.e., a protein without a masking moiety). For example, an attenuated form of a reference protein that is an agonist of a particular receptor would have decreased agonist activity relative to the naturally occurring agonist.

[0074] The term “cancer” refers to the physiological condition in mammals in which a population of cells is characterized by uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and / or certain morphological features. Often cancers can be in the form of a tumor or mass but may exist alone within the subject or may circulate in the bloodstream as independent cells, such as leukemic or lymphoma cells. The term cancer includes all types of cancer and metastases, including hematological malignancy, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic acid, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (e.g., triple negative breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial (e.g., serous) or uterine cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, and various types of head and neck cancers.

[0075] As used herein, the term “half-life extension moiety” in the context of the disclosed masked proteins refers to an element, preferably a protein or polypeptide that increases the serum half-life and improves pharmacokinetics (PK), for example, by altering its size (e.g., to be above the kidney filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and elimination.124920-5746-9564.2Attorney Docket No. 131986-6001

[0076] As used herein, the term “inert” when used in the context of the disclosed cleavable moieties means that the cleavable moiety is relatively stable and not labile to significant amounts of protease degradation. While some baseline cleavage may occur, a majority of cleavable moieties (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) expressed in / by a cell or tissue are not cleaved within that cell or tissue, respectively. Thus, for the purposes of the present disclosure, for a cleavable moiety to be considered “inert,” it does not have to be completely uncleaved / uncleavable within a given tissue. For example, the disclosed cleavable moieties are inert in the liver or spleen, which means that there is relatively less cleavage of these substrates in the liver and spleen compared to a target tissue such as a tumor or infection site (i.e., the cleavable moiety is more stable in the liver or spleen compared to target tissue).

[0077] The term “nucleotide” herein refers to a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1’ carbon of the pentose), and that combination of base and sugar is a nucleoside. When the nucleoside contains a phosphate group bonded to the 3’ or 5’ position of the pentose, it is referred to as a nucleotide. A sequence of polymeric operatively linked nucleotides is typically referred to herein as “base sequence,” “polynucleotide,” “nucleotide sequence,” “nucleic acid strand,” “oligonucleotide,” or “polynucleotide strand,” and is represented herein by a formula whose left to right orientation is in the conventional direction of 5 ’-terminus to 3 ’-terminus, referring to the terminal 5’ phosphate group and the terminal 3’ hydroxyl group at the “5”’ and “3”’ ends of the polymeric sequence, respectively. Nucleic acids comprise a polymer of nucleotides (nucleotide monomers). Thus, nucleic acids are also referred to as polynucleotides. Nucleic acids may be or may include, for example, deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs), ethylene nucleic acids (ENAs), cyclohexenyl nucleic acids (CeNA), and / or chimeras and / or combinations thereof.

[0078] The term “messenger RNA” and “mRNA” refers to any RNA that encodes 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. The skilled artisan will appreciate that, except for 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.134920-5746-9564.2Attorney Docket No. 131986-6001Thus, 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”.

[0079] The term “open reading frame” and “ORF” refers to 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 3’ UTRs, but that those elements, unlike the ORF, need not necessarily be present in an RNA polynucleotide of the present disclosure.

[0080] The terms “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, may perform any function, known or unknown, and may be single- or multi -stranded (e.g., single-stranded, double-stranded, triple-helical, etc.), which contain deoxyribonucleotides, ribonucleotides, and / or analogs or modified forms of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or their analogs. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present invention encompasses polynucleotides which encode a particular amino acid sequence.

[0081] As used herein, the term “operably linked” in the context of an RNA encoding a masked protein refers to the orientation of the components of a polypeptide complex that permits the components to function in their intended manner. For example, a polypeptide comprising an IL-12 subunit as a therapeutic protein and an IL-12 blocking element as a masking moiety are operably linked by a protease cleavable linker in a polypeptide complex when the IL-12 blocking element is capable of inhibiting activity of the IL-12 subunit, but upon cleavage of the protease cleavable linker, the inhibition of the IL-12 subunit is decreased or eliminated, for example, because the IL- 12 blocking element can diffuse away from the IL- 12 subunit.

[0082] As used herein, the terms “peptide,” “polypeptide,” or “protein” are used broadly and interchangeably herein to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used herein interchangeably to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to suggest a144920-5746-9564.2Attorney Docket No. 131986-6001particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more.

[0083] The phrases “similar” in the context of at least two nucleic acids or polypeptides typically means that a polynucleotide or polypeptide includes a sequence that has at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequence identity, in comparison with a reference polynucleotide or polypeptide sequence. The phrases “substantially similar” and “substantially identical” in the context of at least two nucleic acids typically means that a polynucleotide includes a sequence that has at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequence identity, in comparison with a reference polynucleotide sequence. The phrase “substantially similar” in the context of at least two polypeptides typically means that a polynucleotide includes a sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequence identity, in comparison with a reference polypeptide sequence. The phrase “substantially identical” in the context of at least two polynucleotides or polypeptides typically means that a polynucleotide or polypeptide includes a sequence that has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequence identity, in comparison with a reference polynucleotide or polypeptide sequence. Sequence identity may be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters. (See, e.g., Altshul et al. (1990) J. Mol. Biol. 215: 403-410; Henikoff et al. (1989) Proc. Natl. Acad. Sci.89: 10915; Karin et al. (1993) Proc. Natl. Acad. Sci. 90: 5873; and Higgins et al. (1988) Gene 73: 237). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. Also, databases may be searched using FASTA (Person et al. (1988) Proc. Natl. Acad. Sci. 85: 2444-2448). In some embodiments, substantially identical nucleic acid molecules hybridize to each other under stringent conditions (e.g., with a range of medium to high stringency).

[0084] The term “special amino acid” refers to cysteine, proline, and glycine, which possess unique chemical properties that set them apart from the standard 20 amino acids used in protein synthesis, including the ability to form disulfide bonds (cysteine) and create unique flexibility in a protein backbone (proline and glycine).154920-5746-9564.2Attorney Docket No. 131986-6001

[0085] Unless otherwise indicated, nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0086] The term “subject” herein refers to any animal, such as any mammal, including, but not limited to, humans, non-human primates, rodents, and the like. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0087] As used herein, the term “therapeutically effective amount” refers to an amount of a compound described herein that is sufficient to achieve a desired pharmacological or physiological effect under the conditions of administration. For example, a therapeutically effective amount can be an amount that is sufficient to reduce the signs or symptoms of a disease or condition (e.g., a tumor). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject. A therapeutically effective amount of a pharmaceutical composition can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmaceutical composition to elicit a desired response in the individual. An ordinarily skilled clinician can determine appropriate amounts to administer to achieve the desired therapeutic benefit based on these and other considerations.

[0088] The terms “treat,” “treatment,” and “treating” refer to the reduction or amelioration of the progression, severity, and / or duration of a proliferative disorder, e.g., cancer, or the amelioration of a proliferative disorder resulting from the administration of one or more therapies.

[0089] The term “derived from” encompasses the terms “originated from,” “obtained from,” “obtainable from,” “isolated from,” and “created from,” and generally indicates that one specified material (e.g., a biological sample) finds its origin in another specified material or individual or has features that can be described with reference to another specified material.II. Masked Proteins

[0090] Many potentially therapeutic proteins (e.g., cytokines, chemokines, antibodies, receptor ligands, etc.) are difficult to utilize for treating a disease due at least to on-target toxicities, short half-life, or both. For example, certain cytokines (e.g., IL-12, IL-6, IL-1, IL-17, IL- 18, IFN-y, and TNF-a) and other proteins can elicit potentially dangerous cytokine release syndrome (CRS) and can be toxic even in small quantities. Additionally, poor protein164920-5746-9564.2Attorney Docket No. 131986-6001half-life, which can be due to degradation in circulation, filtration by the liver or kidneys, and sequestration in on-target cells, limits the duration of benefit of these therapeutics.

[0091] Masked proteins provide an approach to address the foregoing difficulties by, for example, increasing half-life, limiting protein activity to a desirable location within the body, or both. Masked proteins comprise a “mask” or “masking moiety” attached (e.g., via a linker) to a therapeutic protein (e.g., a cytokine) to attenuate or eliminate the activity of the therapeutic protein. The masking moiety can be removed by cell type-specific or diseasespecific proteases, which allow the mask to separate from the therapeutic protein (e.g., IL-12, IL-6, IL- 1, IL- 17, IL- 18, IFN-y, and TNF-a).

[0092] For the purposes of the present disclosure, a masked protein will generally comprise a therapeutic protein (e.g., IL- 12, IL-6, IL-1, IL- 17, IL- 18, IFN-y, and TNF-a), a masking moiety, and at least one cleavable moiety, which, upon cleavage, allows for separation of the masking moiety from the therapeutic protein. The masked protein can optionally further comprise a half-life extending moiety. Further, the disclosed masked proteins are not directly administered to a subject (e.g., a human with cancer, an infection, or another disease), but rather an mRNA encoding the masked protein is administered to the subject. These mRNAs and the encoded masked proteins can be prepared such that the mRNA is expressed in one type of cell or tissue (e.g., the liver or spleen) and the masked protein is activated by a different cell type or within a different tissue that is not the liver or the spleen (e.g., a tumor or an infection site). To this end, the at least one cleavable moiety is inert in mammalian liver and / or spleen cells where the disclosed mRNAs are generally expressed. The masked protein is activated upon cleavage of the at least one cleavable moiety in a different tissue that is not the liver or the spleen (e.g., a tumor or an infection site). The cleavage of the at least one cleavable moiety results in the production of an activated masked protein, wherein the masking moiety releases and is separated from the therapeutic protein.

[0093] The terms “activated masked protein” and “cleaved masked protein” are used interchangeably herein to refer to the cleavage product that is generated after exposure of the masked protein to a protease that is capable of cleaving the cleavable moiety (i.e., after cleavage of the cleavable moiety by at least one protease). In a cleaved masked protein, cleavage of the cleavable moiety results in the release of the masking moiety from the therapeutic protein.174920-5746-9564.2Attorney Docket No. 131986-6001

[0094] The coupling of two moieties (e.g., a masking moiety and a therapeutic protein) within a masked protein may be direct or indirect. When the two components are coupled directly, the amino acid residue at the C-terminus of a first component forms a peptide bond with the amino acid residue at the N-terminus of a second component. When the two components are coupled indirectly, there may be a stretch of amino acids between the two components, including, but not limited, to a linker. Two components of the masked protein may be indirectly coupled to one another via one or more other components in the masked protein, i.e., one or more other components are between the two indirectly coupled components. For indirect coupling, the masked protein may comprise one or more linkers, one or more therapeutic proteins, one or more cleavable moieties, one or more masking moieties, one or more half-life extending moieties, or any combination or arrangement thereof.

[0095] A masked protein can comprise a variety of structural configurations. Exemplary formulas for masked proteins are provided below. The N-terminal to C-terminal order of the therapeutic protein, masking moiety, and cleavable moiety may be reversed within any of the masked protein configurations provided. For example, exemplary masked proteins can be represented by the following formulas (in order from amino (N) terminal region to carboxyl (C) terminal region):Masking moiety-cleavable moiety-therapeutic protein;Therapeutic protein-cleavable moiety-masking moiety;Masking moiety-cleavable moiety-therapeutic protein-cleavable moiety-masking moiety; Half-life extension domain-masking moiety-cleavable moiety-therapeutic protein;Therapeutic protein-cleavable moiety-masking moiety-half-life extension domain;Half-life extension domain-cleavable moiety-therapeutic protein-cleavable moiety-masking moiety; orHalf-life extension domain-masking moiety-cleavable moiety-therapeutic protein-cleavable moiety-masking moiety.

[0096] As used herein and unless otherwise stated, each dash (-) between the components of the masked protein represents either a direct linkage or indirect linkage via one or more linkers or overlaps between the components. It should be noted that although the masking moiety and cleavable moiety are indicated as functionally distinct components in the formulas above, in all exemplary embodiments disclosed herein (including the formulas above), the amino acid sequences of the masking moiety and the cleavable moiety may184920-5746-9564.2Attorney Docket No. 131986-6001overlap. For example, the cleavable moiety may be partially or completely contained within the masking moiety. Additionally, the formulas provided above can further comprise additional amino acid sequences that may be positioned N-terminal or C-terminal to the masked protein components. Such additional components include, but are not limited to, targeting moieties (e.g., a ligand for a receptor or a cell present in a target tissue).

[0097] In some embodiments, the masked protein comprises one or more peptide chains (e.g., 2, 3, or 4 or more peptide chains). In some embodiments, the masked protein comprises one peptide chain. In some embodiments, the masked protein comprises a first peptide chain and a second peptide chain. For example, a masked protein may comprise two separate peptide chains in which a component of the first peptide chain dimerizes or interacts with a component of the second peptide chain (e.g., two peptide chains that each comprise one half of an Fc domain as a half-life extending moiety may form a masked protein through dimerization of the Fc domains). The number of peptide chains making up the masked protein may be determined based on the specific components of the masked protein, including the half-life extending moiety, the masking moiety, and the therapeutic protein, and the specific structure required to allow the components of the masked protein to have their proper function.

[0098] In some embodiments, the masked protein comprises a structural arrangement from N-terminus to C-terminus as follows: a single peptide chain comprising (i) a therapeutic protein, (ii) a first cleavable moiety, (iii) a masking moiety, (iv) a second cleavable moiety, and (v) a half-life extending moiety. In some embodiments, the masked protein comprises a structural arrangement from N-terminus to C-terminus as follows: (a) a first peptide chain comprising (i) a therapeutic protein, (ii) a first cleavable moiety, (iii) a masking moiety, (iv) a second cleavable moiety, and (v) a first region of a half-life extending moiety, and a second peptide chain comprising a second region of the half-life extending moiety; (b) a first peptide chain comprising (i) a first portion or subunit of a therapeutic protein, (i) a first cleavable moiety, and (iii) a first region of a half-life extending moiety, and a second peptide chain comprising (i) a masking moiety, (ii) a second cleavable moiety, (iii) a second portion or subunit of the therapeutic protein, (iv) a third cleavable moiety, and (v) a second region of the half-life extending moiety; and (c) a first peptide chain comprising (i) a therapeutic protein, (ii) a first cleavable moiety, and (iii) a first region of a half-life extending moiety, and a second peptide chain comprising (i) a masking moiety, (ii) a second cleavable moiety, and (iii) a second region of the half-life extending moiety. In some embodiments, the masked194920-5746-9564.2Attorney Docket No. 131986-6001protein comprises a structural arrangement from N-terminus to C-terminus as follows: a single peptide chain comprising (i) a masking moiety, (ii) a first cleavable moiety, (iii) a therapeutic protein, (iv) a second cleavable moiety, and (v) a half-life extending moiety. In some embodiments, the masked protein comprises a structural arrangement from N-terminus to C-terminus as follows: (a) a first peptide chain comprising (i) a masking moiety, (ii) a first cleavable moiety, (iii) a therapeutic protein, (iv) a second cleavable moiety, and (v) a first region of a half-life extending moiety, and a second peptide chain comprising a second region of the half-life extending moiety; or (b) a first peptide chain comprising: (i) a masking moiety, (ii) a first cleavable moiety, (iii) a first portion or subunit of a therapeutic protein, (iv) a second cleavable moiety, and (v) a first region of a half-life extending moiety, and a second peptide chain comprising (i) a second portion or subunit of the therapeutic protein, (ii) a third cleavable moiety, and (iii) a second region of the half-life extending moiety.A. Masking Moieties

[0099] Masking moieties employed in the encoded masked proteins are polypeptides that are capable of interfering with the activity of a therapeutic protein. The masking moiety may inhibit the activity of the therapeutic protein in any of a variety of ways, such as, for example, by specifically binding to or being bound by the therapeutic protein’s corresponding ligand, receptor, or substrate (i.e., its “target”) via structural changes, competition for a binding domain, steric hinderance, and the like. The coupling of a therapeutic protein with a masking moiety may effect a structural change that reduces or inhibits the ability of the therapeutic protein to specifically bind or be bound by or otherwise interact with its target. The coupling of a therapeutic protein with a masking moiety may sterically block, reduce, or inhibit the ability of the therapeutic protein to specifically bind to or be bound by or otherwise interact with its target. A masking moiety may interact with the therapeutic protein, thus reducing or inhibiting the interaction between the therapeutic protein and its binding partner. When the masked protein is activated via cleavage at the cleavable substrate, the masked moiety does not substantially or significantly interfere with the therapeutic protein’s ability to bind to or be bound by or otherwise interact with its target.

[0100] A masking moiety may be coupled to a therapeutic protein via a cleavable moiety directly or indirectly (e.g., via one or more linkers or additional components). Additionally or alternatively, the masking moiety may be coupled, either directly or indirectly, with a halflife extending moiety, such that in the tertiary or quaternary conformation of the masked204920-5746-9564.2Attorney Docket No. 131986-6001protein, the masked moiety may be in a position (e.g., proximal to the therapeutic protein) that allows the masked moiety to mask the therapeutic protein.

[0101] The masking moiety may comprise at least a partial or complete amino acid sequence of a naturally occurring binding partner of the therapeutic protein, such as an extracellular domain of a cognate receptor of the therapeutic protein. The masking moiety may be a fragment of a naturally occurring binding partner. The masking moiety may be a ligand, a receptor, or a fragment thereof (e.g., an extracellular domain of a receptor) of the therapeutic protein that binds to the therapeutic protein. The fragment may retain at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65% at least 60%, at least 50%, at least 40%, at least 30%, at least 25%, or at least 20% nucleic acid or amino acid sequence homology to the naturally occurring binding partner of the therapeutic protein.

[0102] The masking moiety may be a modified binding partner for the therapeutic protein that contains amino acid changes that decrease or increase affinity and / or avidity of binding to the therapeutic protein. The masking moiety may contain no or substantially no nucleic acid or amino acid homology to the therapeutic protein’s natural binding partner. The masking moiety may not have more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% homology to the natural binding partner of the therapeutic protein.

[0103] The masking moiety may be an antibody or antibody fragment (e.g., a Fab fragment, a F(ab’)2 fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody) that binds to the therapeutic protein. In some embodiments, the masking moiety is a single-chain antibody, such as an scFv, a VHH, a nanobody, or a single domain heavy chain.

[0104] In some embodiments, the masking moiety is a steric mask. As used herein, the term “steric mask” means a polypeptide that does not bind the therapeutic protein but still may interfere with the therapeutic protein’s binding to its binding partner through non-specific interactions, including, but not limited to, steric hindrance. The masking moiety may be positioned in the masked protein such that the tertiary or quaternary structure of the masked protein allows the masking moiety to mask the therapeutic protein through charge-based interaction, thereby holding the masking moiety in place to interfere with the therapeutic protein’s access or capacity to bind to its binding partner. Additionally or alternatively, the masking moiety may recruit a large protein binding partner that sterically interferes with214920-5746-9564.2Attorney Docket No. 131986-6001therapeutic protein-target interactions. Such masking moieties may include, but are not limited to, an albumin (e.g., human serum albumin (HSA)), a fragment crystallizable (Fc) domain, an antibody constant domain (e.g., CH domain), a peptide-based polymer (e.g., a latency associated protein (LAP)), or any polypeptide or other moiety that sterically interferes with therapeutic protein-target interactions.

[0105] Examples of suitable masking moieties include, but are not limited to, a full-length or a therapeutic protein-binding fragment or mutein of a cognate receptor of the therapeutic protein, and therapeutic protein-binding antibodies and fragments thereof (e.g., a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of camelid-type nanobody (VHH), a dAb, and the like). For example, when the therapeutic protein is IL-12, the masking moiety may be an scFv, including, but not limited to, Briakinumab. Other exemplary antigenbinding domains that bind the therapeutic protein may also be used as masking moieties, including, but not limited to, non-immunoglobulin proteins that mimic antibody binding and / or structure such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipcallin and CTLA4 scaffolds. The masking moiety may also be a peptide that is modified to sterically inhibit or prevent binding of the therapeutic protein to its target.

[0106] The masking moiety may have a dissociation constant for binding to the therapeutic protein that is no more than the dissociation constant of the therapeutic protein to its target. The masking moiety may not interfere or compete with the therapeutic protein for binding to the target in the masked protein following cleavage of the cleavable moiety by a protease.

[0107] The structural properties of the masking moieties may be selected according to factors such as the minimum amino acid sequence required for interference with the therapeutic protein binding to a target, the target protein-protein binding pair of interest, the size of the therapeutic protein, the presence or absence of linkers, and the like.

[0108] The masking moiety may be unique for the coupled therapeutic protein. Examples of masking moiety may include masking moieties or fragments thereof (e.g., affinity masks) that are specifically screened to bind to a binding domain of the therapeutic protein.224920-5746-9564.2Attorney Docket No. 131986-6001

[0109] The term “masking efficiency” refers to an ECso of the masked protein divided by the effective dose of a control molecule, wherein the control molecule may be either the cleavage product of the masked protein (i.e., the activated masked protein) or the corresponding therapeutic protein without a mask. A masked protein may have a masking efficiency that is greater than 10. The masked proteins described herein may have a masking efficiency that is greater than 10, 100, 1000, or 5000.

[0110] In some embodiments, the masking moiety may be a polypeptide of about 2 to 50 amino acids in length. More specifically, the masking moiety may be a polypeptide of from 2 to 50 amino acids, from 2 to 40 amino acids, from 2 to 30 amino acids, from 2 to 20 amino acids, from 2 to 10 amino acids, from 5 to 50 amino acids, from 5 to 40 amino acids, from 5 to 30 amino acids, from 5 to 20 amino acids, from 5 to 10 amino acids, from 10 to 50 amino acids, from 10 to 40 amino acids, from 10 to 30 amino acids, from 10 to 20 amino acids, from 15 to 50 amino acids, from 15 to 40 amino acids, from 15 to 30 amino acids, from 15 to 20 amino acids, from 20 to 50 amino acids, from 20 to 40 amino acids, from 20 to 30 amino acids, from 30 to 50 amino acids, from 30 to 40 amino acids, or from 40 to 50 amino acids in length. The masking moiety may be a polypeptide with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In some embodiments, the masking moiety may be a polypeptide of more than 50 amino acids in length, e.g., 100, 200, 300, 400, 500, 600, 700, 800, or more amino acids.

[0111] In some embodiments, in a masked protein, there is no binding or substantially no activity of the therapeutic protein, or no more than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% activity the therapeutic protein to its target, as compared to activity of a counterpart protein without the interfering masking moiety. The activity the therapeutic protein with an interfering masking moiety may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, or 50,000,000 times lower than the activity of the therapeutic protein towards without an interfering masking moiety. The activity the therapeutic protein with an interfering masking moiety may be between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 10-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1,000-10,000,000, 10,000-100,000, 10,000-1,000,000, or 10,000-10,000,000 times lower than the activity of the therapeutic protein when there is no interfering masking moiety. Suitable234920-5746-9564.2Attorney Docket No. 131986-6001measures of activity will vary depending on the therapeutic protein component of the masked protein, and include, but are not limited to, ECso determinations (as shown in Example 1 below), binding assays, and activity assays. Exemplary binding assays include, but are not limited to, surface plasmon resonance (SPR), interferometry (e.g., OCTET® assay), enzyme-linked immunosorbent assay (ELISA), or other comparable methods of measuring molecular interaction.

[0112] The dissociation constant (Kd) of the masking moiety towards the therapeutic protein may be greater than the dissociation constant of the therapeutic protein towards the target. The dissociation constant of the masking moiety may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times greater than the dissociation constant of the therapeutic protein towards the target. Conversely, the binding affinity of the masking moiety towards the therapeutic protein may be lower than the binding affinity of the therapeutic protein towards the target. The binding affinity of the masking moiety towards the therapeutic protein may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times lower than the binding affinity of the therapeutic protein towards the target.

[0113] The Kd of the masked protein comprising a masking moiety and a cleavable moiety towards the therapeutic protein’s target may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 10-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1,000-10,000,000, 10,000-100,000, 10,000-1,000,000, or 10,000-10,000,000 times greater than the Kd of a counterpart molecule that does not comprise the masking moiety or cleavable moiety. Alternatively, the binding affinity of the mask protein comprising a masking moiety and a cleavable moiety towards the therapeutic protein’s target may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 10-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1,000-10,000,000, 10,000-100,000, 10,000-1,000,000, or 10,000-10,000,000 times lower than the binding affinity of a counterpart molecule that does not comprise the masking moiety or cleavable moiety.244920-5746-9564.2Attorney Docket No. 131986-6001

[0114] When the therapeutic protein is coupled with a masking moiety and is in the presence of the target, the specific binding of the therapeutic protein to its target is reduced or inhibited, as compared to the specific binding of the therapeutic protein not coupled with the masking moiety. Compared to the binding of the therapeutic protein not coupled with the masking moiety to the target, the target-binding ability of the therapeutic protein coupled with the masking moiety may be reduced by at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as measured via in vivo or in an in vitro assay for demonstrating activity of the therapeutic protein.ScFv-Based Masking moiety

[0115] In some embodiments, the masking moiety is a single-chain variable fragment (scFv) that is capable of binding to the therapeutic protein. For the purposes of the present disclosure, it should be understood that other single-chain antibodies beyond scFvs (e.g., VH, VHH, etc.) can be used as a masking moiety.

[0116] An scFv contains only the variable domain of a light chain (VL) linked by a stretch of peptide to a variable domain from a heavy chain (VH). The VL and VH regions may be a VL region and a VH region derived from an antibody known to bind to the therapeutic protein. In some embodiments, the scFv may be an scFv previously characterized to bind to the therapeutic protein. In some embodiments, the scFv may be a therapeutic scFv that binds to the endogenous form of the therapeutic protein in a cell or tissue.

[0117] In some embodiments, the therapeutic protein is a cytokine and the masking moiety is an scFv derived from an anti-cytokine antibody (i.e., the scFv comprises variable heavy and light chain sequences of an anti-cytokine antibody). For example, in certain embodiments when the therapeutic protein is IL-12, the masking moiety is an scFv that comprises the heavy and light chain variable domain sequences of briakinumab. Other cytokines that may be masked according to the present disclosure include, but are not limited to, TNFa, IL-6, IL-23 and IL- 17, and scFvs for masking these therapeutic proteins can be derived from, for example, for TNFa: infliximab, etanercept, adalimumab, and certolizumab; for IL-6: golimumab, tocilizumab, siltuximab, sarilumab, olokizumab, and sirukumab; for IL-23: ustekinumab, tildrakizumab, guselkumab, BL655066, and LY3074828; for IL-17: secukinumab, ixekizumab, brodalumab, CNTO6785, bimekkizumab, and SCH-900117. Extracellular Domain-Based Masking moiety254920-5746-9564.2Attorney Docket No. 131986-6001

[0118] In some embodiments, the masking moiety is an extracellular domain that is capable of binding to the therapeutic protein. In some embodiments, the extracellular domain may be derived from a protein that is known to bind to the therapeutic protein, such as a transmembrane receptor.Peptide Masking moiety

[0119] In some embodiments, the masking moiety may be a protein aptamer that is capable of binding to the therapeutic protein. In some embodiments, the protein aptamer may be an artificial or non-natural oligopeptide that is capable of binding to the therapeutic protein. In some embodiments, the protein aptamer may be an affinity peptide mask, which exhibits high affinity to the therapeutic protein. In some embodiments, the protein aptamer may be derived from an endogenous receptor for the therapeutic protein. In some embodiments, the artificial or non-natural peptide derived from an endogenous receptor for the therapeutic protein may be modified to enhance or reduce the affinity the peptide has for the therapeutic protein to modulate the masking behavior of the masked protein.Steric Masking moiety

[0120] In some embodiments, the masking moiety may be a steric mask. A steric mask may act to structurally mask the active site of the therapeutic protein by inhibiting the therapeutic protein from having the proper steric conformation to be active. When the steric mask is released from the therapeutic protein upon protease cleavage at the cleavable moiety, the therapeutic protein is allowed to adopt the proper confirmation at the active site and bind or be bound by the target within the cell.B. Half-Life Extending Moieties

[0121] To improve the half-life of the masked protein after administration, the masked protein may comprise a half-life extension moiety or domain. The half-life extending moiety may be serum half-life extending moiety, i.e., capable of extending the serum half-life of the molecule attached to the half-life extension domain.

[0122] In some embodiments, the half-life extending moiety may include a fragment crystallizable region (Fc domain) of an antibody. The half-life extending moiety may be the Fc domain of an IgG (e.g., IgGl, IgG2, IgG3, or IgG4). In some embodiments, the Fc domain may be a wild-type Fc domain. In other embodiments, the Fc domain is a mutated or engineered Fc domain. The half-life extending moiety may be a dimer formed by two Fc 264920-5746-9564.2Attorney Docket No. 131986-6001domains. The first Fc domain may comprise a first CH2 region and a first CH3 region. The second Fc domain may comprise a second CH2 region and a second CH3 region. When the half-life extending moiety is a dimer formed by two Fc domains, the masked protein having the half-life extending moiety may comprise a first peptide chain and a second peptide chain.

[0123] The half-life extending moiety may be a dimer formed by two naturally occurring Fc domains or two mutant Fc domains. In some embodiments, the two mutant Fc domains may be an Fc domain hole mutant and a Fc domain knob mutant. The Fc domain hole mutant and the Fc domain knob mutant may interact with one another to facilitate dimerization of the two Fc domains. The Fc domain knob mutant and the Fc domain hole mutant may include one or more amino acid modifications within the interface between the two Fc domains (e.g., in the CH3 domain). The modifications may include amino acid substitution T366W and, optionally, the amino acid substitution S354C in one IgGFc domain and the amino acid substitutions T366S, L368A, and Y407V, and, optionally, Y349C in the other IgGFc domain, the amino acid numbering according to EU numbering system. In some embodiments, the Fc mutants have the amino acid sequence of ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK (SEQ ID NO: 1) for the first Fc domain, and:GSSKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK (SEQ ID NO: 2) for the second Fc domain.

[0124] Additional examples of mutant Fc domains also include those described in U. S. Patent No. 7,695,936, which is incorporated herein by reference in its entirety. The modifications may include the amino acid substitution T366Y in one IgG Fc domain and the amino acid substitution Y407T in the other IgG Fc domain. The modifications may include the amino acid substitution T366W in one IgG domain and the amino acid substitution Y407A in the other IgG Fc domain. The modifications may include the amino acid substitution F405A in one IgG Fc domain and the amino acid substitution T394W in the other IgG Fc domain. The modifications may include the amino acid substitutions T366Y and F405A in one IgGFc274920-5746-9564.2Attorney Docket No. 131986-6001domain and the amino acid substitutions T394W and Y407T in the other IgGFc domain. The modifications may include the amino acid substitutions T366W and F405W in one IgG Fc domain and the amino acid substitutions T394S and Y407A in the other IgG Fc domains. The modifications may include F405W and Y407A in one IgG Fc domain and the amino acid substitutions T336W and T394S in the other IgG Fc domain. The modifications may include the amino acid substitution F405W in one IgG Fc domain and the amino acid substitution T394S in the other IgG Fc domain. The mutation positions in the Fc domains are numbered according to EU numbering systems. The IgG Fc domain may have a sequence of any one of the following sequences, wherein amino acids 1-107 correspond to EU 341-447 of the IgG: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (IgGl, SEQ ID NO: 3);ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (IgG2, SEQ ID NO: 4); ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRC PEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKS RWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (IgG3, SEQ ID NO: 5); or ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY284920-5746-9564.2Attorney Docket No. 131986-6001TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (IgG4, SEQ ID NO: 6).

[0125] The mutant Fc domains may have reduced effector function, such as IgGl modifications K326W / E333S, F243L / R292P / Y300L / V305I / P396L, S239D / I332E, S239D / I332E / A330L, S298A / E333A / K334A, G236A / S239D / I332E, K326W / E333S, S267E / H268F / S324T, E345R / E430G / S440Y, N297A, N297Q, N297G, L235E, L234A / L235A, M252Y / S254T / T256E, M428L / N434S, S267E / L328F, and N325S / L328F. Similarly, IgG4 modifications include, but are not limited to F234A / L235A, and IgG2 modifications include, but are not limited to H268Q / V309L / A330S / P331S and V234A / G237A / P238S / H268A / V309L / A330S / P331S. Further examples of modified Fc domains include those disclosed in US 20190135943, which is incorporated herein by reference in its entirety.

[0126] In some embodiments, the half-life extending moiety may be an immunoglobulin (IgG) or an Fc domain thereof, a serum albumin (including, but not limited to, human serum albumin (HSA)), an antibody or fragment thereof (e.g., scFv) that binds to a serum albumin, a hexa-hat glutathione S-transferase (GST), a glutathione affinity moiety, a calmodulin-binding peptide (CGP), a strep-tag, a cellulose binding domain, a maltose binding protein, an s-peptide tag, a chitin binding tag, an immune-reactive epitope, or an epitope tag (including, but not limited to, E2 tag, HA epitope tag, myc epitope, FLAG epitope, AU1 and AU5 epitopes, glu-glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope, and VSV epitope).

[0127] The serum half-life of the masked protein having the half-life extending moiety is longer than that of a counterpart molecule that is substantially the same as the masked protein but does not include the half-life extending moiety, e.g., the pharmacokinetics (PK) of the masked protein with the half-life extending moiety is longer than that of the reference molecule without the half-life extending moiety. The masked protein with the half-life extending moiety may have a serum half-life that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% longer than the serum half-life of a masked protein without the half-life extending moiety. The masked protein with the half-life extending moiety may have a serum half-life that is at least 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold longer than the serum half-life of a masked protein without the half-life extending moiety. The serum half-life of the masked294920-5746-9564.2Attorney Docket No. 131986-6001protein having the half-life extending moiety may be at least 15 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour when administered to a subject.C. Cleavable moieties

[0128] Masked proteins encoded by the mRNA agents of the present disclosure employ cleavable substrates that exhibit reduced cleavability in liver and spleen tissue as compared to tissue that is not liver or spleen tissue. The masked protein may include one or more cleavable substrates. In some embodiments, each cleavable substrate may be cleaved by the same protease. In some embodiments, each cleavable substrate may be cleaved by different proteases. In some embodiments, a subset of cleavable substrates may be cleaved by the same protease. In some embodiments, one of the cleavable substrates are selectively cleavable by certain proteases but have reduced or no cleavability by other proteases. More specifically, one or more cleavable substrates may be cleavable by proteases expressed in a specific cell type or tissue but have reduced or no cleavability by proteases expressed in all other cell types or tissues. Resistance of cleavable substrates to protease cleavage in non-target cells or tissues may reduce systemic toxicities by limiting the binding of the therapeutic protein to targets that are present in the non-target cells or tissues, which may lead to a greater therapeutic window and safety profile with less on-target toxicities occurring in the non-target cells or tissues.

[0129] For the purposes of the present disclosure, the one or more cleavable substrates are more stable in liver or spleen cells and tissue (i.e., mammalian or, more specifically, human liver or spleen), but labile and subject to cleavage by proteases expressed in a target tissue, such as a tumor or an infection site. This platform design allows for mRNA encoding the masked protein to be administered systemically, such that the mRNA is ultimately localized to and expressed by liver or spleen cells. Accordingly, to maintain the benefits of masking the therapeutic protein, the cleavable substrate(s) are generally not cleaved (i.e., they are more stable) when expressed in liver or spleen cells, but can be cleaved to allow activation of the therapeutic protein at a distal target tissue.

[0130] The one or more proteases that cleave the one or more cleavable moieties may include, but are not limited to MMPs (e.g., MMP2 or MMP9) or serine proteases (e.g., a membrane type serine protease I (MT-SP1) or uPA).304920-5746-9564.2Attorney Docket No. 131986-6001

[0131] The cleavable moiety may include one or a plurality of protease cleavage moieties. The protease cleavage moiety may be an amino acid sequence that includes a target site for a sequence-specific protease. In some embodiments, the protease cleavage moiety may comprise of consist of the amino acid sequence of any one of SEQ ID NOs: 7-9. In some embodiments, the cleavable moiety may have the amino acid sequence of any one of SEQ ID NOs: 7-9. In some embodiments, the cleavable moiety may have the amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 7-9. In some embodiments, the cleavable moiety may include a combination, a C-terminal truncation variant, a C-terminal extension variant, an N-terminal truncation variant, or an N-terminal extension variant of the amino acid sequences of any of SEQ ID NOs: 7-9. Truncation variants of the aforementioned amino acid sequences that are suitable for use as a cleavable moiety may be any that retain the recognition site for the corresponding protease. In some embodiments, the truncation variant may have a C-terminal deletion or an N-terminal deletion of one amino acid residue from an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-9. These include C-terminal and / or N-terminal truncation variants comprising at least 1, 2, 3, 4, 5, or more contiguous amino acids of any one of SEQ ID NOs: 7-9 that retain a recognition site for a protease. Extension variants of the aforementioned amino acid sequences that are suitable for use as a cleavable moiety may be any that have one or more (e.g., 1, 2, 3, 4, 5, or more) additional amino acids and retain the recognition site for the corresponding protease. The additional amino acids may be coupled to the C-terminus or the N-terminus of the amino acid sequence of any one of SEQ ID NOs: 7-9. The extension variants may have additional amino acids coupled to both the C-terminus and the N-terminus of the amino acid sequence of any one of SEQ ID NOs: 7-9. The C-terminus or N-terminus extension variants can have a C-terminal glycine and / or an N-terminal serine amino acid.

[0132] The cleavable moiety may include one, two, three, four, five, six, or more amino acids in addition to the amino acid sequence of any one of SEQ ID NOs: 7-9. The cleavable moiety may include one, two, three, four, five, six, or more additional amino acids to the N-terminus of the amino acid sequence of any one of SEQ ID NOs: 7-9. The cleavable moiety may include one, two, three, four, five, six, or more additional amino acids to the C-terminus of the amino acid sequence of any one of SEQ ID NOs: 7-9. The cleavable moiety may include one, two, three, four, five, six, or more additional amino acids to the N-terminus and one,314920-5746-9564.2Attorney Docket No. 131986-6001two, three, four, five, six, or more additional amino acids to the C-terminus of any one of SEQ IDNOs: 7-9.

[0133] The cleavable moiety may have a sequence with mutation(s) of one or more amino acids of the amino acid sequence of any one of SEQ ID NOs: 7-9. The cleavable moiety may have one amino acid, two amino acid, three amino acid, four amino acid, or five amino acid mutation(s) in the amino acid sequence of any one of SEQ ID NOs: 7-9. The cleavable moiety may have the amino acid sequence of any one of SEQ ID NOs: 7-9 and one conservative substitution.

[0134] The cleavable moiety may comprise a total of 3 amino acids to 25 amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids). The cleavable moiety may comprise a total of 3-25 amino acids, 3-20 amino acids, 3-15 amino acids, 3-12 amino acids, 4-25 amino acids, 4-20 amino acids, 4-15 amino acids, 4-12 amino acids, 5-25 amino acids, 5-20 amino acids, 5-15 amino acids, 5-12 amino acids, 6-25 amino acids, 6-20 amino acids, 6-15 amino acids, 6-12 amino acids, 7-25 amino acids, 7-20 amino acids, 7-15 amino acids, 7-12 amino acids, 8-25 amino acids, 8-20 amino acids, 8-15 amino acids, or 8-12 amino acids.

[0135] The cleavable moiety may comprise consensus sequences that comprise characteristic amino acids. For example, the cleavable moiety may comprise a consensus sequence comprising: a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid; wherein special amino acids include C, G, or P and hydrophobic amino acids include A, V, I, L, M, F, Y, or W. Additionally or alternatively, the cleavable moiety may comprise a consensus sequence comprising: a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid; wherein polar amino acids include S, T, N, or Q, special amino acids include C, G, or P, and positive amino acids include R, H, or K. In some embodiments, the cleavable moiety may comprise both a consensus sequence comprising a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid and a consensus sequence comprising a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid. Substrate- 1 and Substrate2 are exemplary such embodiments and comprise both of the foregoing consensus sequences.

[0136] In some embodiments, the cleavable moiety may comprise 3 or fewer (e.g., 3, 2, 1, or 0) negatively charged amino acids (e.g., D or E). In some embodiments, the cleavable moiety324920-5746-9564.2Attorney Docket No. 131986-6001may comprise 2 or more (e.g., 2, 3, 4, or 5) special amino acids (e.g., C, G, or P). In some embodiments, the cleavable moiety may comprise 2 or more (e.g., 2, 3, 4, or 5) hydrophobic amino acids (e.g., A, V, I, L, M, F, Y, or W). In some embodiments, the cleavable moiety may comprise 2 or more (e.g., 2, 3, 4, or 5) polar amino acids (e.g., S, T, N, or Q). In some embodiments, the cleavable moiety may comprise 1 or more (e.g., 1, 2, 3, 4, or 5) positive amino acids (e.g., R, H, or K).

[0137] In some embodiments, the cleavable moiety may comprise 3 or fewer (e.g., 3, 2, 1, or 0) negatively charged amino acids; 2 or more (e.g., 2, 3, 4, or 5) special amino acids; 2 or more (e.g., 2, 3, 4, or 5) hydrophobic amino acids; 2 or more (e.g., 2, 3, 4, or 5) polar amino acids; and 1 or more (e.g., 1, 2, 3, 4, or 5) positive amino acids. Substrate-1 and Substrate2 are exemplary such embodiments and comprise 3 or fewer (e.g., 3, 2, 1, or 0) negatively charged amino acids; 2 or more (e.g., 2, 3, 4, or 5) special amino acids; 2 or more (e.g., 2, 3, 4, or 5) hydrophobic amino acids; 2 or more (e.g., 2, 3, 4, or 5) polar amino acids; and 1 or more (e.g., 1, 2, 3, 4, or 5) positive amino acids.Table 1 - Examples of Cleavable moietiesID Number SEQ ID NO Amino Acid Sequence Substrate-2 7 PWGLSGRSSubstrate-1 8 DHQSRSGPWGLLSubstrate-3 9 QNQALRSA

[0138] Additional potentially suitable cleavable moieties are disclosed in WO 2024 / 030850 and WO 2024 / 03085, each of which is incorporated herein by reference with respect to the cleavable moieties disclosed therein.

[0139] The cleavable moiety may be specifically cleaved by one or more proteases (e.g., MMP2, MMP9, MP-ST1, or uPA) at a desired rate. The rate may be measured as cleavable moiety cleavage kinetics (kcat / Ku) as disclosed in WO 2016 / 118629, which is incorporated by reference. In brief, kcat is the turnover number and describes how many cleavable moiety molecules are transformed into cleavage products per unit time by a protease. The KM value describes the affinity of the cleavable moiety to the active site of the protease. The kcat / K ratio provides a measurement of cleavability of the cleavable moiety by the protease.Generally, the greater the ratio, the higher the rate of cleavability is; conversely, the lower the ratio, the slower the rate of cleavability is. The kcat / K value may be determined with the following equation:= —In (1 — C / (t X p), where C is the cleavage product conversion,334920-5746-9564.2Attorney Docket No. 131986-6001t is time in seconds, and p is protease concentration in moles, which assumes that the substrate concentration is below the KM value and in excess of the protease concentration.

[0140] The cleavable moiety may be cleaved by the protease at a rate that has a kcat / K value from 1x10 to IxlO6M’1, e.g., from 1x10 to SxlOM’1, from 5x10 to IxlO2M^s’1, from IxlO2to 5xl02M^s’1, from 5xl02to IxlO3M^s’1, from IxlO3to 5xl03M^s’1, from 5xl03to IxlO4M’1, from IxlO4to 5xl04M’1, from 5xl04to IxlO5M’1, from IxlO5to 5xl05M^s’1, or from 5xl05to IxlO6M’1. For example, the cleavable moiety may be cleaved by MMP2 at a rate that has a kcat K\ / va\ue from 1x10 to IxlO6M^s’1, e.g., from 1x10 to SxlOM^s'1, from 5x10 to IxlO2M^s’1, from IxlO2to 5xl02M^s’1, from 5xl02to IxlO3M^s’1, from IxlO3to 5xl03M^s’1, from 5xl03to IxlO4M’1, from IxlO4to 5xl04M’1, from 5xl04to IxlO5M^s’1, from IxlO5to 5xl05M’1, or from 5xl05to IxlO6M-1s' h Additionally or alternatively, the cleavable moiety may be cleaved by MMP9 at a rate that has a kcat / K value from 1x10 to IxlO6M^s’1, e.g., from 1x10 to SxlOM^s’1, from 5x10 to IxlO2M^s’1, from IxlO2to 5xl02M^s’1, from 5xl02to IxlO3M’1, from IxlO3to 5xl03M’1, from 5xl03to IxlO4M^s’1, from IxlO4to 5xl04M’1, from 5xl04to IxlO5M’1, from 1 x 105to 5x 105M’1, or from 5x 105to 1x106M^s'1. Additionally or alternatively, the cleavable moiety may be cleaved by MP-ST1 at a rate that has a kcat / KM value from 1 x 10 to IxlO6M^s’1, e.g., from 1x10 to SxlOM’1, from 5x10 to IxlO2M’1, from IxlO2to 5xl02M^s’1, from 5xl02to IxlO3M^s’1, from IxlO3to 5xl03M’1, from 5xl03to IxlO4M’1, from IxlO4to 5xl04M^s’1, from 5xl04to IxlO5M’1, from IxlO5to 5xl05M’1, or from 5xl05to IxlO6M’1. Additionally or alternatively, the cleavable moiety may be cleaved by uPA at a rate that has a kcat / KMNa\ue, from 1x10 to IxlO6M^s’1, e.g., from 1x10 to SxlOM^s’1, from 5x10 to IxlO2M^s’1, from IxlO2to 5xl02M’1, from 5xl02to IxlO3M’1, from IxlO3to 5xl03M^s’1, from 5xl03to IxlO4M’1, from IxlO4to 5xl04M’1, from 5xl04to IxlO5M’1, from IxlO5to 5xl05M’1, or from 5xl05to IxlO6M’1.

[0141] The cleavable moiety may be cleaved by a tissue-specific protease, e.g., a tumorspecific protease, at a rate that has a kcat / KM a\\iQ from 1x10 to IxlO6M^s’1, e.g., from 1x10 to SxlOM^s’1, from 5x10 to IxlO2M^s’1, from IxlO2to 5xl02M’1, from 5xl02to IxlO3M’1, from IxlO3to 5xl03M^s’1, from 5xl03to IxlO4M’1, from IxlO4to 5xl04M’1, from 5xl04to IxlO5M^s’1, from IxlO5to 5xl05M^s’1, or from 5xl05to IxlO6M^s'1.

[0142] The cleavability of the cleavable moiety by a protease is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%. Specifically, the cleavability of the substrate by a protease is at least 50%, at least 60%, at least 70%, at least 80%, at least 344920-5746-9564.2Attorney Docket No. 131986-600190%, at least 95%, or 100% when 500 nM activatable antibody containing a pro-domain with the cleavable moiety being tested was included with 10 nM of a protease for 1.5 hours or 4 hours at 37 °C.

[0143] To determine whether a cleavable moiety is a desirable candidate for the disclosed mRNA-encoded masked proteins, assessment of the cleavability of the cleavable moiety can be performed with reference to proteases found in the spleen / liver and proteases found in the target tissue. The cleavage by a certain proteases in the target tissue in situ of the cleavable moiety may be less than 99%, less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% compared to the cleavage of a desired protease (i.e., a protease found specifically or preferentially at the site in which activation is desired). The different proteases may be other proteases in the target tissue or other normal tissues, as well as other proteases involved in processes including, but not limited to, inflammation and wound healing. A cleavable moiety that is resistant to cleavage by a protease, or sample or tissue comprising the protease, refers to (i) a cleavable moiety in which no peptide bond is hydrolyzed by the protease, or no peptide bond is hydrolyzed when incubated in the sample or tissue comprising the protease, or (ii) a cleavable moiety in which a reduced level of peptide bonds is hydrolyzed by the protease, or reduced level of peptide bonds is hydrolyzed when incubated in the sample or tissue comprising the protease.

[0144] The cleavable moiety may be cleaved by more than two proteases. For example, the cleavable moiety may be cleaved by proteases including, but not limited to, MMP2, MMP9, MP-ST1, uPA, or any combination thereof. Specific cleavable moieties are described, for example in WO2010 / 081173, WO2015 / 048329, WO2015 / 116933, WO2016 / 118629, WO 2024 / 030850, and WO 2024 / 03085 each of which is incorporated herein by reference with respect to the cleavable moieties disclosed therein.

[0145] In some embodiments, at least a portion of the first protease cleavage moiety in a cleavable moiety may overlap with at least a portion of the second protease cleavage moiety in the cleavable moiety, such that one or more amino acids in the cleavable moiety belongs to both the first and second protease cleavage moieties. For example, a cleavable moiety with the sequence X1X2X3X4X5X6, wherein each X is an amino acid, may comprise overlapping first and second protease cleavage moieties, in which the first protease cleavage moiety is X1X2X3X4 and the second protease cleavage moiety is X3X4X5X6.354920-5746-9564.2Attorney Docket No. 131986-6001

[0146] In some embodiments, the first and second protease cleavage moieties do not overlap in amino acid sequence such that no amino acid in the cleavable moiety belongs to both the first protease cleavage moiety and the second protease cleavage moiety. For example, a cleavable moiety with the sequence X1X2X3X4X5X6X7X8, wherein each X is an amino acid, may comprise non-overlapping first and second protease cleavage moieties, in which the first protease cleavage moiety is X1X2X3X4 and the second protease cleavage moiety is X5X6X7X8. The non-overlapping first and second protease cleavage moieties may be coupled directly. The non-overlapping first and second protease cleavage moieties may be coupled indirectly, e.g., via a linking peptide.

[0147] The first protease cleavage moiety and the second protease cleavage moiety may have a structural arrangement from N-terminus to C-terminus as first protease cleavage moiety and second protease cleavage moiety or second protease cleavage moiety and first protease cleavage moiety. As used herein, the first and second protease cleavage moieties in the arrangement of first protease cleavage moiety and second protease cleavage moiety or second protease cleavage moiety and first protease cleavage moiety may be overlapping first and second protease cleavage moieties, non-overlapping first and second protease cleavage moieties coupled directly, or non-overlapping first and second protease cleavage moieties coupled indirect, e.g., via a linking peptide.D. Therapeutic Proteins

[0148] The disclosed masked proteins include a masking moiety coupled with a therapeutic protein via the cleavable moiety.

[0149] For the purposes of the present disclosure, a “therapeutic protein” is any protein polypeptide that specifically binds to, is bound by, or otherwise interacts with a target, such as a ligand, receptor, or substrate. The activity of the therapeutic protein may treat, slow the progression of, or reduce one or more symptoms of a disease or condition. The therapeutic protein may include, but is not limited to, a cytokine, an antibody, an antigen binding fragment, a chimeric antigen receptor, or an immunogenic fragment of any one of these.

[0150] As used herein, the terms “specific binding,” “specifically binds,” and / or “specifically bound by” refers to the non-covalent interactions of the type that occur between a therapeutic protein and its target, e.g., a cytokine and its receptor or an immunoglobulin molecule and its antigen. The strength or affinity of binding interactions can be expressed in terms of the dissociation constant (Ka) of the interaction, wherein a smaller Ka represents a greater364920-5746-9564.2Attorney Docket No. 131986-6001affinity. Unless indicated otherwise, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1: 1 interaction between members of a therapeutic protein and its target. Affinity can be measured by common methods known in the art. A statement that a therapeutic protein “specifically binds” to its target refers to a therapeutic protein that binds its target with a dissociation constant (Ka) of less than 100 pM (e.g., less than 5 pM or 10 pM). The therapeutic protein may specifically bind to its target with a Kd of about 0.01 nM to about 500 nM. a therapeutic protein may be said to specifically bind the target when the Kd is < 1 pM, < 100 nM, < 10 nM, or < 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

[0151] In some embodiments, the therapeutic protein may be endogenous to a mammal. In some embodiments, the therapeutic protein may be naturally expressed in mice, rats, nonhuman primates, and / or humans. In some embodiments, the therapeutic protein may be a protein that is not endogenous to a mammal. In some embodiments, the therapeutic protein may be an artificial protein not found in any animal. In some embodiments, the therapeutic protein may be a recombinant protein.Cytokines as Therapeutic proteins

[0152] Cytokines, which are small glycoproteins and polypeptides, are known to regulate both innate and adaptive immune responses. Cytokines are potent signaling molecules, which means that cytokine-based therapies have the potential to cause dangerous side effects. This is problematic for patients because cytokine therapies are generally administered at high dosages to stimulate the desired immunological response (e.g., an anti-tumor response), but these high dosages may also result in significant side effects, including extreme nausea, organ failure, hypotension, and cytopenias. Thus, utilizing a masked format of a cytokine may improve cytokine-based treatments by reducing the negative side effects associated with cytokine-based therapeutics.

[0153] Cytokines may be divided into categories including tumor necrosis factors (TNFs), interleukins (ILs), lymphokines, monokines, interferons (IFNs), colony stimulating factors (CSFs), and transforming growth factors (TGFs). Cytokines may also be broadly classified as pro-inflammatory or anti-inflammatory, based on their ability to facilitate inflammatory reactions when released and either active or suppress immune cells, although a single cytokine may have both pro- and anti-inflammatory activities, depending on the context of the cytokine activity. Pro-inflammatory cytokines generally include IL-ip, IL-6, IL-8, IL-12,374920-5746-9564.2Attorney Docket No. 131986-6001IL-17, IL-18, IFN- a / y, and TNF-a. Anti-inflammatory cytokines generally include, IL-IRA, IL-4, IL-6, IL-10, IL-11, IL-13, IL-35, and TGF-p.

[0154] Cytokines may also be associated with various biological conditions, including various diseases. IL-1, IL-6, IL-12, IL-15, IL-16, IL-17, IL-18, IL-23, TNF-a, IFN-a, and IFN-y are associated with autoimmune diseases. TNF-a, TGF-P, IL-1, IL-6, IL-10, IL-17, and IL-18 are associated with cardiovascular disorders. TNF- a, TRAIL, IL-6, IL-10, IL-12, IL-17, and IL-23 are associated with cancer.

[0155] In order to modulate inflammatory responses to treat a disease or a disorder or to treat a biological condition associated with a cytokine, the therapeutic protein of the masked peptide may be the relevant cytokine or a fragment thereof, including, but not limited to, the cytokines disclosed above. For example, the therapeutic protein may be IL-12 or an immunogenic fragment thereof; or IL-15 or an immunogenic fragment thereof.

[0156] In some embodiments, the therapeutic protein comprising a cytokine is incorporated in a masked protein for use as a treatment of a cancer. The cancer may be, but is not limited to, any one of adenocarcinoma, melanoma, colon cancer, breast cancer, lymphoma, pancreatic cancer, prostate cancer, bladder cancer, urothelial cancer, and Kaposi sarcoma.E. Linkers

[0157] The masked protein may comprise one or more linkers. The linkers may be linking peptides that comprise a stretch of amino acid sequence that link two components in the masked protein. The linkers may be non-cleavable by any protease. One or more linkers may be introduced into the masked protein to provide flexibility at one or more of the junctions between domains, between moi eties, between moi eties and domains, or at any other junctions where a linker would be beneficial. When the masked protein is provided in a conformationally constrained construct, a flexible linker may be inserted to facilitate formation and maintenance of a structure in the masked protein. Any of the linkers described herein may provide the desired flexibility to facilitate the inhibition of the binding of a target, or to facilitate cleavage of a cleavable moiety by a protease. Linkers in the masked protein may be all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexibility to provide for a desired masked protein conformation. Some linkers may include cysteine residues, which may form disulfide bonds and reduce flexibility of the construct.384920-5746-9564.2Attorney Docket No. 131986-6001

[0158] A linker coupled to a masking moiety may have a length that allows the masking moiety to be in a position in the tertiary or quaternary structure to effectively mask a therapeutic protein, (e.g., proximal to the therapeutic protein to be masked) that allows the masking moiety to mask the therapeutic protein.

[0159] The linker’s length may be determined by counting, in a N- to C-terminal direction, the number of amino acids from the N-terminus of the linker adjacent to the C-terminal amino acid of the preceding component, to the C-terminus of the linker adjacent to the N-terminal amino acid of the following component (i.e., where the linker length does not include either the C-terminal amino acid of the preceding component or the N-terminal amino acid of the following component).

[0160] A linker may include a total of 1 to 50, 1 to 40, 1 to 30, or 1 to 25 amino acids (e.g., 1 to 24, 1 to 22, 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 25, 2 to 24, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 15, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 4 to 25, 4 to 24, 4 to 22, 4 to 20, 4 to 18, 4 to 16, 4 to 15, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 4 to 5, 5 to 25, 5 to 24, 5 to 22, 5 to 20, 5 to 18, 5 to 16, 5 to 15, 5 to 14, 5 to 12, 5 to 10, 5 to 8, 5 to 6, 6 to 25, 6 to 24, 6 to 22, 6 to 20, 6 to 18, 6 to 16, 6 to 15, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 6 to 7, 8 to 25, 8 to 24, 8 to 22, 8 to 20, 8 to 18, 8 to 16, 8 to 15, 8 to 14, 8 to 12, 8 to 10, 8 to 9, 10 to 25, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 15, 10 to 14, 10 to 12, 10 to 11, 12 to 25, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 15, 12 to 14, 12 to 13, 14 to 25, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 14 to 15, 15 to 25, 15 to 24, 15 to 22, 15 to 20, 15 to 18, 15 to 16, 16 to 25, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to 25, 18 to 24, 18 to 22, 18 to 20, 18 to 19, 20 to 25, 20 to 24, 20 to 22, 20 to 21, 22 to 25, 22 to 24, 22 to 23, or 24 to 25 amino acids). The linker may include a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0161] The linker may be rich in glycine (Gly or G) residues. The linker may be rich in serine (Ser or S) residues. The linker may be rich in glycine and serine residues (i.e., include more glycine or serine residues than any other respective amino acid). The linker may have one or more glycine-serine residue pairs (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs).

[0162] The linker may have one or more Gly-Gly-Gly-Ser (GGGS) (SEQ ID NO: 10) sequences, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more GGGS sequences. The linker may have one or more Gly-Gly-Gly-Gly-Ser (GGGGS) (SEQ ID NO: 11) sequences, i.e., 1, 2, 3, 4, 5, 6, 7,394920-5746-9564.2Attorney Docket No. 131986-60018, 9, 10 or more GGGGS sequences. The linker may have one or more Gly-Gly-Ser-Gly (GGSG) (SEQ ID NO: 12) sequences, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more GGSG sequences. The linker may include glycine polymers (e.g., (G)n), glycine-serine polymer (e.g., (GS)n, (GGS)n, (GSGG)n, and (GGGS)n, where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may be relatively unstructured, and therefore, may be able to serve as a neutral link between components. Exemplary flexible linkers include one of or any combination of one or more of: GGSG, GGSGG, GSGSG, GSGGG, GGGSG, GSSSG, GSSGGSGGSGG, GGGS, GGGSGGGS, GGGSGGGSGGGS, GGGGSGGGGSGGGGS, GGGGS GGGGS GGGGS GGGGS GGGGS, GGGGSGGGGS, GGGGS, GS, GGGGSGGGGSGGGGSGS, GGSLDPKGGGGS, PKSCDKTHTCPPCPAPELLG, SKYGPPCPPCPAPEFLG, GKSSGSGSESKS, GSTSGSGKSSEGKG, GSTSGSGKSSEGSGSTKG, GSTSGSGKPGSGEGSTKG, GSTSGSGJPGSSEGST, GGGSSGGS, GGGGSGGGGSS, GGGSSGGSGGSSGGS, and GSTSGSGKPGSSEGST.

[0163] Examples of linkers may further include a sequence that is at least 70% identical (e.g., at least 70%, at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97% at least 98%, at least 99%, or 100% identical) to the exemplary linkers described herein.

[0164] The design of a masked protein can include linkers that are all or partially flexible. Thus, the linker can include a flexible portion as well as one or more portions that confer less flexible structure.

[0165] A masked protein may include one, two, three, four, five, six, seven, eight, nine, or ten linker sequence(s) (e.g., the same or different linker sequences of any of the exemplary linker sequences described herein or known in the art).III. mRNAs Encoding Masked Proteins

[0166] Provided herein are polynucleotides that encode the masked protein disclosed above. The polynucleotide may comprise ribonucleotides (RNA). More particularly, the polynucleotide may be a messenger RNA (mRNA). The compositions, as provided herein, may include an mRNA or multiple mRNAs encoding any of the masked proteins described above.404920-5746-9564.2Attorney Docket No. 131986-6001

[0167] Messenger RNA (mRNA) is RNA that encodes at least one protein or a fragment thereof and can be translated to produce the encoded protein or fragment in vitro, in vivo, in situ, or ex vivo. mRNA of the present disclosure may comprise an open reading frame (ORF) encoding a masked protein disclosed herein. In some embodiments, the mRNA further comprises a 5' untranslated region (UTR), 3' UTR, a polyA tail and / or a 5' cap analog.

[0168] The disclosed mRNA may include nucleotides that are not chemically modified, nucleotides that are chemically modified, or both. Nucleotides that are not chemically modified are standard ribonucleotides comprising the unmodified nucleobases of adenine, guanine, cytosine, and uracil (e.g., A, G, C, or U). In some embodiments, mRNA of the present disclosure comprise standard nucleoside residues. Chemically modified nucleosides are discussed in more detail below.

[0169] The disclosed mRNA may be polycistronic constructs, which encode more than one protein separately within the same mRNA molecule.

[0170] 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 herein also discloses the corresponding mRNA sequence complementary to the DNA, where each “T” of the DNA sequence is substituted with “U ”A. Open Reading Frame (ORF)

[0171] 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, TGA, UAA, UAG, UGA, UGAUGA (SEQ ID NO: 13) or UGAUAAUAG (Seq ID NO: 14)). 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.B. Untranslated Regions (UTRs)

[0172] 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” (5' UTR) is a region of an mRNA that is directly upstream (i.e., 5') from the start codon and does not414920-5746-9564.2Attorney Docket No. 131986-6001encode a polypeptide. A 3' untranslated region” (3'UTR) is a region of an mRNA that is directly downstream ( / .<., 3') from the stop codon and also does not encode a polypeptide.

[0173] The 5' UTR may start at the transcription start site and continues to the start codon but does not include the start codon. The 3' UTR may start immediately following the stop codon and continue until the transcriptional termination signal. A variety of 5' UTR and 3' UTR sequences are known, and therefore the mRNA of the present disclosure may include any 5' UTR and / or any 3' UTR. Exemplary UTR sequences include SEQ ID NOs: 15-78, which are shown in Tables 2 and 3; however, other UTR sequences may be used or exchanged for any of the UTR sequences described herein.Table 2 - Examples of 5' UTRsSEQ ID SequenceNO:15 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC 16 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGC GCCGCCACC17 GAGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUC UCGCAACUAGCAAGCUUUUUGUUCUCGCC18 GGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUCG CAACUAGCAAGCUUUUUGUUCUCGCC19 GGGAGAGUCCCGCAGUCGGCGUCCAGCGGCUCUGCUUGUUCGUGUGUGU GUCGUUGCAGGCCUUAUUCAAGCUUACC20 GUCCCGCAGUCGGCGUCCAGCGGCUCUGCUUGUUCGUGUGUGUGUCGUU GCAGGCCUUAUUC21 GGGAGAAAGCUUACC22 AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC 23 GAGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCG CCACC24 GAGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCA ACCUCAAACAGACACCGCCACC25 GGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUCG CAACUAGCAAGCUUUUUGUUCUCGCC26 GGAAAUCCCCACAACCGCCUCAUAUCCAGGCUCAAGAAUAGAGCUCAGU GUUUUGUUGUUUAAUCAUUCCGACGUGUUUUGCGAUAUUCGCGCAAAG CAGCCAGUCGCGCGCUUGCUUUUAAGUAGAGUUGUUUUUCCACCCGUUU GCCAGGCAUCUUUAAUUUAACAUAUUUUUAUUUUUCAGGCUAACCUACG CCGCCACC27 GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAUCUCCCUG AGCUUCAGGGAGCCCCGGCGCCGCCACC28 GGAAACCCCCCACCCCCGUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAU AAGAUCUCCCUGAGCUUCAGGGAGCCCCGGCGCCGCCACC29 GGAGAACUUCCGCUUCCGUUGGCGCAAGCGCUUUCAUUUUUUCUGCUAC CGUGACUAAG30 GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC 31 GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCCACC424920-5746-9564.2Attorney Docket No. 131986-600132 GGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUC GCAACUAGCAAGCUUUUUGUUCUCGCCGCCGCC33 GGAAAUCGCAAAAUUUUCUUUUCGCGUUAGAUUUCUUUUAGUUUUCUU UCAACUAGCAAGCUUUUUGUUCUCGCCGCCGCC34 GGAAAAUUUUAGCCUGGAACGUUAGAUAACUGUCCUGUUGUCUUUAUA UACUUGGUCCCCAAGUAGUUUGUCUUCCAAA35 GGAAACUUUAUUUAGUGUUACUUUAUUUUCUGUUUAUUUGUGUUUCUU CAGUGGGUUUGUUCUAAUUUCCUUGGCCGCC36 GGAAAAUCUGUAUUAGGUUGGCGUGUUCUUUGGUCGGUUGUUAGUAUU GUUGUUGAUUCGUUUGUGGUCGGUUGCCGCC37 GGAAAAUUAUUAACAUCUUGGUAUUCUCGAUAACCAUUCGUUGGAUUU UAUUGUAUUCGUAGUUUGGGUUCCUGCCGCC38 GGAAAUUAUUAUUAUUUCUAGCUACAAUUUAUCAUUGUAUUAUUUUAG CUAUUCAUCAUUAUUUACUUGGUGAUCAACA39 GGAAAUAGGUUGUUAACCAAGUUCAAGCCUAAUAAGCUUGGAUUCUGG UGACUUGCUUCACCGUUGGCGGGCACCGAUC40 GGAAAUCGUAGAGAGUCGUACUUAGUACAUAUCGACUAUCGGUGGACA CCAUCAAGAUUAUAAACCAGGCCAGA41 GGAAACCCGCCCAAGCGACCCCAACAUAUCAGCAGUUGCCCAAUCCCAA CUCCCAACACAAUCCCCAAGCAACGCCGCC42 GGAAAGCGAUUGAAGGCGUCUUUUCAACUACUCGAUUAAGGUUGGGUA UCGUCGUGGGACUUGGAAAUUUGUUGUUUCC43 GGAAACUAAUCGAAAUAAAAGAGCCCCGUACUCUUUUAUUUCUAUUAG GUUAGGAGCCUUAGCAUUUGUAUCUUAGGUA44 GGAAAUGUGAUUUCCAGCAACUUCUUUUGAAUAUAUUGAAUUCCUAAU UCAAAGCGAACAAAUCUACAAGCCAUAUACC45 GGAAAUCGUAGAGAGUCGUACUUACGUGGUCGCCAUUGCAUAGCGCGCG AAAGCAACAGGAACAAGAACGCGCC46 GGAAAUCGUAGAGAGUCGUACUUAGAAUAAACAGAGUCGGGUCGACUU GUCUCUGAUACUACGACGUCACAAUC47 GGAAAAUUUGCCUUCGGAGUUGCGUAUCCUGAACUGCCCAGCCUCCUGA UAUACAACUGUUCCGCUUAUUCGGGCCGCC48 GGAAAUCUGAGCAGGAAUCCUUUGUGCAUUGAAGACUUUAGAUUCCUC UCUGCGGUAGACGUGCACUUAUAAGUAUUUG49 GGAAAGCGAUUGAAGGCGUCUUUUCAACUACUCGAUUAAGGUUGGGUA UCGUCGUGGGACUUGGAAAUUUGUUGCCACC50 GGAAAUUUUUUUUUGAUAUUAUAAGAGUUUUUUUUUGAUAUUAAGAAA AUUUUUUUUUGAUAUUAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCG CCGCCACC51 GGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCAAAAA AAAAAAACC52 GGAAAUCUCCCUGAGCUUCAGGGAGUAAGAGAGAAAAGAAGAGUAAGA AGAAAUAUAAGACCCCGGCGCCGCCACC53 GGACUCACUAUUUGUUUUCGCGCCCAGUUGCAAAAA54 GCCRCC, wherein R= A or G55 GGAAA U C G CAAAA (N2)x(N3)xC U (N4)x(N5)xC G C GU UA GA UU U C U U U U A G U U U U C U NeNyC A A C U A G C A A G C U U U U U G U U C U C G C C (Ns C C)x(N2)Xis a uracil and x is an integer from 0 to 5, e.g., wherein x =3 or 4;(Ns)x is a guanine and x is an integer from 0 to 1;(N4)xis a cytosine and x is an integer from 0 to 1;(Ns)x is a uracil and x is an integer from 0 to 5, e.g., wherein x =2 or 3;Ne is a uracil or cytosine;434920-5746-9564.2Attorney Docket No. 131986-6001N7is a uracil or guanine;Ns is adenine or guanine and x is an integer from 0 to 1.Table 3 - Examples of 3' UTRs (stop cassette is italicized; miR binding sites are boldened)SEQ ID SequenceNO:UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCC56 CCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUA AAGUCUGAGUGGGCGGC UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCC57 CCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUA AAGUCUGAGUGGGCGGC UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCC58 CCCCAGCCCCUCCUCCCCUUCCUGCAGGAGAUUGAGUGUAGUGACUAGUGG UCUUUGAAUAAAGUCUGAGUGGGCGGC UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCC59 CCCCAGCCCCUCCUCCCCUUCCUGCAGGAUUGAGACUACGGGUGGUCUUUG AAUAAAGUCUGAGUGGGCGGC UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCC60 CCCCAGCCCCUCCUCCCCUUCCUGCAGCAUAGACACUACGUGGUCUUUGAA UAAAGUCUGAGUGGGCGGC UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCC61 CCCCAGCCCCUCCUCCCCUUCCUGCAGGAGAUUGAGUGUAGUGGUGGUCUU UGAAUAAAGUCUGAGUGGGCGGC UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCC62 CCCCAGCCCCUCCUCCCCUUCCUGCAGGAGAUUGAGUGUAGUGACGUGGUC UUUGAAUAAAGUCUGAGUGGGCGGC UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCC63 CCCCAGCCCCUCCUCCCCUUCCUGCAGUGGUCUUUGAAUAAAGUCUGAGUG GGCGGC AGGACUAGUCCCUGUUCCCAGAGCCCACUUUUUUUUCUUUUUUUGAAAUA64AAAUAGCCUGUCUUUCAGAUCU GGACUAGUUAUAAGACUGACUAGCCCGAUGGGCCUCCCAACGGGCCCUCCU65CCCCUCCUUGCACCGAGAUUAAU CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACC CCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCC CCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGC66AGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUA ACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUU GGUCAAUUUCGUGCCAGCCACACC CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUG GGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCA CCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAG67CAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAG UGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCC AGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACC CCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCC68CCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUA444920-5746-9564.2Attorney Docket No. 131986-6001ACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUU GGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC GCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAA69 GUCCAACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAU UCUGCCUAAUAAAAAACAUUUAUUUUCAUUGC MUL4GCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCC 70 CCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUA AAGUCUGAGUGGGCGGC UA4GUCU2L4GCUGGAGCCUCCUGAGAGACCUGUGUGAACUAUUGAGAAGAU 71 CGGAACAGCUCCUUACUCUGAGGAAGUUGGUACCCCCGUGGUCUUUGAAU AAAGUCUGAGUGGGCGGCU2LU4GCUCCCCGGGGCAAACACCAUUGUCACACUCCAGCCUCGGUGGCCUA GCUUCUUGCCCCUUGGGCCCAAACACCAUUGUCACACUCCAUCCCCCCAG72 CCCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUCACACUC CAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(miR122 binding sites boldened) U2LU4GCUCCCCGGGGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCG GUGGCCUAGCUUCUUGCCCCUUGGGCCCAAACACCAUUGUCACACUCCAU73 CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAU AAAGUCUGAGUGGGCGGC(miR-142-3p and miR122 binding sites boldened) UAU4GCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCC CCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUC74ACACUCCAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(miR122 binding site boldened) UA4GCCCCUCCGGGGCAAACACCAUUGUCACACUCCAGCCUCGGUGGCCU AGCUUCUUGCCCCUUGGGCCCAAACACCAUUGUCACACUCCAUCCCCCCA75 GCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUCACACU CCAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(miR122 binding sites boldened) UA4GCCCCUCCGGGGUCCAUAAAGUAGGAAACACUACAGCCUCGGUGGCC UAGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCC76 CAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCGCAUUAUUACUCACGG UACGAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(miR-142-3p and miR-126-3p binding sites boldened) UA4GCCCCUCCGGGGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCG GUGGCCUAGCUUCUUGCCCCUUGGGCCCAAACACCAUUGUCACACUCCAU77 CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAU AAAGUCUGAGUGGGCGGC(miR-142-3p and miR122 binding sites boldened) UA4GCCCCUCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCC CCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUC78ACACUCCAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(miR122 binding site boldened)

[0174] In some embodiments, a 3' UTR comprises, in 5 '-to-3 ' order: (a) the nucleic acid sequence: UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGC CUCCCCCCAGCCCCUCCUCCCCUUCCUGCAG (SEQ ID NO: 79), (b) an identification and ratio determination (IDR) sequence, and (c) the nucleic acid sequence UGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 80). IDR sequences are454920-5746-9564.2Attorney Docket No. 131986-6001described herein in the section entitled “Identification and Ratio Determination (IDR) Sequences.” UTRs may also be omitted from the mRNA provided herein.

[0175] Non-UTR sequences may also be used as regions or subregions within a nucleic acid. For example, introns or portions of intron 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.

[0176] 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 / 085318 A2.

[0177] It should be understood that any UTR from any gene may be incorporated into the regions of a nucleic acid (e.g., mRNA) of the present disclosure. 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 term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence.

[0178] 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.

[0179] 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.

[0180] For the purposes of the present disclosure, a UTR may also include one or more 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.464920-5746-9564.2Attorney Docket No. 131986-6001C. 5' End Cap

[0181] The disclosed mRNA may comprise a 5' end cap or a “5' terminal cap.” A cap analog may be, for example, a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. In some embodiments, a cap analog is a dinucleotide cap. In some embodiments, a cap analog is a trinucleotide cap. In some embodiments, a cap analog is a tetranucleotide cap.

[0182] 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'-0-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). A 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. A 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. A 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, such as a 7mG(5')ppp(5')NlmpNp cap, may be used.D. Polyadenylation Tailing

[0183] A “polyA tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3'), from the 3' UTRthat contains multiple, consecutive adenosine monophosphates. A polyA tail may contain 10 to 300 adenosine monophosphates. It can, in some instances, comprise up to about 400 adenosine nucleotides. For example, a polyA 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 nucleotides. In some embodiments, a polyA tail contains 50 to 250 adenosine nucleotides. In some embodiments, a polyA 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 polyA tail has a length of 100 nucleotides.

[0184] In some embodiments, an mRNA may comprise two polyA sequences separated by an intervening nucleotide sequence. In some embodiments, the intervening nucleotide sequence 474920-5746-9564.2Attorney Docket No. 131986-6001comprises no more than 3, no more than two, no more than 1, or no adenosine nucleotides. In some embodiments, the intervening sequence comprises 3 adenosine nucleotides. In some embodiments, the intervening sequence is no more than 30, no more than 25, no more than 20, no more than 15, or no more than 10 nucleotides long. In some embodiments, the intervening sequence consists of 10 nucleotides. In some embodiments, the intervening sequence comprises the sequence of GCAUAUGACU (SEQ ID NO: 81). In some embodiments, the intervening sequence does not begin with an adenosine nucleotide, and does not end with an adenosine nucleotide. In some embodiments, the first polyA sequences comprises at least 15, at least 20, at least 25, or at least 30 consecutive adenosine nucleotides. In some embodiments, the second polyA sequences comprises at least 55, at least 60, at least 65, or at least 70 consecutive adenosine nucleotides. In some embodiments, the first polyA sequence comprises 30 consecutive adenosine nucleotides. In some embodiments, the second polyA sequence comprises 70 adenosine nucleotides.

[0185] In some embodiments, an mRNA of the present disclosure may comprise the nucleotide sequence:AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAA (SEQ ID NO: 82).

[0186] Additionally or alternatively, the disclosed mRNA may also comprise a polyC region, which comprises multiple, consecutive cytidine monophosphates. The polyC region may be downstream of the polyA tail or within the polyA tail. A polyC region may comprise or consist of at least 10, at least 20, at least 30, at least 40, or at least 50 consecutive cytidine nucleotides. In some embodiments, the mRNA further comprises a polyC sequence comprising about 30 consecutive cytidine nucleotides.E. Additional Stabilizing Elements

[0187] mRNA provided herein may comprise one or more additional stabilizing elements. Stabilizing elements may include, for example, a histone stem-loop. The histone stem-loop sequence may comprise 15 to 45 nucleotides. In some embodiments, an mRNA includes the combination of a polyA sequence or polyadenylation signal and at least one histone stemloop, even though both represent alternative mechanisms in nature, they act synergistically to increase the protein expression beyond the level observed with either of the individual484920-5746-9564.2Attorney Docket No. 131986-6001elements. The synergistic effect of the combination of polyA and a histone stem-loop does not depend on the order of the elements or the length of the polyA sequence.

[0188] 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.

[0189] 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.

[0190] 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.F. Chemically Modified Nucleotides

[0191] For the purposes of the present disclosure, the nucleic acids (e.g., mRNA) disclosed herein may comprise nucleotides and / or nucleosides that can be standard (i.e., unmodified) or modified as is known in the art. For example, mRNA encoding a masked protein may comprise one or more 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.

[0192] 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.494920-5746-9564.2Attorney Docket No. 131986-6001

[0193] A non-naturally-occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such non-naturally-occurring modified nucleotides and nucleosides can be found, inter alia, in international publication numbers WO2013052523A1; WO2014093924 Al; W02015051173A2;W02015051169A2; W02015089511A2; or WO2017153936A1.

[0194] Hence, nucleic acids of the disclosure (e.g., mRNA) can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof. More specifically, nucleic acids of the disclosure (e.g., mRNA) may comprise various (more than one) different types of standard and / or 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.

[0195] An mRNA comprising modified nucleotides or nucleosides may exhibit reduced degradation or immunogenicity in a cell or organism relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

[0196] Modified nucleotide base pairing encompasses 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. 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.

[0197] Modified nucleobases in nucleic acids e.g., RNA, such as mRNA) may comprise 1-methyl-pseudouridine (mly), 1-ethyl-pseudouridine (ely), 5 -methoxy -uridine (mo5U), 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 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.504920-5746-9564.2Attorney Docket No. 131986-6001

[0198] In some embodiments, an mRNA of the disclosure comprises 1-methyl-pseudouridine (mly) substitutions at one or more (e.g., 2, 3, 4, etc.) or all uridine positions of the nucleic acid.

[0199] In some embodiments, an mRNA of the disclosure comprises 5-methyl-uridine substitutions at one or more (e.g., 2, 3, 4, etc.) or all uridine positions of the nucleic acid.

[0200] In some embodiments, an mRNA of the disclosure comprises 5-methyl cytidine substitutions at one or more (e.g., 2, 3, 4, etc.) or all cytidine positions of the nucleic acid.

[0201] In some embodiments, an mRNA of the disclosure comprises 1-methyl-pseudouridine (mly) substitutions at one or more (e.g., 2, 3, 4, etc.) or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more (e.g., 2, 3, 4, etc.) or all cytidine positions of the nucleic acid.

[0202] In some embodiments, an mRNA of the disclosure comprises pseudouridine (y) substitutions at one or more (e.g., 2, 3, 4, etc.) or all uridine positions of the nucleic acid.

[0203] In some embodiments, an mRNA of the disclosure comprises pseudouridine (y) substitutions at one or more (e.g., 2, 3, 4, etc.) or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more (e.g., 2, 3, 4, etc.) or all cytidine positions of the nucleic acid. In some embodiments, an mRNA of the disclosure comprises nucleosides consisting of 1-methyl-pseudouridine, adenosine, guanosine, and cytidine. In some embodiments, an mRNA of the disclosure comprises 5-methyl-uridine substitutions at all uridine positions of the nucleic acid and 5-methyl-cytidine substitutions at all cytidine positions of the nucleic acid.

[0204] In some embodiments, an mRNA of the disclosure comprises modified nucleotides in the open reading frame (ORF) portion of the mRNA. In some embodiments, an mRNA of the disclosure comprises 1-methyl-pseudouridine, adenosine, guanosine, and cytidine. In some embodiments, an mRNA of the disclosure comprises an ORF comprising nucleosides consisting of 1-methyl-pseudouridine, adenosine, guanosine, and cytidine. In some embodiments, an mRNA of the disclosure comprises an ORF comprising nucleosides consisting of 1-methyl-pseudouridine, adenosine, guanosine, and 5-methyl cytidine. In some embodiments, an mRNA of the disclosure comprises 5-methyl-uridine substitutions at all uridine positions within the ORF and 5-methyl cytidine substitutions at all cytidine positions within the ORF.514920-5746-9564.2Attorney Docket No. 131986-6001

[0205] In some embodiments, an mRNA of the disclosure comprises uridine at one or more or all uridine positions of the nucleic acid.

[0206] The disclosed RNAs (e.g., mRNAs) may be 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.

[0207] 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 polyA tail).

[0208] The nucleic acids of the present disclosure may comprise an ORF that contains 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%).

[0209] 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 different524920-5746-9564.2Attorney Docket No. 131986-6001structures (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 cytosine (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).G. Codon Optimization

[0210] An open reading frame encoding a masked protein of the disclosure may be codon optimized. Codon optimization 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.

[0211] In some embodiments, a codon optimized sequence shares less than 95%, less than 90%, less than 85%, less than 80%, or less than 75% sequence identity to a non-codon optimized open reading frame. In some embodiments, a codon optimized sequence shares between 65% and 85% sequence identity to a non-codon optimized sequence.

[0212] In some embodiments, a codon optimized mRNA may be one in which the levels of G / C are enhanced. The G / C-content of nucleic acid molecules (e.g., mRNA) may influence the stability of the RNA. mRNA having an increased amount of guanine (G) and / or cytosine (C) residues may be functionally more stable than RNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. As an example, WO02 / 098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modifications in the translated region. Due to the degeneracy of the genetic code, the modifications work by substituting existing codons for those that promote greater RNA stability without changing the resulting amino acid. This approach is limited to coding regions of the mRNA.534920-5746-9564.2Attorney Docket No. 131986-6001IV. Lipid Compositions

[0213] The mRNAs of the present disclosure may be formulated with or combined with a lipid composition, such as a lipid nanoparticle, a liposome, or a lipoplex. In some embodiments, the mRNAs of the present disclosure are formulated with or combined with a lipid nanoparticle (LNP).

[0214] Lipid nanoparticles typically comprise ionizable amino lipid, non-cationic or neutral lipid, structural lipid (e.g., a sterol), and PEG-lipid components along with the nucleic acid cargo (e.g., RNA, such as mRNA) of interest. Lipid nanoparticles of the present disclosure can be generated using components, compositions, and methods as are generally known in the art, see for example PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551; PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016000129; PCT / US2016 / 014280; PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 52117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575; PCT / US2016 / 069491; PCT / US2016 / 069493; and PCT / US2014 / 66242.

[0215] In some embodiments, a lipid nanoparticle (LNP) comprises at least one ionizable amino lipid, at least one non-cationic or neutral lipid, at least one sterol, and / or at least one polyethylene glycol (PEG)-lipid. In some embodiments, the LNP comprises or consists of an ionizable amino lipid, a neutral lipid, a sterol, and a PEG-lipid. For example, the LNP may comprise 40-55 mol% ionizable amino lipid, 5-15 mol% neutral / non-cationic lipid, 30-50 mol% sterol, and 0.5-5 mol% PEG-modified lipid. Alternatively, the LNP may comprise 20-60 mol% ionizable amino lipid, 5-25 mol% non-cationic lipid, 25-55 mol% structural lipid, and 0.5-15 mol% PEG-lipid; or 20-60 mol% ionizable amino lipid, 5-30 mol% non-cationic lipid, 10-55 mol% structural lipid, and 0.5-15 mol% PEG-modified lipid.

[0216] For the purposes of the present disclosure, an ionizable amino lipid may comprise the structure of Formula (I):or a salt or isomer thereof, wherein:Ri is R”M’R’ or C5-20 alkenyl;R2 and R3 are each independently selected from C1-14 alkyl and C2-14 alkenyl;544920-5746-9564.2Attorney Docket No. 131986-6001R4 is -(CH2)nQ, wherein Q is OH and n is selected from 3, 4, and 5;M and M’ are each independently -OC(O)- or -C(O)O-;Rs, Re, and R? are each H;R’ is a linear C1-12 alkyl, or C1-12 alkyl substituted with Ce-9 alkyl;R” is C3-14 alkyl;m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0217] More specifically, the ionizable amino lipid may comprise the structure of Compound (I):

[0218] In some embodiments, the neutral (i.e., non-cationic) lipid may be a phospholipid. In some embodiments the neutral (i.e., non-cationic) lipid may be distearoylphosphatidylcholine (DSPC). In some embodiments, the structural lipid or sterol may be cholesterol. In some embodiments, the PEG lipid may be 1,2 dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG-DMG). In some embodiments, the LNP may comprise or consist of an ionizable amino lipid of Compound (I), DSPC, cholesterol, and PEG-DMG.

[0219] In some embodiments, the lipid nanoparticle comprises 49 mol% ionizable amino lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG. In some embodiments, the lipid nanoparticle comprises 49 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG. In some embodiments, the lipid nanoparticle comprises 48 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG.

[0220] In some embodiments, a LNP comprises a wt / wt ratio of the ionizable amino lipid component to the RNA of from about 10:1 to about 100: 1. For example, a LNP may comprise a wt / wt ratio of the ionizable amino lipid component to the RNA of about 20: 1 or of about 10:1.

[0221] Generally, LNPs have 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, 90 nm, 80 nm, 70554920-5746-9564.2Attorney Docket No. 131986-6001nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm or less. In some embodiments, the LNP may have a mean diameter from about 30nm to about 150nm, or a mean diameter from about 60nm to about 120nm.

[0222] Additional discussion of concentrations and alternative ionizable lipids, neutral / non-cationic lipids, structural lipids, and PEG-lipids that can be incorporated into LNPs or other lipid compositions for the purposes of the present disclosure are provided below.A. Ionizable Amino Lipids

[0223] A lipid nanoparticle or other lipid composition of the present disclosure may comprise 20-60 mol% ionizable amino lipid. For example, the LNP may comprise 20-50 mol%, 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 amino lipid. In some embodiments, a lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% ionizable amino lipid. In some embodiments, the LNP may comprise 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% ionizable amino lipid. In some embodiments, the LNP 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 amino lipid.

[0224] The ionizable amino lipid that is incorporated into an LNP or other lipid composition may be any known ionizable amino lipid, including, but not limited to, those described below.

[0225] In some embodiments, the ionizable lipid is a compound of Formula (IL*)R2R1HTM'X lRx3(IL*)or a salt thereof, wherein:564920-5746-9564.2Attorney Docket No. 131986-6001R1is -OH, -NRN-C4-IO cycloalkenyl optionally substituted with one or more oxo or - N(RN’RN”);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 -(Ci-6 alkylene)-(C3-8 cycloalkyl)-Ci-6 alkyl;R2ais -H or Ci-io alkyl;R2bis -H or Ci-io alkyl;R2Cis C1-8 alkyl or C2-8 alkenyl;R'R3is R3aR3b;R3ais H or Ci-10 alkyl;R3bis H or C1-8 alkyl; andR3Cis Ci-10 alkyl or C2-8 alkenyl.

[0226] In some embodiments, the ionizable lipid is of Formula (IL**-I):(IL**-I)or a salt thereof, wherein:R1is -OH;o is 2, 3, or 4;n is 4, 5, 6, 7, or 8;M is -C(=O)-O-*, wherein * indicates attachment to R2;m is 6, 7, or 8;574920-5746-9564.2Attorney Docket No. 131986-6001M’ is -C(=0)-0-*, wherein * indicates attachment to R3;R2Cis C4-8 alkyl;R3ais C7-10 alkyl; andR3Cis C3-5 alkyl.

[0227] In some embodiments, the ionizable lipid is of Formula (IL**-III):R2aR33(IL* Mil)or a salt thereof, wherein:R1is NRN-C4-IO cycloalkenyl optionally substituted with one or more oxo or - N(RN’RN”);RNis H;RNis C1-2 alkyl;RN” is H;o is 2, 3, or 4;n is 6, 7, or 8;M is -C(=O)-O-*, wherein * indicates attachment to R2;m is 6, 7, or 8;M’ is -C(=O)-O-*, wherein * indicates attachment to R3;R2ais C7-10 alkyl;R2Cis C4-6 alkyl;R3ais C1-3 alkyl; andR3Cis C4-6 alkyl.

[0228] In some embodiments, the ionizable lipid is of Formula (IL**-IV):R3a(IL* MV)584920-5746-9564.2Attorney Docket No. 131986-6001or a salt thereof, wherein:R1is OH;o is 2, 3, or 4;n is 6, 7, or 8;M is -C(=O)-O-*, wherein * indicates attachment to R2;m is 6, 7, or 8;M’ is -C(=O)-O-*, wherein * indicates attachment to R3;R2bis C3-5 alkyl;R2Cis C2-4 alkyl;R3ais C7-10 alkyl; andR3Cis C4-6 alkyl.

[0229] In some embodiments, the ionizable lipid is of Formula (IL*-I):R2CR’R3a(IL*-Ia)or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; and R3ais C1-8 alkyl.

[0230] In some embodiments, ionizable lipid is of Formula (IL*-Ia):(IL*-Ia)or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and R3ais C1-8 alkyl.

[0231] In some embodiments, the ionizable lipid is of Formula (IL*-Ia’):594920-5746-9564.2Attorney Docket No. 131986-6001or a salt thereof, wherein:o, M, M’, R2Cand R3care as defined for variable IL*; andR3ais C1-8 alkyl.

[0232] In some embodiments, the ionizable lipid is of Formula (IL*-IIa):or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and R3ais C1-8 alkyl.

[0233] In some embodiments, the ionizable lipid is of Formula (IL*-II’):or a salt thereof, wherein:o, M, M’, R2Cand R3care as defined for variable IL*; andR3ais C1-8 alkyl.

[0234] In some embodiments, the ionizable lipid is of Formula (IL*-III):(IL*-III)or a salt thereof, wherein:604920-5746-9564.2Attorney Docket No. 131986-6001R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8 alkyl; andR3ais C1-8 alkyl.

[0235] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa):or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; R2bis a C1-8 alkyl; andR3ais C1-8 alkyl.

[0236] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa):or a salt thereof, wherein:R1, o, M, M’, R2C, and R3care as defined for variable IL*;R2ais a C1-8 alkyl; andR3ais C1-8 alkyl.

[0237] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa’):or a salt thereof, wherein:R1, o, M, M’, R2C, and R3care as defined for variable IL*;R2ais a C1-8 alkyl; andR3ais C1-8 alkyl.

[0238] In some embodiments, the ionizable lipid is of Formula (IL*-IIIb):614920-5746-9564.2Attorney Docket No. 131986-6001or a salt thereof, wherein:R1, o, M, M’, R2C, and R3care as defined for variable IL*;R2ais a C1-8 alkyl; andR3ais C1-8 alkyl.

[0239] In some embodiments, the ionizable lipid is of Formula (IL*-IIIb’):or a salt thereof, wherein:R1, o, M, M’, R2C, and R3care as defined for variable IL*;R2ais a C1-8 alkyl; andR3ais C1-8 alkyl.

[0240] In some embodiments, the ionizable lipid is of Formula (IL*-IV):(IL*-IV)or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;R2bis a C1-8 alkyl; andR3ais C1-8 alkyl.

[0241] In some embodiments, the ionizable lipid is of Formula (IL*-IVa):624920-5746-9564.2Attorney Docket No. 131986-6001R2bR2CR1MR3aR3C(IL*-IVa)or a salt thereof, wherein:R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;R2bis a C1-8 alkyl; andR3ais C1-8 alkyl.

[0242] In some embodiments, the ionizable lipid is of Formula (IL*-Iva’):R2bHO ° N MR3a(IL*-IVa)or a salt thereof, wherein:o, M, M’, R2C, and R3care as defined for variable IL*;R2ais a C1-8 alkyl; andR3ais C1-8 alkyl.Variables o, R1, R RN', RN'' of Ionizable Lipid

[0243] In some embodiments of the ionizable lipid, o is 1.

[0244] In some embodiments of the ionizable lipid, o is 2.

[0245] In some embodiments of the ionizable lipid, o is 3.

[0246] In some embodiments of the ionizable lipid, o is 4.

[0247] In some embodiments of the ionizable lipid, R1is -OH.

[0248] In some embodiments of the ionizable lipid, RNis H.

[0249] In some embodiments of the ionizable lipid, RNis methyl.

[0250] In some embodiments of the ionizable lipid, RNis ethyl.

[0251] In some embodiments of the ionizable lipid, R1is -NRN-cyclobutenyl, wherein the cyclobutenyl is optionally substituted with one or more oxo or -N(RNRN”).

[0252] In some embodiments of the ionizable lipid, RNis H.634920-5746-9564.2Attorney Docket No. 131986-6001

[0253] In some embodiments of the ionizable lipid, RNis methyl.

[0254] In some embodiments of the ionizable lipid, RNis ethyl.

[0255] In some embodiments of the ionizable lipid, RNis H.

[0256] In some embodiments of the ionizable lipid, RNis methyl.

[0257] In some embodiments of the ionizable lipid, RNis ethyl.

[0258] In some embodiments of the ionizable lipid, RNis H and RNis methyl.

[0259] In some embodiments of the ionizable lipid,R1is

[0260] In some embodiments of the ionizable lipid,R1is Variables m and n of the Ionizable Lipid

[0261] In some embodiments of the ionizable lipid, m is 4.

[0262] In some embodiments of the ionizable lipid, m is 5.

[0263] In some embodiments of the ionizable lipid, m is 6.

[0264] In some embodiments of the ionizable lipid, m is 7.

[0265] In some embodiments of the ionizable lipid, m is 8.

[0266] In some embodiments of the ionizable lipid, m is 4.

[0267] In some embodiments of the ionizable lipid, n is 5.

[0268] In some embodiments of the ionizable lipid, n is 6.

[0269] In some embodiments of the ionizable lipid, n is 7.

[0270] In some embodiments of the ionizable lipid, n is 8.

[0271] In some embodiments of the ionizable lipid, n is 5 and m is 7.

[0272] In some embodiments of the ionizable lipid, n is 7 and m is 7.

[0273] In some embodiments of the ionizable lipid, m is 6 and n is 6.644920-5746-9564.2Attorney Docket No. 131986-6001Variables M and M ’

[0274] In some embodiments of the ionizable lipid, M is -O-C(=O)-*, wherein * indicates attachment to R2.

[0275] In some embodiments of the ionizable lipid, M is -C(=O)-O-* wherein * indicates attachment to R2.

[0276] In some embodiments of the ionizable lipid, M’ is -O-C(=O)-*, wherein * indicates attachment to R3.

[0277] In some embodiments of the ionizable lipid, M’ is -C(=O)-O-* wherein * indicates attachment to R3.

[0278] In some embodiments of the ionizable lipid, M is -O-C(=O)-*, wherein * indicates attachment to R2, and M’ is -C(=O)-O-* wherein * indicates attachment to R3Variables R2, R2a, R2b, R2c

[0279] In some embodiments of the ionizable lipid, R2is

[0280] In some embodiments of the ionizable lipid, R2ais hydrogen.

[0281] In some embodiments of the ionizable lipid, R2ais methyl.

[0282] In some embodiments of the ionizable lipid, R2ais ethyl.

[0283] In some embodiments of the ionizable lipid, R2ais propyl.

[0284] In some embodiments of the ionizable lipid, R2ais butyl.

[0285] In some embodiments of the ionizable lipid, R2ais pentyl.

[0286] In some embodiments of the ionizable lipid, R2ais hexyl.

[0287] In some embodiments of the ionizable lipid, R2ais heptyl.

[0288] In some embodiments of the ionizable lipid, R2ais octyl.

[0289] In some embodiments of the ionizable lipid, R2bis hydrogen.

[0290] In some embodiments of the ionizable lipid, R2bis methyl.

[0291] In some embodiments of the ionizable lipid, R2bis ethyl.

[0292] In some embodiments of the ionizable lipid, R2bis propyl.

[0293] In some embodiments of the ionizable lipid, R2bis butyl.654920-5746-9564.2Attorney Docket No. 131986-6001

[0294] In some embodiments of the ionizable lipid, R2bis pentyl.

[0295] In some embodiments of the ionizable lipid, R2bis hexyl.

[0296] In some embodiments of the ionizable lipid, R2bis heptyl.

[0297] In some embodiments of the ionizable lipid, R2bis octyl.

[0298] In some embodiments of the ionizable lipid, R2ais hydrogen and R2bis hydrogen.

[0299] In some embodiments of the ionizable lipid, R2ais hexyl and R2bis hydrogen.

[0300] In some embodiments of the ionizable lipid, R2ais octyl and R2bis hydrogen.

[0301] In some embodiments of the ionizable lipid, R2ais hydrogen and R2bis butyl.

[0302] In some embodiments of the ionizable lipid, R2cis methyl.

[0303] In some embodiments of the ionizable lipid, R2cis ethyl.

[0304] In some embodiments of the ionizable lipid, R2cis propyl.

[0305] In some embodiments of the ionizable lipid, R2cis butyl.

[0306] In some embodiments of the ionizable lipid, R2cis pentyl.

[0307] In some embodiments of the ionizable lipid, R2cis hexyl.

[0308] In some embodiments of the ionizable lipid, R2cis heptyl.

[0309] In some embodiments of the ionizable lipid, R2cis octyl.

[0310] In some embodiments of the ionizable lipid, R2is -(Ci-6 alkylene)-(C3-s cycloalkyl)-Ci-6 alkyl.

[0311] In some embodiments of the ionizable lipid, R2is -(Ci-6 alkylene)-(cyclohexyl)-Ci-6 alkyl.

[0312] In some embodiments of the ionizable lipid, R2is -(Ci-6 alkylene)-(cyclopentyl)-Ci-6 alkyl.Variables R3, R3a, R3b, and R3cR'

[0313] In some embodiments of the ionizable lipid, R3is R3aR3b

[0314] In some embodiments of the ionizable lipid, R3ais hydrogen.

[0315] In some embodiments of the ionizable lipid, R3ais methyl.664920-5746-9564.2Attorney Docket No. 131986-6001

[0316] In some embodiments of the ionizable lipid, R3ais ethyl.

[0317] In some embodiments of the ionizable lipid, R3ais propyl.

[0318] In some embodiments of the ionizable lipid, R3ais butyl.

[0319] In some embodiments of the ionizable lipid, R3ais pentyl.

[0320] In some embodiments of the ionizable lipid, R3ais hexyl.

[0321] In some embodiments of the ionizable lipid, R3ais heptyl.

[0322] In some embodiments of the ionizable lipid, R3ais octyl.

[0323] In some embodiments of the ionizable lipid, R3bis hydrogen.

[0324] In some embodiments of the ionizable lipid, R3bis methyl.

[0325] In some embodiments of the ionizable lipid, R3bis ethyl.

[0326] In some embodiments of the ionizable lipid, R3bis propyl.

[0327] In some embodiments of the ionizable lipid, R3bis butyl.

[0328] In some embodiments of the ionizable lipid, R3bis pentyl.

[0329] In some embodiments of the ionizable lipid, R3bis hexyl.

[0330] In some embodiments of the ionizable lipid, R3bis heptyl.

[0331] In some embodiments of the ionizable lipid, R3bis octyl.

[0332] In some embodiments of the ionizable lipid, R3ais octyl and R3bis hydrogen.

[0333] In some embodiments of the ionizable lipid, R3ais ethyl and R3bis hydrogen.

[0334] In some embodiments of the ionizable lipid, R3ais hexyl and R3bis hydrogen.

[0335] In some embodiments of the ionizable lipid, R3cis methyl.

[0336] In some embodiments of the ionizable lipid, R3cis ethyl.

[0337] In some embodiments of the ionizable lipid, R3cis propyl.

[0338] In some embodiments of the ionizable lipid, R3cis butyl.

[0339] In some embodiments of the ionizable lipid, R3cis pentyl.

[0340] In some embodiments of the ionizable lipid, R3cis hexyl.

[0341] In some embodiments of the ionizable lipid, R3cis heptyl.674920-5746-9564.2Attorney Docket No. 131986-6001

[0342] In some embodiments of the ionizable lipid, R3cis octyl.

[0343] 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 R3ccan be combined, where applicable, with any group described herein for one or more of the remainder of variables o, R1, RN, RN, RN’, m, n, M, M’, R2, R2a, R2b, R2c, R3, R3a, R3b, and R3c.

[0344] In some embodiments, the ionizable lipid is a compound selected from:

[0345] In some embodiments, the ionizable lipid is18).

[0346] In some embodiments, the ionizable lipid is

[0347] In some embodiments, the ionizable lipid is301).684920-5746-9564.2Attorney Docket No. 131986-6001

[0348] In some embodiments, the ionizable lipid is° (II-6).

[0349] 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 lipids may be referred to as cationic or ionizable (amino)lipids. Lipids may also be zwitterionic, / .<., neutral molecules having both a positive and a negative charge.B. Non-Cationic Lipids

[0350] For the purposes of the present disclosure, non-cationic or neutral lipids may be phospholipids, such as one or more saturated or (poly)unsaturated phospholipids or a combination thereof. Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin. In some embodiments, the phospholipid may be one or more of the phospholipids described in PCT Application No. PCT / US2018 / 037922.

[0351] A lipid nanoparticle or other lipid composition of the present disclosure may comprise 5-25 mol% non-cationic lipid. For example, the LNP 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 LNP comprises 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% non-cationic lipid.

[0352] Non-cationic or neutral lipids that can be incorporated into LNPs or other lipid compositions for the purposes of the present disclosure include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-diundecanoyl-sn-gly cero-phosphocholine (DUPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl -sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), 1, 2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1,2-diarachidonoyl-sn-glycero-3 -phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3-694920-5746-9564.2Attorney Docket No. 131986-6001phosphocholine, l,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), sphingomyelin, or mixtures thereof.

[0353] In some embodiments, a LNP or other lipid composition of the present disclosure may comprise 5-15 mol%, 5-10 mol%, or 10-15 mol% DSPC. For example, the LNP may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% DSPC.

[0354] In some embodiments, the non-cationic lipid may be an analog or variant of DSPC.

[0355] In some embodiments, the lipid nanoparticle comprises a molar ratio of 5-25% noncationic 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.

[0356] 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.Phospholipids

[0357] Phospholipids, as defined herein, are any lipids that comprise a phosphate group. Phospholipids are a subset of non-cationic lipids. The lipid component of a lipid nanoparticle composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety may be selected from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety may be selected 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,704920-5746-9564.2Attorney Docket No. 131986-6001docosapentaenoic acid, and docosahexaenoic acid. Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group may undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions may 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).

[0358] In some embodiments, the nanoparticle described herein comprises about 5 mol% to about 15 mol% of phospholipid. In some embodiments, the nanoparticle comprises about 8 mol% to about 13 mol% of phospholipid. In some embodiments, the nanoparticle comprises about 10 mol% to about 12 mol% of phospholipid.

[0359] Phospholipids useful or potentially useful in the compositions and methods may be selected from the non-limiting group consisting of1.2-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),l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC),1.2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC),l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 Lyso PC),1.2-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-diphytanoyl-sn-glycero-3 -phosphocholine (4ME 16:0 PC),l,2-diphytanoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (sodium salt) (4ME 16:0 PG), l,2-diphytanoyl-sn-glycero-3-phospho-L-serine714920-5746-9564.2Attorney Docket No. 131986-6001(sodium salt) (4ME 16:0 PS), l,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, and1.2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), and sphingomyelin. Each possibility represents a separate embodiment of the present invention.

[0360] In some embodiments, a lipid nanoparticle composition includes DSPC. In certain embodiments, a lipid nanoparticle composition includes DOPE. In some embodiments, a lipid nanoparticle composition includes both DSPC and DOPE. In some embodiments, the lipid nanoparticle includes: l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (4ME 16:0 PE)CH3CH3CH3CH3O • 1,2-diphytanoyl-sn-glycero-3 -phosphocholine (4ME 16:0 PC)CH3CH3CH3CH3O • 1,2-diphytanoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (sodium salt) (4ME 16:0 PG), or1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0 PS)CH3CH3CH3CH3o or a mixture thereof.724920-5746-9564.2Attorney Docket No. 131986-6001

[0361] Examples of phospholipids include, but are not limited to, the following:o 00X '°T,,0 H O’ (Compound 432),o' H o (Compound 433), 9 o X0N.d H 6" o (Compound 434), o 'O' '<,,0 H d"(Compound 435), O•0" P"-0^Nt d" o (Compound 436), 0 0Nt. O H O"(Compound 437), o O A a O"Pi " O 'NH-.0 H (Compound 438), p, A o* 'NH;A o (Compound 439),0 0t:Q -P-Q,o (Compound 440)," NHgO'(Compound 441), o a" O"P'O.6“(Compound 442),734920-5746-9564.2Attorney Docket No. 131986-6001O ii. P i> N O© (Compound 448).

[0362] In certain embodiments, a phospholipid useful or potentially useful in the present invention is an analog or variant of DSPC.

[0363] In certain embodiments, a phospholipid useful or potentially useful in the present R1©. \© OR Nv / UXk AI (Jn PR1"invention is a compound of Formula (IX): O (IX), or a salt thereof, wherein:each R1is independently H or 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;744920-5746-9564.2Attorney Docket No. 131986-6001n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;L2- R2B4-(R2)PA is of the formula: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)-;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-;each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; andep is 1 or 2; provided that the compound is not of the formula:, wherein each instance of R2is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.754920-5746-9564.2Attorney Docket No. 131986-6001

[0364] In certain embodiments, a phospholipid useful or potentially useful in the present R1©. \® OR I Un P MnR1"invention is a compound of Formula (IX): O (IX), or a salt thereof, wherein: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;n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;L2- R2(R2)PL2— RA is of the formula:2oreach 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)-;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-;each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;764920-5746-9564.2Attorney Docket No. 131986-6001Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is 1 or 2;provided that the compound is not of the formula:wherein each instance of R2is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0365] In some embodiments, the phospholipid is selected from: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),l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC),1.2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC),l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 Lyso PC),1.2-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-diphytanoyl-sn-glycero-3 -phosphocholine (4ME 16:0 PC), l,2-diphytanoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (sodium salt) (4ME 16:0 PG), l,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0 PS), l,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,774920-5746-9564.2Attorney Docket No. 131986-6001l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), and Sphingomyelin.

[0366] In some embodiments, the phospholipid is DSPC, DOPE, or combinations thereof. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DOPE. In some embodiments, the phospholipid is 4ME 16:0 PE, 4ME 16:0 PC, 4ME 16:0 PG, 4ME 16:0 PS, or combination thereof.

[0367] In some embodiments, the phospholipid is N-lauroyl-D-erythro-sphinganylphosphorylcholine.

[0368] Phospholipid Head Modifications

[0369] In certain embodiments, a phospholipid useful or potentially useful in the present invention comprises a modified phospholipid head (e.g., a modified choline group). In certain embodiments, a phospholipid with a modified head is DSPC, or analog thereof, with a modified quaternary amine. For example, in embodiments of Formula (IX), at least one of R1is not methyl. In certain embodiments, at least one of R1is not hydrogen or methyl. In certain embodiments, the compound of Formula (IX) is of one of the following formulae:^ )uQ_ © 0I, or a salt thereof, wherein: each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each v is independently 1, 2, or 3.

[0370] In certain embodiments, the compound of Formula (IX) is of one of thefollowing formulae:784920-5746-9564.2Attorney Docket No. 131986-60010 0 Ox ixCL J. A ’ f?0, or a salt thereof.

[0371] In certain embodiments, a compound of Formula (IX) is one of the following:(Compound 401),(Compound 403),794920-5746-9564.2Attorney Docket No. 131986-6001(Compound 407),(Compound 409), or a salt thereof.

[0372] In certain embodiments, a compound of Formula (IX) is of Formula (IX-a):R1O L2-R2R1-N^o. 1,02_2'1 ™ P L KO (IX-a), or a salt thereof.

[0373] In certain embodiments, phospholipids useful or potentially useful in the present invention comprise a modified core. In certain embodiments, a phospholipid with a modified core described herein is DSPC, or analog thereof, with a modified core structure.804920-5746-9564.2Attorney Docket No. 131986-6001 For example, in certain embodiments of Formula (IX-a), group A is not of the followingformula:

[0374] In certain embodiments, the compound of Formula (IX-a) is of one of thefollowing formulae:

[0375] In certain embodiments, a compound of Formula (IX) is one of the following:(Compound 449),(Compound 450),(Compound 451).814920-5746-9564.2Attorney Docket No. 131986-6001(Compound 452).(Compound 453), or salts thereof.

[0376] In certain embodiments, a phospholipid useful or potentially useful in the present invention comprises a cyclic moiety in place of the glyceride moiety. In certain embodiments, a phospholipid useful in the present invention is DSPC, or analog thereof, with a cyclic moiety in place of the glyceride moiety. In certain embodiments, the compound ofFormula (IX) is of Formula (IX -b):, (IX-b), or a salt thereof.

[0377] In certain embodiments, the compound of Formula (IX-b) is of Formula (IX-(IX-b-1), or a salt thereof, wherein: w is 0, 1, 2, or 3.

[0378] In certain embodiments, the compound of Formula (IX-b) is of Formula (IX-(IX-b-2), or a salt thereof.

[0379] In certain embodiments, the compound of Formula (IX-b) is of Formula (IX-(IX-b-3), or a salt thereof.824920-5746-9564.2Attorney Docket No. 131986-6001

[0380] In certain embodiments, the compound of Formula (IX-b) is of Formula (IX-b-4) (IX -b-4), or a salt thereof.salts thereof.C. Structural Lipids

[0382] The disclosed LNPs and lipid compositions can comprise one or more structural lipids. As used herein, the term “structural lipid” includes sterols and also to lipids containing sterol moieties.

[0383] A lipid nanoparticle or other lipid composition of the present disclosure may comprise 30-45 mol% sterol. For example, the LNP may comprise 35-40 mol%, 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% sterol. In some embodiments, the LNP comprises 25-55 mol% sterol. For example, the lipid nanoparticle may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 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.

[0384] Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol,834920-5746-9564.2Attorney Docket No. 131986-6001tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. 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.

[0385] In some embodiments, the LNP comprises 35-40 mol% cholesterol. For example, the LNP may comprise 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, or 40 mol% cholesterol.

[0386] 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.D. Polyethylene glycol (PEG) and PEG-modified Lipid

[0387] In general, some of the other lipid components (e.g., PEG lipids) of various formulae, described herein may be synthesized as described International Patent Application No.PCT / US2016 / 000129, filed December 10, 2016, entitled “Compositions and Methods for Delivery of Therapeutic Agents,” which is incorporated by reference in its entirety.

[0388] 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 lipid is a lipid modified with polyethylene glycol. A PEG 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 lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments, a PEG lipid is DMG-PEG 2k or Compound 428.844920-5746-9564.2Attorney Docket No. 131986-6001

[0389] In some embodiments, the PEG lipid is PEG-DMG. In some embodiments, the PEG lipid is PEG-DMG 2k. In some embodiments, a PEG lipid has the structure:0

[0390] DMG-PEG 2k has the following structure:00

[0391] In some embodiments, the PEG-modified lipids are a modified form of PEG DMG. PEG-DMG has the following structure:0

[0392] In some embodiments, the nanoparticle described herein comprises about 1 mol% to about 5 mol% of PEG-lipid. In some embodiments, the nanoparticle comprises about 1 mol% to about 2.5 mol% of PEG-lipid.

[0393] In one embodiment, PEG lipids useful in the present invention can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG 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 of the present invention.854920-5746-9564.2Attorney Docket No. 131986-6001

[0394] In certain embodiments, a PEG lipid useful in the present invention is a compound of R37 L1-D^ A Formula (VII). Provided herein are compounds of Formula (VII):r(VII), or salts thereof,wherein: R3is -OR°; R° is hydrogen, optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive;L1is optionally substituted Ci-io alkylene, wherein at least one methylene of the optionally substituted Ci-io alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -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)-; D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;A is of the formula: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)-; 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-;864920-5746-9564.2Attorney Docket No. 131986-6001each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; andp is 1 or 2.

[0395] In certain embodiments, the compound of Formula (VII) is a PEG-OH lipid ( / .<., R3is -OR°, and R° is hydrogen). In certain embodiments, the compound of Formula (VII) is of HO LI-VFormula (VII-OH):r m(VII-OH), or a salt thereof.

[0396] In certain embodiments, D is a moiety obtained by click chemistry (e.g., triazole). In certain embodiments, the compound of Formula (VII) is of Formula (VII-a-1) or (VII-a-2):N-N(VII-a-1) or ' A (VII-a-2), or a salt thereof.

[0397] In certain embodiments, the compound of Formula (VII) is of one of the followingformulae:salt thereof, wherein s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0398] In certain embodiments, the compound of Formula (VII) is of one of the followingformulae:874920-5746-9564.2Attorney Docket No. 131986-6001

[0399] In certain embodiments, a compound of Formula (VII) is of one of the followingthereof.

[0400] In certain embodiments, a compound of Formula (VII) is of one of the following formulae, wherein r is 1-100:884920-5746-9564.2Attorney Docket No. 131986-6001

[0401] In certain embodiments, D is a moiety cleavable under physiological conditions (e.g., ester, amide, carbonate, carbamate, urea). In certain embodiments, a compound of Formular y vin (VII) is of Formula (VII-b-1) or (VII-b-2): O (VII-b-1) or0(VII-b-2), or a salt thereof.

[0402] In certain embodiments, a compound of Formula (VII) is of Formula (VII-b-l-OH)or (VII-b-2-OH): O (VII-b-1 -OH) or0HO'{^O^L" OA^Ar(VII-b-2-OH), or a salt thereof.

[0403] In certain embodiments, the compound of Formula (VII) is of one of the following894920-5746-9564.2Attorney Docket No. 131986-6001

[0404] In certain embodiments, a compound of Formula (VII) is of one of the following

[0405] In certain embodiments, a compound of Formula (VII) is of one of the followingthereof.

[0406] In certain embodiments, a compound of Formula (VII) is of one of the following431), or salts thereof.

[0407] In certain embodiments, a PEG lipid useful in the present invention is a PEGylated fatty acid. In certain embodiments, a PEG lipid useful in the present invention is a compound904920-5746-9564.2Attorney Docket No. 131986-6001of Formula (VIII). Provided herein are compounds of Formula (VIII):r(VIII), or a salts thereof, wherein:R3is-OR°; R° is hydrogen, optionally substituted alkyl or an oxygen protecting group;r is an integer between 1 and 100, inclusive;R5is 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(0)N(RN)-, -NRNC(0)-, -NRNC(0)N(RN)-, -C(O)O-, -OC(O)-, -OC(O)O-, -0C(0)N(RN)-, -NRNC(0)0-, -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(0)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(0)2N(RN)-, -OS(O)2N(RN)- or -N(RN)S(O)2O-; andeach instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.

[0408] In certain embodiments, the compound of Formula (VIII) is of Formula (VIII-OH):(VIII-OH), or a salt thereof.

[0409] In certain embodiments, a compound of Formula (VIII) is of one of the following O07 formulae: ' r (Compound 419),07 'r (Compound 420),914920-5746-9564.2Attorney Docket No. 131986-6001H0 (Compound 424), HO(Compound 425), or a salt thereof. In some embodiments, r is 45.

[0410] In certain embodiments, a compound of Formula (VIII) is of one of the followingformulae:0(Compound 420),HO(Compound 423), or a salt thereof. In some embodiments, r is 45.924920-5746-9564.2Attorney Docket No. 131986-6001

[0411] In yet other embodiments the compound of Formula (VIII) is:(Compound 427), or a salt thereof.

[0412] In some embodiments, the compound of Formula (VIII) is:(Compound 428),(Compound 410).

[0413] In certain embodiments, the PEG lipid is one of the following formula:(Compound 424),(Compound 425), or a salt thereof. In some embodiments, r is 45.

[0414] In one embodiment, PEG-lipids useful in the present invention can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety.

[0415] Any of the PEG-lipids described herein may be modified to comprise one or more hydroxyl group on the PEG chain (OH-PEG-lipids) or one or more hydroxyl group on the lipid (PEG-lipid-OH). In some embodiments, the PEG-lipid is an OH-PEG-lipid. In some embodiments, the OH-PEG-lipid comprises a hydroxyl group at the terminus of the PEG chain. In some embodiments, the PEG-lipids described herein may be modified to comprise one or more alkyl group on the PEG chain (alkyl-PEG-lipid). In some embodiments, the alkyl-PEG-lipid is a methoxy -PEG-lipid.

[0416] In some embodiments, the LNP comprises about 0.1 mol% to about 5.0 mol%, about 0.5 mol% to about 5.0 mol%, about 1.0 mol% to about 5.0 mol%, about 1.0 mol% to about 2.5 mol%, about 0.5 mol% to about 2.0 mol%, or about 1.0 mol% to about 1.5 mol% of PEG-934920-5746-9564.2Attorney Docket No. 131986-6001lipid. In some embodiments, the LNP comprises about 1.5 mol % or about 3.0 mol % PEG-lipid.

[0417] Certain of the LNPs provided herein comprise no or low levels of PEG-lipid. Some LNPs comprise less than 0.5 mol % PEG-lipid.

[0418] In some embodiments, PEG is used as a stabilizer. In some embodiments, the PEG stabilizer is a PEG-lipid. In some embodiments, the LNP comprises less than 0.5 mol% PEG stabilizer.

[0419] Other non-limiting examples of PEG lipids can be found in, e.g., International PCT Application Publication Nos. WO 2020 / 061284, published March 26, 2020; and WO 2020 / 061295, published March 26, 2020, the entire contents of each of which (including any generic or specific structures disclosed therein) is incorporated herein by reference.V. Pharmaceutical Compositions, Dosing, and Administration

[0420] The present disclosure provides pharmaceutical compositions comprising mRNA that encode a masked protein. The pharmaceutical compositions provided herein can be used as therapeutic or prophylactic agents. They may be used in medicine to prevent and / or treat diseases such as cancer or an infection.

[0421] The disclosed pharmaceutical compositions do not comprise masked protein per se, but rather comprise mRNA that encode a masked protein that, once delivered to a cell, tissue or subject, can be translated and ultimately produce the masked protein. 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.

[0422] The disclosed pharmaceutical compositions may be formulated for parenteral administration, particularly intravenous administration.

[0423] Pharmaceutical compositions of the disclosed herein may comprise more than one mRNA. For example, the pharmaceutical composition may comprise a first mRNA encoding a first masked protein and a second mRNA encoding a second masked protein. Alternatively,944920-5746-9564.2Attorney Docket No. 131986-6001the pharmaceutical composition may comprise a first mRNA encoding a first polypeptide chain of a masked protein and a second mRNA encoding a second polypeptide chain of the masked protein.

[0424] For the purposes of embodiments that include more than one mRNA (e.g., a first and second mRNA), the mRNAs may be present in the composition in an equal amount (e.g., a 1: 1 weight / weight ratio or a 1:1 molar ratio), for example, a ratio of 1:1 ( 1: 1: 1: 1: 1 ) of mRNA. In some embodiments, the first and second mRNAs are present in the composition in a 1:1, 1:2, 1:3, or 1:4 weight ratio. In some embodiments, the first and second mRNAs are present in the composition in a 2: 1, 3: 1, or 4:1 weight ratio. In some embodiments, the first and second mRNAs are present in the composition in a 1:1 weight ratio. In some embodiments, the ratio is 1:1, 1:2, 1:3, 1:4, 2:1, 3:1, or 4:1 weight ratio. In some embodiments, the first and second mRNAs are present in the composition in a 1: 1, 1:2, 1:3, or 1:4 molar ratio. In some embodiments, the first and second mRNAs are present in the composition in a 2: 1, 3: 1, or 4:1 molar ratio. In some embodiments, the first and second mRNAs are presents in the composition in a 1: 1 molar ratio. In embodiments, the ratio is 1:1, 1:2, 1:3, 1:4, 2:1, 3:1, or 4:1 molar ratio.

[0425] A pharmaceutical composition as described herein may be formulated with mRNA alone (i.e., as the sole active agent) or administered alone or in conjunction with one or more other components. For example, pharmaceutical composition may comprise other components including, but not limited to, an additional anti-cancer or anti-infection agent. A pharmaceutical composition described herein may also be independently administered with other prophylactic or therapeutic compounds. A pharmaceutical composition described herein may be administered concurrently or sequentially with other prophylactic or therapeutic compounds.

[0426] RNA is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the RNA may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or954920-5746-9564.2Attorney Docket No. 131986-6001coincidental with the specific compound employed; and like factors well known in the medical arts.

[0427] The effective amount (e.g., effective dose) of the RNA, as provided herein, may be as low as 0.1 pg / kg. In some embodiments, the effective amount (e.g., effective dose) comprises one or more administrations of a dose of 0.1-10 pg / kg, 0.2-10 pg / kg, 0.3-10 pg / kg, 0.4-10 pg / kg, 0.5-10 pg / kg, 0.6-10 pg / kg, 0.7-10 pg / kg, 0.8-10 pg / kg, 0.9-10 pg / kg, 1-10 pg / kg, 1.5-10 pg / kg, 2-10 pg / kg, 2.5-10 pg / kg, 3-10 pg / kg, 3.5-10 pg / kg, 4-10 pg / kg, 4.5-10 pg / kg, 5-10 pg / kg, 5.5-10 pg / kg, 6-10 pg / kg, 6.5-10 pg / kg, 7-10 pg / kg, 7.5-10 pg / kg, 8-10 pg / kg, 8.5-10 pg / kg, 9-10 pg / kg, 9.5-10 pg / kg, 0.1-9 pg / kg, 0.2-9 pg / kg, 0.3-9 pg / kg, 0.4-9 pg / kg, 0.5-9 pg / kg, 0.6-9 pg / kg, 0.7-9 pg / kg, 0.8-9 pg / kg, 0.9-9 pg / kg, 1-9 pg / kg, 1.5-9 pg / kg, 2-9 pg / kg, 2.5-9 pg / kg, 3-9 pg / kg, 3.5-9 pg / kg, 4-9 pg / kg, 4.5-9 pg / kg, 5-9 pg / kg, 5.5-9 pg / kg, 6-9 pg / kg, 6.5-9 pg / kg, 7-9 pg / kg, 7.5-9 pg / kg, 8-9 pg / kg, 8.5-9 pg / kg, 0.1-8 pg / kg, 0.2-8 pg / kg, 0.3-8 pg / kg, 0.4-8 pg / kg, 0.5-8 pg / kg, 0.6-8 pg / kg, 0.7-8 pg / kg, 0.8-8 pg / kg, 0.9-8 pg / kg, 1-8 pg / kg, 1.5-8 pg / kg, 2-8 pg / kg, 2.5-8 pg / kg, 3-8 pg / kg, 3.5-8 pg / kg, 4-8 pg / kg, 4.5-8 pg / kg, 5-8 pg / kg, 5.5-8 pg / kg, 6-8 pg / kg, 6.5-8 pg / kg, 7-8 pg / kg, 7.5-8 pg / kg, of 0.1-7 pg / kg, 0.2-7 pg / kg, 0.3-7 pg / kg, 0.4-7 pg / kg, 0.5-7 pg / kg, 0.6-7 pg / kg, 0.7-7 pg / kg, 0.8-7 pg / kg, 0.9-7 pg / kg, 1-7 pg / kg, 1.5-7 pg / kg, 2-7 pg / kg, 2.5-7 pg / kg, 3-7 pg / kg, 3.5-7 pg / kg, 4-7 pg / kg, 4.5-7 pg / kg, 5-7 pg / kg, 5.5-7 pg / kg, 6-7 pg / kg, 6.5-7 pg / kg, 0.1-6 pg / kg, 0.2-6 pg / kg, 0.3-6 pg / kg, 0.4-6 pg / kg, 0.5-6 pg / kg, 0.6-6 pg / kg, 0.7-6 pg / kg, 0.8-6 pg / kg, 0.9-6 pg / kg, 1-6 pg / kg, 1.5-6 pg / kg, 2-6 pg / kg, 2.5-6 pg / kg, 3-6 pg / kg, 3.5-6 pg / kg, 4-6 pg / kg, 4.5-6 pg / kg, 5-6 pg / kg, 5.5-6 pg / kg, 0.1-5 pg / kg, 0.2-5 pg / kg, 0.3-5 pg / kg, 0.4-5 pg / kg, 0.5-5 pg / kg, 0.6-5 pg / kg, 0.7-5 pg / kg, 0.8-5 pg / kg, 0.9-5 pg / kg, 1-5 pg / kg, 1.5-5 pg / kg, 2-5 pg / kg, 2.5-5 pg / kg, 3-5 pg / kg, 3.5-5 pg / kg, 4-5 pg / kg, or 4.5-5 pg / kg. In some embodiments, the effective amount (e.g., effective dose) comprises one or more administrations of a dose of 0.1 pg / kg, 0.15 pg / kg, 0.2 pg / kg, 0.25 pg / kg, 0.3 pg / kg, 0.35 pg / kg, 0.4 pg / kg, 0.45 pg / kg, 0.5 pg / kg, 0.55 pg / kg, 0.6 pg / kg, 0.65 pg / kg, 0.7 pg / kg, 0.75 pg / kg, 0.8 pg / kg, 0.85 pg / kg, 0.9 pg / kg, 0.95 pg / kg, 1 pg / kg, or any value in between 0.1 pg / kg and 1 pg / kg. In some embodiments, the effective amount (e.g., effective dose) comprises one or more administrations of a dose of 1 pg / kg, 1.5 pg / kg, 2 pg / kg, 2.5 pg / kg, 3 pg / kg, 3.5 pg / kg, 4 pg / kg, 4.5 pg / kg, 5 pg / kg, 5.5 pg / kg, 6 pg / kg, 6.5 pg / kg, 7 pg / kg, 7.5 pg / kg, 8 pg / kg, 8.5 pg / kg, 9 pg / kg, 9.5 pg / kg, 10 pg / kg, or any value in between 1 pg / kg and 10 pg / kg. In some embodiments, the effective amount (e.g., effective dose) comprises one or more administrations of a dose of 5 pg / kg, 10 pg / kg, 15 pg / kg, 20 pg / kg, 25 pg / kg, 30 pg / kg, 35964920-5746-9564.2Attorney Docket No. 131986-6001pg / kg, 40 pg / kg, 45 pg / kg, 50 pg / kg, 55 pg / kg, 60 pg / kg, 65 pg / kg, 70 pg / kg, 75 pg / kg, 80 pg / kg, 85 pg / kg, 90 gg / kg, 95 gg / kg, 100 gg / kg, 105 gg / kg, 110 gg / kg, 115 gg / kg, 120 gg / kg, 125 gg / kg, 130 gg / kg, 135 gg / kg, 140 gg / kg, 145 gg / kg, 150 gg / kg, 155 gg / kg, 160 gg / kg, 165 gg / kg, 170 gg / kg, 175 gg / kg, 180 gg / kg, 185 gg / kg, 190 gg / kg, 195 gg / kg, 200 gg / kg, 205 gg / kg, 210 gg / kg, 215 gg / kg, 220 gg / kg, 225 gg / kg, 230 gg / kg, 235 gg / kg, 240 gg / kg, 245 gg / kg, 250 gg / kg, 255 gg / kg, 260 gg / kg, 265 gg / kg, 270 gg / kg, 275 gg / kg, 280 gg / kg, 285 gg / kg, 290 gg / kg, 295 gg / kg, 300 gg / kg, or any value in between 5 gg / kg and 300 gg / kg. In some embodiments, the effective amount (e.g., effective dose) comprises one or more administrations of a dose of 0.1 gg / kg to 3 mg / kg, 0.1 gg / kg to 2.75 mg / kg, 0.1 gg / kg to 2.5 mg / kg, 0.1 gg / kg to 2.25 mg / kg, 0.1 gg / kg to 2 mg / kg, 0.1 gg / kg to 1.75 mg / kg, 0.1 gg / kg to 1.5 mg / kg, 0.1 gg / kg to 1.25 mg / kg, 0.1 gg / kg to 1 mg / kg, 0.1 gg / kg to 0.75 mg / kg, 0.1 gg / kg to 0.5 mg / kg, or 0.1 gg / kg to 0.25 mg / kg. In some embodiments, the effective amount (e.g., effective dose) comprises one or more administrations of a dose of 3 mg / kg, 2.75 mg / kg, 2.5 mg / kg, 2.25 mg / kg, 2 mg / kg, 1.75 mg / kg, 1.5 mg / kg, 1.25 mg / kg, 1 mg / kg, 0.75 mg / kg, 0.5 mg / kg, 0.25 mg / kg or any value in between 0.25 mg / kg and 3 mg / kg.

[0428] In some embodiments, the effective amount (e.g., effective dose) of an mRNA disclosed herein may comprise one or more administrations of a dose of about 0.2 pg / kg, about 0.25 pg / kg, or about 0.3 pg / kg. In some embodiments, the effective amount (e.g., effective dose) of an mRNA disclosed herein may comprise one or more administrations of a dose of about 0.75 pg / kg, about 0.8 pg / kg, or about 0.85 pg / kg. In some embodiments, the effective amount (e.g., effective dose) of an mRNA disclosed herein may comprise one or more administrations of a dose of about 2.0 pg / kg, about 2.5 pg / kg, or about 2.75 pg / kg. In some embodiments, the effective amount (e.g., effective dose) of an mRNA disclosed herein may comprise one or more administrations of a dose of about 7.5 pg / kg, about 8.0 pg / kg, or about 8.5 pg / kg. In some embodiments, the effective amount (e.g., effective dose) of an mRNA disclosed herein may comprise one or more administrations of a dose of about 20 pg / kg, about 25 pg / kg, or about 30 pg / kg.VI. Methods of Using mRNA-Encoded Masked Protein Therapeutics

[0429] Provided herein are methods using mRNA-encoded masked proteins. In particular, the disclosed mRNA encoding masked proteins can be used to deliver the encoded therapeutic protein (which is a component of the masked peptide) to a particular tissue, stimulate an immune response in a subject, or treat cancer.974920-5746-9564.2Attorney Docket No. 131986-6001A. Delivery of a therapeutic protein

[0430] The mRNA encoding the masked protein may be administered systemically to a subject in need thereof, which will generally involve intravenous administration. Without being bound by theory, it is believed that when the mRNA encoding the masked protein is administered systemically, the majority of the administered mRNA is translated in the subject’s liver and spleen, resulting in production of the masked protein in the liver and spleen. In some embodiments, the liver and / or spleen lack or express only at low levels the tissue-specific protease(s) that is / are capable of efficiently cleaving the cleavable moiety. While some cleavage of the cleavable moiety may occur in the liver or spleen following expression of the masked protein, the majority of the masked proteins (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) expressed in the liver or spleen will remain intact, such that the masked proteins can be secreted into the subject’s bloodstream and thereafter contact the target cell, cell population, tissue, or organ.

[0431] In some embodiments, the target cell, cell population, tissue, or organ express one or more proteases that can cleave the cleavable moiety. Thus, in the target cell, cell population, tissue, or organ, the masked protein is cleaved at the cleavable moiety, releasing the therapeutic protein from the masking moiety, thereby delivering the therapeutic protein to its intended target site. The therapeutic protein may then bind to or be bound by the target within the target cell, cell population, tissue, or organ in order to mediate the cellular response underlying the therapy.B. Stimulating an Immune Response

[0432] As demonstrated herein in the Examples, mRNA constructs of the present disclosure that contained an immune-stimulating protein, such as a cytokine (e.g., IL-12) were effective at inducing the desired immune response. As described in Example 2, MC38 syngeneic mice treated with mRNA encoding masked IL-2 protein at low to relatively high dosage levels exhibited reduced tumor volume relative to a vehicle control with no significant side effects across all dosage levels. These results demonstrate that mRNA encoding the masked protein was not only effectively translated in vivo, but that premature activation did not occur during the process to the extent of rendering the treatment ineffective.

[0433] Cytokines that may be incorporated as the therapeutic protein into the disclosed masked proteins including, but are not limited to, tumor necrosis factors (TNFs), interleukins (ILs), lymphokines, monokines, interferons (IFNs), colony stimulating factors (CSFs), and 984920-5746-9564.2Attorney Docket No. 131986-6001transforming growth factors (TGFs). For the purposes of the present disclosure, the mode of immune stimulation may be pro-inflammatory or anti-inflammatory, based on the therapeutic protein express and its ability to facilitate inflammatory reactions when released and either active or suppress immune cells, although a single cytokine may have both pro- and antiinflammatory activities, depending on the context of the cytokine activity. Pro-inflammatory cytokines generally include IL-ip, IL-6, IL-8, IL- 12, IL- 17, IL- 18, IFN- a / y, and TNF-a. Anti-inflammatory cytokines generally include, IL-IRA, IL-4, IL-6, IL-10, IL-11, IL-13, IL-35, and TGF-p.

[0434] The disclosed mRNA encoding a masked proteins provide benefits of (a) reduced side effects relative to an unmasked cytokine, and (b) more targeted delivery relative to an unmasked cytokine, and the masked protein is only “unmasked” and activated in a desired cell, tissue, or microenvironment. Accordingly, the disclosed masked protein constructs provide for a wide therapeutic window relative to administration of a corresponding unmasked cytokine.

[0435] For example, an unmasked cytokine may only be administered up to the level that it causes negative immune responses and / or systemic side effects. The disclosed mRNA encoding masked proteins may be administered at a higher dose, if needed, relative to a corresponding unmasked cytokine because the encoded masked protein will result in less systemic toxicity. Alternatively, because the masked protein is also more targeted, a lower does relative to a corresponding unmasked cytokine, in some circumstances (i.e., the masked cytokine is potent enough to yield the desired immune response even at a low dose). The desired immune response in the subject may be induced 1 day to 10 weeks or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more weeks).

[0436] Thus, in some embodiments, the mRNA encoding the masked protein may be administered at dosages that are 2 to 1,000 times higher than the acceptable dosage for an unmasked version of the protein. In some embodiments, the mRNA encoding the masked protein may be administered at an acceptable dosage that is at 4 times, 5 times, 10 times, 50 times, 100 times, 200 time, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, or 1000 times higher than an acceptable dose of the unmasked cytokine.

[0437] Alternatively, in some embodiments, the mRNA encoding the masked protein may be administered at dosages that are 2 to 1,000 times lower than the acceptable dosage for an994920-5746-9564.2Attorney Docket No. 131986-6001unmasked version of the protein. In some embodiments, the mRNA encoding the masked protein may be administered at an acceptable dosage that is at 4 times, 5 times, 10 times, 50 times, 100 times, 200 time, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, or 1000 times lower than an acceptable dose of the unmasked cytokine.C. Treatment of Cancer

[0438] In some embodiments, the method of using the mRNA encoding a masked protein may be used as a treatment of a disease, such as cancer. The cancer may be, but is not limited to, any one of adenocarcinoma, melanoma, colon cancer, breast cancer, lymphoma, pancreatic cancer, prostate cancer, bladder cancer, urothelial cancer, and Kaposi sarcoma. In some embodiments, the method of treating a cancer may include administering to a subject with cancer the mRNA encoding the masked protein, wherein the masked protein is not efficiently cleaved or is inert in the liver and / or spleen but is efficiently cleaved and therefore activated in by cells affected by the cancer. The cells affected by the cancer may include cancer cells, tumor cells, or other cells within a tumor microenvironment (TME). In some embodiments, activation of the masked protein by cells affected by the cancer may mediate treatment of the cancer, including, but not limited to, triggering an immunogenic response to the tumor. The immunogenic response to the tumor may include, but is not limited to, activating immune cells to target the cells affected by the cancer for removal.VII. Specific Embodiments

[0439] Provided herein is an mRNA encoding a masked protein, the masked protein comprising: (a) a therapeutic protein; (b) a masking moiety; (c) at least one cleavable moiety; and (d) a half-life extending moiety; wherein the at least one cleavable moiety is inert in mammalian liver and spleen, and wherein the therapeutic protein is activated upon cleavage of the at least one cleavable moiety.

[0440] Also provided herein is an mRNA encoding a first peptide chain of a masked protein and a second peptide chain of the masked protein; the first peptide chain of the masked protein comprising, in the following order: (a) a therapeutic protein, (b) a first cleavable moiety, (c) a masking moiety, (d) a second cleavable moiety, and (e) a first Fc peptide comprising a first CH2 region and a first CH3 region; and the second peptide chain of the masked protein comprising a second Fc peptide comprising a second CH2 region and a second CH3 region; wherein the at first cleavable moiety and optionally the second cleavable moiety are inert in mammalian liver and spleen, and wherein the first cleavable moiety and 1004920-5746-9564.2Attorney Docket No. 131986-6001optionally the second cleavable moiety are cleaved in the tissue of interest, thereby delivering the therapeutic protein.

[0441] Further, provided herein is a method of delivering a therapeutic protein to a tissue of interest, comprising administering to a subject an mRNA encoding a masked protein, wherein the masked protein comprises: (a) the therapeutic protein; (b) a masking moiety; (c) at least one cleavable moiety; and (d) a half-life extending moiety; wherein the masked protein is expressed from the mRNA in the subject’s liver or spleen, and wherein the at least one cleavable moiety is cleaved in the tissue of interest, thereby delivering the therapeutic protein. Provided herein also is a method of delivering a therapeutic protein to a tissue of interest, comprising administering to a subject an mRNA encoding a first peptide chain of a masked protein and a second peptide chain of the masked protein: the first peptide chain of the masked protein comprising, in the following order: (a) the therapeutic protein, (b) a first cleavable moiety, (c) a masking moiety, (d) a second cleavable moiety, and (e) a first Fc peptide comprising a first CH2 region and a first CH3 region; and the second peptide chain of the masked protein comprising a second Fc peptide comprising a second CH2 region and a second CH3 region, wherein the wherein the masked protein is expressed from the mRNA in the subject’s liver or spleen, and wherein the first cleavable moiety and optionally the second cleavable moiety are cleaved in the tissue of interest, thereby delivering the therapeutic protein.

[0442] Additionally provided herein is a method of stimulating an immune response, comprising administering to a subject an mRNA encoding a masked protein, the masked protein comprising: (a) a cytokine or an immunogenic fragment thereof as a therapeutic protein; (b) a masking moiety; (c) at least one cleavable moiety; and (d) a half-life extending moiety; wherein the at least one cleavable moiety is inert in mammalian liver and spleen, and wherein the cytokine or the immunogenic fragment is activated upon cleavage of the at least one cleavable moiety. Provided herein is a method of stimulating an immune response, comprising administering to a subject an mRNA encoding a first peptide chain of a masked protein and a second peptide chain of the masked protein: the first peptide chain of the masked protein comprising, in the following order: (a) a cytokine or immunogenic fragment thereof as a therapeutic protein, (b) a first cleavable moiety, (c) a masking moiety, (d) a second cleavable moiety, and (e) a first Fc peptide comprising a first CH2 region and a first CH3 region; and the second peptide chain of the masked peptide comprising a second Fc peptide comprising a second CH2 region and a second CH3 region; wherein the first1014920-5746-9564.2Attorney Docket No. 131986-6001cleavable moiety, and optionally the second cleavable moiety, are inert in the mammalian liver and spleen, and wherein the cytokine or the immunogenic fragment is activated upon cleavage of the first cleavable moiety and optionally the second cleavable moiety.

[0443] Provided herein is a method of treating cancer, comprising administering to a subject with cancer an mRNA encoding a masked protein, the masked protein comprising: (a) a cytokine or an immunogenic fragment thereof as a therapeutic protein; (b) a masking moiety; (c) at least one cleavable moiety; and (d) a half-life extending moiety; wherein the at least one cleavable moiety is inert in mammalian liver and spleen, and wherein the cytokine or immunogenic fragment is activated upon cleavage of the at least one cleavable moiety.Provided herein is a method of treating cancer, comprising administering to a subject with cancer an mRNA encoding a first peptide chain of a masked protein and a second peptide chain of the masked protein: the first peptide chain of the masked protein comprising, in the following order: (a) a cytokine or immunogenic fragment thereof as a therapeutic protein, (b) a first cleavable moiety, (c) a masking moiety, (d) a second cleavable moiety, and (e) a first Fc peptide comprising a first CH2 region and a first CH3 region; and the second peptide chain of the masked peptide comprising a second Fc peptide comprising a second CH2 region and a second CH3 region; wherein the first cleavable moiety, and optionally the second cleavable moiety, are inert in the mammalian liver and spleen, and wherein the cytokine or the immunogenic fragment is activated upon cleavage of the first cleavable moiety and optionally the second cleavable moiety.

[0444] The following list of embodiments is non-limiting and merely an example of the scope of the subject matter disclosed herein.

[0445] Embodiment 1. An mRNA encoding a masked protein, the masked protein comprising:(a) a therapeutic protein;(b) a masking moiety;(c) at least one cleavable moiety; and(d) a half-life extending moiety;wherein the at least one cleavable moiety is inert in mammalian liver and spleen, and wherein the therapeutic protein is activated upon cleavage of the at least one cleavable moiety.

[0446] Embodiment 2. The mRNA of Embodiment 1, wherein the therapeutic protein is a cytokine.1024920-5746-9564.2Attorney Docket No. 131986-6001

[0447] Embodiment 3. The mRNA of Embodiment 2, wherein the cytokine is a pro-inflammatory cytokine.

[0448] Embodiment 4. The mRNA of Embodiment 3, wherein the pro-inflammatory cytokine is selected from IL-ip, IL-6, IL-8, IL-12, IL-17, IL-18, IFN- a, IFN- y, and TNF-a.

[0449] Embodiment 5. The mRNA of Embodiment 2, wherein the cytokine is an antiinflammatory cytokine.

[0450] Embodiment 6. The mRNA of Embodiment 5, wherein the anti-inflammatory cytokine is selected from IL-IRA, IL-4, IL-6, IL-10, IL-11, IL-13, IL-35, and TGF-p.

[0451] Embodiment 7. The mRNA of any one of Embodiments 1-6, wherein the masking moiety is selected from a single-chain antibody (e.g., an scFv, VH, VHH, etc.), an extracellular domain-based masking moiety, a protein aptamer-based masking moiety, and a steric masking moiety.

[0452] Embodiment 8. The mRNA of Embodiment 7, wherein the single-chain antibody is an scFv comprising a heavy chain variable sequence and a light chain variable sequence of briakinumab, infliximab, etanercept, adalimumab, certolizumab, golimumab, tocilizumab, siltuximab, sarilumab, olokizumab, sirukumab, ustekinumab, tildrakizumab, guselkumab, BL655066, LY3074828, secukinumab, ixekizumab, brodalumab, CNTO6785, bimekkizumab, or SCH-900117.

[0453] Embodiment 9. The mRNA of any one of Embodiments 1-8, wherein the halflife extending moiety comprises an immunoglobulin (IgG) or an Fc domain thereof, a serum albumin, a single-chain antibody that binds to a serum albumin, a single-chain antibody, a hexa-hat glutathione S-transferase (GST), a glutathione affinity moiety, a calmodulin-binding peptide (CGP), a strep-tag, a cellulose binding domain, a maltose binding protein, an s-peptide tag, a chitin binding tag, an immune-reactive epitope, or an epitope tag.

[0454] Embodiment 10. The mRNA of any one of Embodiments 1-9, wherein the halflife extending moiety increases serum half-life of the masked protein by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the serum half-life of an otherwise identical masked protein without the half-life extending moiety.

[0455] Embodiment 11. The mRNA of any one of Embodiments 1-10, wherein the at least one cleavable moiety is cleavable by proteases found in a tumor or in an infection site.1034920-5746-9564.2Attorney Docket No. 131986-6001

[0456] Embodiment 12. The mRNA of any one of Embodiments 1-11, wherein the at least one cleavable moiety can be cleaved by MMP2, MMP9, MP-ST1, uPA, or any combination thereof.

[0457] Embodiment 13. The mRNA of any one of Embodiments 1-12, wherein the at least one cleavable moiety comprises 3-25 amino acids, 3-20 amino acids, 3-15 amino acids, 3-12 amino acids, 4-25 amino acids, 4-20 amino acids, 4-15 amino acids, 4-12 amino acids, 5-25 amino acids, 5-20 amino acids, 5-15 amino acids, 5-12 amino acids, 6-25 amino acids, 6-20 amino acids, 6-15 amino acids, 6-12 amino acids, 7-25 amino acids, 7-20 amino acids, 7-15 amino acids, 7-12 amino acids, 8-25 amino acids, 8-20 amino acids, 8-15 amino acids, or 8-12 amino acids.

[0458] Embodiment 14. The mRNA of any one of Embodiments 1-13, wherein the at least one cleavable moiety comprises a consensus sequence comprising (i) a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid; (ii) a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid; or (ii) both a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid and a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid.

[0459] Embodiment 15. The mRNA of any one of Embodiments 1-14, wherein the at least one cleavable moiety comprises 3 or fewer negatively charged amino acids; 2 or more special amino acids; 2 or more hydrophobic amino acids; 2 or more polar amino acids; 1 or more positive amino acids; or any combination thereof.

[0460] Embodiment 16. The mRNA of any one of Embodiments 1-15, wherein the at least one cleavable moiety comprises or consists of an amino acid sequence selected from PWGLSGRS (SEQ ID NO: 7), DHQSRSGPWGLL (SEQ ID NO: 8), and QNQALRSA (SEQ ID NO: 9).

[0461] Embodiment 17. An mRNA encoding a first peptide chain of a masked protein and a second peptide chain of the masked protein;the first peptide chain of the masked peptide comprising, in the following order:(a) a therapeutic protein,(b) a first cleavable moiety,(c) a masking moiety,(d) a second cleavable moiety, and1044920-5746-9564.2Attorney Docket No. 131986-6001(e) a first IgG Fc domain; andthe second peptide chain of the masked peptide comprising a second IgGFc domain.

[0462] Embodiment 18. The mRNA of Embodiment 17, wherein the therapeutic protein is a cytokine.

[0463] Embodiment 19. The mRNA of Embodiment 18, wherein the cytokine is a pro-inflammatory cytokine.

[0464] Embodiment 20. The mRNA of Embodiment 19, wherein the pro-inflammatory cytokine is selected from IL-ip, IL-6, IL-8, IL-12, IL-17, IL-18, IFN- a, IFN- y, and TNF-a.

[0465] Embodiment 21. The mRNA of Embodiment 18, wherein the cytokine is an antiinflammatory cytokine.

[0466] Embodiment 22. The mRNA of Embodiment 21, wherein the anti-inflammatory cytokine is selected from IL-IRA, IL-4, IL-6, IL-10, IL-11, IL-13, IL-35, and TGF-p.

[0467] Embodiment 23. The mRNA of any one of Embodiments 17-22, wherein the masking moiety is selected from a single-chain antibody (e.g., an scFv, VH, VHH, etc.), an extracellular domain-based masking moiety, a protein aptamer-based masking moiety, and a steric masking moiety.

[0468] Embodiment 24. The mRNA of Embodiment 23, wherein the single-chain antibody is an scFv comprising a heavy chain variable sequence and a light chain variable sequence of briakinumab, infliximab, etanercept, adalimumab, certolizumab, golimumab, tocilizumab, siltuximab, sarilumab, olokizumab, sirukumab, ustekinumab, tildrakizumab, guselkumab, BL655066, LY3074828, secukinumab, ixekizumab, brodalumab, CNTO6785, bimekkizumab, or SCH-900117.

[0469] Embodiment 25. The mRNA of any one of Embodiments 17-24, wherein the first cleavable moiety and the second cleavable moiety each comprise or consist of the same amino acid sequence.

[0470] Embodiment 26. The mRNA of any one of Embodiments 17-24, wherein the first cleavable moiety and the second cleavable moiety each independently comprise or consist of a different amino acid sequence.

[0471] Embodiment 27. The mRNA of any one of Embodiments 17-26, wherein the first cleavable moiety and the second cleavable moiety are each independently cleavable by proteases found in a tumor or in an infection site.1054920-5746-9564.2Attorney Docket No. 131986-6001

[0472] Embodiment 28. The mRNA of any one of Embodiments 17-27, wherein the first cleavable moiety and the second cleavable moiety are each independently cleavable by MMP2, MMP9, MP-ST1, uPA, or any combination thereof.

[0473] Embodiment 29. The mRNA of any one of Embodiments 17-28, wherein the first cleavable moiety and the second cleavable moiety each independently comprise or consist of 3-25 amino acids, 3-20 amino acids, 3-15 amino acids, 3-12 amino acids, 4-25 amino acids, 4-20 amino acids, 4-15 amino acids, 4-12 amino acids, 5-25 amino acids, 5-20 amino acids, 5-15 amino acids, 5-12 amino acids, 6-25 amino acids, 6-20 amino acids, 6-15 amino acids, 6-12 amino acids, 7-25 amino acids, 7-20 amino acids, 7-15 amino acids, 7-12 amino acids, 8-25 amino acids, 8-20 amino acids, 8-15 amino acids, or 8-12 amino acids.

[0474] Embodiment 30. The mRNA of any one of Embodiments 17-29, wherein the first cleavable moiety and the second cleavable moiety each independently comprise a consensus sequence comprising (i) a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid; (ii) a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid; or (ii) both a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid and a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid.

[0475] Embodiment 31. The mRNA of any one of Embodiments 17-30, wherein the first cleavable moiety and the second cleavable moiety each independently comprise 3 or fewer negatively charged amino acids; 2 or more special amino acids; 2 or more hydrophobic amino acids; 2 or more polar amino acids; 1 or more positive amino acids; or any combination thereof.

[0476] Embodiment 32. The mRNA of any one of Embodiments 17-31, wherein the first cleavable moiety and the second cleavable moiety each independently comprise or consist of an amino acid sequence selected from PWGLSGRS (SEQ ID NO: 7), DHQSRSGPWGLL (SEQ ID NO: 8), and QNQALRSA (SEQ ID NO: 9).

[0477] Embodiment 33. The mRNA of any one of Embodiments 17-32, wherein the first cleavable moiety and the second cleavable moiety are each independently inert in mammalian liver and spleen.

[0478] Embodiment 34. The mRNA of any one of Embodiments 17-33, wherein the first IgG Fc domain and the second IgG Fc domain are each independently selected from an IgGl Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, and an IgG4 Fc domain.1064920-5746-9564.2Attorney Docket No. 131986-6001

[0479] Embodiment 35. The mRNA of any one of Embodiments 17-34, wherein the first IgG Fc domain comprises a knob mutation and the second IgG Fc domain comprises a hole mutation; or wherein the first IgG Fc domain comprises a hole mutation and the second IgGFc domain comprises a knob mutation.

[0480] Embodiment 36. The mRNA of any one of Embodiments 17-35, wherein the wherein the first IgG Fc domain and the second IgG Fc domain are each independently comprise or consist of an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.

[0481] Embodiment 37. An mRNA comprising a nucleic acid sequence comprising or consisting of any one of SEQ ID NOs: 151-153 or encoding a masked protein comprising an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 135, 136, or 138-148.

[0482] Embodiment 38. The mRNA of any one of Embodiments 1-37, wherein the mRNA comprises at least one chemical modification.

[0483] Embodiment 39. The mRNA of Embodiment 38, wherein the at least one chemical modification comprises a 1 -methylpseudouridine, pseudouridine, 5 -methylcytidine, 5-methyluridine, or any combination thereof.

[0484] Embodiment 40. The mRNA of any one of Embodiments 1-39, wherein all uracil nucleotides in the mRNA are chemically modified.

[0485] Embodiment 41. The mRNA of any one of Embodiments 1-40, wherein the mRNA comprises a 5' untranslated region (UTR), wherein the 5' UTR comprises a nucleotide sequence with at least 90% sequence identity to a nucleotide sequence selected from SEQ ID NOs: 15-55.

[0486] Embodiment 42. The mRNA of Embodiment 41, wherein the 5' UTR comprises a nucleotide sequence selected from SEQ ID NOs: 15-55.

[0487] Embodiment 43. The mRNA of any one of Embodiments 1-42, wherein the mRNA comprises a 3' untranslated region (UTR), wherein the 3' UTR comprises a nucleotide sequence with at least 90% sequence identity to a nucleotide sequence selected from SEQ ID NOs: 56-80.

[0488] Embodiment 44. The mRNA of Embodiment 43, wherein the 3' UTR comprises a nucleotide sequence selected from SEQ ID NOs: 56-80.1074920-5746-9564.2Attorney Docket No. 131986-6001

[0489] Embodiment 45. The mRNA of any one of Embodiments 1-44, wherein the mRNA comprises one or more stop codons immediately downstream from the masked protein sequence.

[0490] Embodiment 46. The mRNA of Embodiment 45, wherein the one or more stop codons comprise the nucleotide sequence UAA, UAG, UGA, UGAUGA (SEQ ID NO: 13) or UGAUAAUAG (SEQ ID NO: 14).

[0491] Embodiment 47. The mRNA of any one of Embodiments 1-46, wherein the mRNA comprises a poly adenosine (poly A) sequence comprising 20 or more consecutive adenosine nucleotides.

[0492] Embodiment 48. The mRNA of Embodiment 47, wherein the poly A sequence comprises 100 consecutive adenosine nucleotides.

[0493] Embodiment 49. The mRNA of Embodiment 47, wherein the polyA sequence comprises, in 5 '-to-3 ' order, a first nucleotide sequence comprising 30 consecutive adenosine nucleotides, an intervening sequence comprising no more than three adenosine nucleotides, and a second nucleotide sequence comprising 70 consecutive adenosine nucleotides.

[0494] Embodiment 50. The mRNA of Embodiment 47, wherein the polyA sequence comprises the nucleotide sequence of SEQ ID NO: 82.

[0495] Embodiment 51. The mRNA of any one of Embodiments 47-50, wherein the mRNA further comprises a poly cytidine (polyC) sequence comprising 20 or more consecutive cytidine nucleotides.

[0496] Embodiment 52. The mRNA of Embodiment 51, wherein the polyC sequence is downstream from the polyA sequence.

[0497] Embodiment 53. The mRNA of any one of Embodiments 1-52, wherein the mRNA comprises a 5' cap.

[0498] Embodiment 54. The mRNA of Embodiment 53, wherein the 5' cap is a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap.

[0499] Embodiment 55. A composition comprising the mRNA of any one of Embodiments 1-54 and a lipid nanoparticle.1084920-5746-9564.2Attorney Docket No. 131986-6001

[0500] Embodiment 56. The composition of Embodiment 55, wherein the lipid nanoparticle comprises an ionizable amino lipid, a neutral lipid, a sterol, and a PEG-modified lipid.

[0501] Embodiment 57. The composition of Embodiment 55 or 56, wherein the lipid nanoparticle comprises 40-55 mol% ionizable amino lipid, 5-15 mol% neutral lipid, 30-50 mol% sterol, and 0.5-5 mol% PEG-modified lipid.

[0502] Embodiment 58. The composition of Embodiment 56 or 57, wherein the ionizable amino lipid comprises a compound of Formula (I):or a salt or isomer thereof, wherein:Ri is R”M’R’ or C5-20 alkenyl;R2 and R3 are each independently selected from C1-14 alkyl and C2-14 alkenyl;R4 is -(CH2)nQ, wherein Q is OH and n is selected from 3, 4, and 5;M and M’ are each independently -OC(O)- or -C(O)O-;Rs, Re, and R7 are each H;R’ is a linear C1-12 alkyl, or C1-12 alkyl substituted with Ce-9 alkyl;R” is C3-14 alkyl;m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0503] Embodiment 59. The composition of Embodiment 56 or 57, wherein the ionizable amino lipid comprises a structure of Compound (I):OHO^N

[0504] Embodiment 60. The composition of any one of Embodiments 56-59, wherein the neutral lipid is distearoylphosphatidylcholine (DSPC).1094920-5746-9564.2Attorney Docket No. 131986-6001

[0505] Embodiment 61. The composition of any one of Embodiments 56-60, wherein the sterol is cholesterol.

[0506] Embodiment 62. The composition of any one of Embodiments 56-61, wherein the PEG-modified lipid is 1,2 dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG-DMG).

[0507] Embodiment 63. A method of delivering a therapeutic protein to a tissue of interest, comprising administering to a subject an mRNA encoding a masked protein, wherein the masked protein comprises:(a) a therapeutic protein;(b) a masking moiety;(c) at least one cleavable moiety; and(d) a half-life extending moiety;wherein the masked protein is expressed from the mRNA in the subject’s liver or spleen; and wherein the at least one cleavable moiety is cleaved in the tissue of interest, thereby delivering the therapeutic protein.

[0508] Embodiment 64. The method of Embodiment 63, wherein the tissue of interest is a tumor or infection site.

[0509] Embodiment 65. The method of Embodiment 63 or 64, wherein the subject has cancer.

[0510] Embodiment 66. The method of Embodiment 65, wherein the cancer is selected from adenocarcinoma, melanoma, colon cancer, breast cancer, lymphoma, pancreatic cancer, prostate cancer, bladder cancer, urothelial cancer, and Kaposi sarcoma.

[0511] Embodiment 67. The method of any one of Embodiments 63-66, wherein the therapeutic protein is a cytokine.

[0512] Embodiment 68. The method of Embodiment 67, wherein the cytokine is a pro-inflammatory cytokine.

[0513] Embodiment 69. The method of Embodiment 68, wherein the pro-inflammatory cytokine is selected from IL-ip, IL-6, IL-8, IL-12, IL-17, IL-18, IFN- a, IFN- y, and TNF-a.

[0514] Embodiment 70. The method of any one of Embodiments 63-69, wherein the masking moiety is selected from a single-chain antibody (e.g., an scFv, VH, VHH, etc.), an1104920-5746-9564.2Attorney Docket No. 131986-6001extracellular domain-based masking moiety, a protein aptamer-based masking moiety, and a steric masking moiety.

[0515] Embodiment 71. The method of Embodiment 70, wherein the single-chain antibody is an scFv comprising a heavy chain variable sequence and a light chain variable sequence of briakinumab, infliximab, etanercept, adalimumab, certolizumab, golimumab, tocilizumab, siltuximab, sarilumab, olokizumab, sirukumab, ustekinumab, tildrakizumab, guselkumab, BI-655066, LY3074828, secukinumab, ixekizumab, brodalumab, CNTO6785, bimekkizumab, or SCH-900117.

[0516] Embodiment 72. The method of any one of Embodiments 63-71, wherein the half-life extending moiety comprises an immunoglobulin (IgG) or an Fc domain thereof, a serum albumin, an scFv (or other single-chain antibody) that binds to a serum albumin, a hexa-hat glutathione S-transferase (GST), a glutathione affinity moiety, a calmodulin-binding peptide (CGP), a strep-tag, a cellulose binding domain, a maltose binding protein, an s-peptide tag, a chitin binding tag, an immune-reactive epitope, or an epitope tag.

[0517] Embodiment 73. The method of any one of Embodiments 63-72, wherein the half-life extending moiety increases serum half-life of the masked protein by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the serum half-life of an otherwise identical masked protein without the half-life extending moiety.

[0518] Embodiment 74. The method of any one of Embodiments 63-73, wherein the at least one cleavable moiety is cleavable by proteases found in a tumor or in an infection site.

[0519] Embodiment 75. The method of any one of Embodiments 63-74, wherein the at least one cleavable moiety can be cleaved by MMP2, MMP9, MP-ST1, uPA, or any combination thereof.

[0520] Embodiment 76. The method of any one of Embodiments 63-75, wherein the at least one cleavable moiety comprises 3-25 amino acids, 3-20 amino acids, 3-15 amino acids, 3-12 amino acids, 4-25 amino acids, 4-20 amino acids, 4-15 amino acids, 4-12 amino acids, 5-25 amino acids, 5-20 amino acids, 5-15 amino acids, 5-12 amino acids, 6-25 amino acids, 6-20 amino acids, 6-15 amino acids, 6-12 amino acids, 7-25 amino acids, 7-20 amino acids, 7-15 amino acids, 7-12 amino acids, 8-25 amino acids, 8-20 amino acids, 8-15 amino acids, or 8-12 amino acids.1114920-5746-9564.2Attorney Docket No. 131986-6001

[0521] Embodiment 77. The method of any one of Embodiments 63-76, wherein the at least one cleavable moiety comprises a consensus sequence comprising (i) a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid; (ii) a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid; or (ii) both a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid and a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid.

[0522] Embodiment 78. The method of any one of Embodiments 63-77, wherein the at least one cleavable moiety comprises 3 or fewer negatively charged amino acids; 2 or more special amino acids; 2 or more hydrophobic amino acids; 2 or more polar amino acids; 1 or more positive amino acids; or any combination thereof.

[0523] Embodiment 79. The method of any one of Embodiments 63-78, wherein the at least one cleavable moiety comprises or consists of an amino acid sequence selected from PWGLSGRS (SEQ ID NO: 7), DHQSRSGPWGLL (SEQ ID NO: 8), and QNQALRSA (SEQ ID NO: 9).

[0524] Embodiment 80. A method of stimulating an immune response, comprising administering to a subject an mRNA encoding a masked protein, the masked protein comprising:(a) a cytokine or an immunogenic fragment thereof,(b) a masking moiety,(c) at least one cleavable moiety, and(d) a half-life extending moiety;wherein the at least one cleavable moiety is inert in mammalian liver and spleen, and wherein the therapeutic protein is activated upon cleavage of the at least one cleavable moiety.

[0525] Embodiment 81. The method of Embodiment 80, wherein the cytokine is a pro-inflammatory cytokine.

[0526] 82. The method of Embodiment 81, wherein the pro-inflammatory cytokine is selected from IL-ip, IL-6, IL-8, IL- 12, IL- 17, IL- 18, IFN- a, IFN- y, and TNF-a.

[0527] Embodiment 83. The method of Embodiment 80, wherein the cytokine is an anti-inflammatory cytokine.

[0528] Embodiment 84. The method of Embodiment 83, wherein the anti-inflammatory cytokine is selected from IL-IRA, IL-4, IL-6, IL-10, IL-11, IL-13, IL-35, and TGF-p.1124920-5746-9564.2Attorney Docket No. 131986-6001

[0529] Embodiment 85. The method of any one of Embodiments 80-84, wherein the subject has cancer or an infection.

[0530] Embodiment 86. The method of Embodiment 85, wherein the cancer is selected from adenocarcinoma, melanoma, colon cancer, breast cancer, lymphoma, pancreatic cancer, prostate cancer, bladder cancer, urothelial cancer, and Kaposi sarcoma.

[0531] Embodiment 87. The method of any one of Embodiments 63-69, wherein the masking moiety is selected from a single-chain antibody (e.g., an scFv, VH, VHH, etc.), an extracellular domain-based masking moiety, a protein aptamer-based masking moiety, and a steric masking moiety.

[0532] Embodiment 88. The method of Embodiment 87, wherein the single-chain antibody is a single-chain antibody is an scFv comprising a heavy chain variable sequence and a light chain variable sequence of briakinumab, infliximab, etanercept, adalimumab, certolizumab, golimumab, tocilizumab, siltuximab, sarilumab, olokizumab, sirukumab, ustekinumab, tildrakizumab, guselkumab, BI-655066, LY3074828, secukinumab, ixekizumab, brodalumab, CNTO6785, bimekkizumab, or SCH-900117.

[0533] Embodiment 89. The method of any one of Embodiments 80-88, wherein the half-life extending moiety comprises an immunoglobulin (IgG) or an Fc domain thereof, a serum albumin, an scFv (or other single-chain antibody) that binds to a serum albumin, a hexa-hat glutathione S-transferase (GST), a glutathione affinity moiety, a calmodulin-binding peptide (CGP), a strep-tag, a cellulose binding domain, a maltose binding protein, an s-peptide tag, a chitin binding tag, an immune-reactive epitope, or an epitope tag.

[0534] Embodiment 90. The method of any one of Embodiments 80-89, wherein the half-life extending moiety increases serum half-life of the masked protein by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the serum half-life of an otherwise identical masked protein without the half-life extending moiety.

[0535] Embodiment 91. The method of any one of Embodiments 80-90, wherein the at least one cleavable moiety is cleavable by proteases found in a tumor or in an infection site.

[0536] Embodiment 92. The method of any one of Embodiments 80-91, wherein the at least one cleavable moiety can be cleaved by MMP2, MMP9, MP-ST1, uPA, or any combination thereof.1134920-5746-9564.2Attorney Docket No. 131986-6001

[0537] Embodiment 93. The method of any one of Embodiments 80-92, wherein the at least one cleavable moiety comprises 3-25 amino acids, 3-20 amino acids, 3-15 amino acids, 3-12 amino acids, 4-25 amino acids, 4-20 amino acids, 4-15 amino acids, 4-12 amino acids, 5-25 amino acids, 5-20 amino acids, 5-15 amino acids, 5-12 amino acids, 6-25 amino acids, 6-20 amino acids, 6-15 amino acids, 6-12 amino acids, 7-25 amino acids, 7-20 amino acids, 7-15 amino acids, 7-12 amino acids, 8-25 amino acids, 8-20 amino acids, 8-15 amino acids, or 8-12 amino acids.

[0538] Embodiment 94. The method of any one of Embodiments 80-93, wherein the at least one cleavable moiety comprises a consensus sequence comprising (i) a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid; (ii) a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid; or (ii) both a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid and a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid.

[0539] Embodiment 95. The method of any one of Embodiments 80-94, wherein the at least one cleavable moiety comprises 3 or fewer negatively charged amino acids; 2 or more special amino acids; 2 or more hydrophobic amino acids; 2 or more polar amino acids; 1 or more positive amino acids; or any combination thereof.

[0540] Embodiment 96. The method of any one of Embodiments 80-95, wherein the at least one cleavable moiety comprises or consists of an amino acid sequence selected from PWGLSGRS (SEQ ID NO: 7), DHQSRSGPWGLL (SEQ ID NO: 8), and QNQALRSA (SEQ ID NO: 9).

[0541] Embodiment 97. A method of treating cancer, comprising administering to a subject with cancer an mRNA encoding a first peptide chain of a masked protein and a second peptide chain of the mask protein;the first peptide chain of the masked peptide comprising, in the following order:(a) a cytokine or an immunogenic fragment thereof,(b) a first cleavable moiety,(c) a masking moiety,(d) a second cleavable moiety, and(e) a first IgG Fc domain; andthe second peptide chain of the masked peptide comprising a second IgGFc domain.1144920-5746-9564.2Attorney Docket No. 131986-6001

[0542] Embodiment 98. The method of Embodiment 97, wherein the cancer is selected from adenocarcinoma, melanoma, colon cancer, breast cancer, lymphoma, pancreatic cancer, prostate cancer, bladder cancer, urothelial cancer, and Kaposi sarcoma.

[0543] Embodiment 99. The method of Embodiment 97 or 98, wherein the cytokine is a pro-inflammatory cytokine.

[0544] Embodiment 100. The method of Embodiment 99, wherein the pro-inflammatory cytokine is selected from IL-ip, IL-6, IL-8, IL-12, IL-17, IL-18, IFN- a, IFN- y, and TNF-a.

[0545] Embodiment 101. The method of any one of Embodiments 17-22, wherein the masking moiety is selected from a single-chain antibody (e.g., an scFv, VH, VHH, etc.), an extracellular domain-based masking moiety, a protein aptamer-based masking moiety, and a steric masking moiety.

[0546] Embodiment 102. The method of Embodiment 101, wherein the single-chain antibody is an scFv comprising a heavy chain variable sequence and a light chain variable sequence of briakinumab, infliximab, etanercept, adalimumab, certolizumab, golimumab, tocilizumab, siltuximab, sarilumab, olokizumab, sirukumab, ustekinumab, tildrakizumab, guselkumab, BL655066, LY3074828, secukinumab, ixekizumab, brodalumab, CNTO6785, bimekkizumab, or SCH-900117.

[0547] Embodiment 103. The method of any one of Embodiments 97-102, wherein the first cleavable moiety and the second cleavable moiety each comprise or consist of the same amino acid sequence.

[0548] Embodiment 104. The method of any one of Embodiments 97-102, wherein the first cleavable moiety and the second cleavable moiety each independently comprise or consist of a different amino acid sequence.

[0549] Embodiment 105. The method of any one of Embodiments 97-104, wherein the first cleavable moiety and the second cleavable moiety are each independently cleavable by proteases found in a tumor or in an infection site.

[0550] Embodiment 106. The method of any one of Embodiments 97-105, wherein the first cleavable moiety and the second cleavable moiety are each independently cleavable by MMP2, MMP9, MP-ST1, uPA, or any combination thereof.

[0551] Embodiment 107. The method of any one of Embodiments 97-106, wherein the first cleavable moiety and the second cleavable moiety each independently comprise or1154920-5746-9564.2Attorney Docket No. 131986-6001consist of 3-25 amino acids, 3-20 amino acids, 3-15 amino acids, 3-12 amino acids, 4-25 amino acids, 4-20 amino acids, 4-15 amino acids, 4-12 amino acids, 5-25 amino acids, 5-20 amino acids, 5-15 amino acids, 5-12 amino acids, 6-25 amino acids, 6-20 amino acids, 6-15 amino acids, 6-12 amino acids, 7-25 amino acids, 7-20 amino acids, 7-15 amino acids, 7-12 amino acids, 8-25 amino acids, 8-20 amino acids, 8-15 amino acids, or 8-12 amino acids.

[0552] Embodiment 108. The method of any one of Embodiments 97-107, wherein the first cleavable moiety and the second cleavable moiety each independently comprise a consensus sequence comprising (i) a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid; (ii) a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid; or (ii) both a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid and a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid.

[0553] Embodiment 109. The method of any one of Embodiments 97-108, wherein the first cleavable moiety and the second cleavable moiety each independently comprise 3 or fewer negatively charged amino acids; 2 or more special amino acids; 2 or more hydrophobic amino acids; 2 or more polar amino acids; 1 or more positive amino acids; or any combination thereof.

[0554] Embodiment 110. The method of any one of Embodiments 97-109, wherein the first cleavable moiety and the second cleavable moiety each independently comprise or consist of an amino acid sequence selected from PWGLSGRS (SEQ ID NO: 7), DHQSRSGPWGLL (SEQ ID NO: 8), and QNQALRSA (SEQ ID NO: 9).

[0555] Embodiment 111. The method of any one of Embodiments 97-110, wherein the first cleavable moiety and the second cleavable moiety are each independently inert in mammalian liver and spleen.

[0556] Embodiment 112. The method of any one of Embodiments 97-111, wherein the first IgG Fc domain and the second IgG Fc domain are each independently selected from an IgGl Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, and an IgG4 Fc domain.

[0557] Embodiment 113. The method of any one of Embodiments 97-112, wherein the first IgG Fc domain comprises a knob mutation and the second IgG Fc domain comprises a hole mutation; or wherein the first IgG Fc domain comprises a hole mutation and the second IgGFc domain comprises a knob mutation.1164920-5746-9564.2Attorney Docket No. 131986-6001

[0558] Embodiment 114. The method of any one of Embodiments 97-113, wherein the wherein the first IgG Fc domain and the second IgG Fc domain are each independently comprise or consist of an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.

[0559] Embodiment 115. The method of any one of Embodiments 63-114, wherein the mRNA comprises a nucleic acid sequence comprising or consisting of any one of SEQ ID NOs: 151-153 or encodes a masked protein comprising an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 135, 136, or 138-148.

[0560] Embodiment 116. The method of any one of Embodiments 63-115, wherein the mRNA is administered to the subject via intravenous administration.

[0561] Embodiment 117. The method of any one of Embodiments 63-116, wherein the mRNA is formulated in a composition comprising a lipid nanoparticle.

[0562] Embodiment 118. The mRNA according to any one of Embodiments 1-55 or the composition according to any one of Embodiments 56-62 for use in the manufacture of a medicament for delivering a therapeutic protein to a tissue of interest.

[0563] Embodiment 119. The mRNA according to any one of Embodiments 1-55 or the composition according to any one of Embodiments 56-62 for use in the manufacture of a medicament for stimulating an immune response in a subject.

[0564] Embodiment 120. The mRNA according to any one of Embodiments 1-55 or the composition according to any one of Embodiments 56-62 for use in the manufacture of a medicament for treating cancer in a subject.

[0565] Embodiment 121. Use of the mRNA according to any one of Embodiments 1-55 or the composition according to any one of Embodiments 56-62 for delivering a therapeutic protein to a tissue of interest.

[0566] Embodiment 122. Use of the mRNA according to any one of Embodiments 1-55 or the composition according to any one of Embodiments 56-62 for stimulating an immune response in a subject.

[0567] Embodiment 123. Use of the mRNA according to any one of Embodiments 1-55 or the composition according to any one of Embodiments 56-62 for treating cancer in a subject.1174920-5746-9564.2Attorney Docket No. 131986-6001VIII. Sequences of Disclosed Masked Proteins and mRNAProtein SequencesMasking Half-LifeCleavableund ExtendingMoiety Substrate Format Chain 1 sequence Chain 2 sequence Mask sequence MoietyCRTSECCFQDPPYPDADSGSASGPRDLRC YRISSDRYECSWQYEGPTAGVSHFLRCCL SSGRCCYFAAGSATRLQFSDQAGVSVLY TVTLWVESWARNQTEKSPEVTLQLYNSV KYEPPLGDIKVSKLAGQLRMEWETPDNQ VGAEVQFRHRTPSSPWKLGDCGPQDDDT ESCLCPLEMNVAQEFQLRRRQLGSQGSS WSKWSSPVCVPPENPGGGGSGGGGSPWG LSGRSGGGGSGGGGSGGGGSIWELKKDV CRTSECCFQDPPYPDAD YWELDWYPDAPGEMWLTCDTPEEDGI SGSASGPRDLRCYRISS TWTLDQSSEVLGSGKTLTIQVKEFGDAG DRYECSWQYEGPTAGV QYTCHKGGEVLSHSLLLLHKKEDGIWST SHFLRCCLSSGRCCYFA DILKDQKEPKNKTFLRCEAKNYSGRFTC AGSATRLQFSDQAGVS WWLTTISTDLTFS VKS SRGS SDPQGVTCG VLYTVTLWVESWARN AATLSAERVRGDNKEYEYSVECQEDSAC QTEKSPEVTLQLYNSVK IL-12 HSA- rol Substrate-2 HSA PAAEESLPIEVMVDAVHKLKYENYTSSFF N / A YEPPLGDIKVSKLAGQL ECD-pi fusionIRDIIKPDPPKNLQLKPLKNSRQVEVSWEY RMEWETPDNQVGAEV PDTWSTPHSYFSLTFCVQVQGKSKREKK QFRHRTPSSPWKLGDC DRVFTDKTSATVICRKNASISVRAQDRYY GPQDDDTESCLCPLEM SSSWSEWASVPCSGGGGSGGGGSGGGGS NVAQEFQLRRRQLGSQ RNLPVATPDPGMFPCLHHSQNLLRAVSN GSSWSKWSSPVCVPPE MLQKARQTLEFYPCTSEEIDHEDITKDKT NP STVEACLPLELTKNESCLNSRETSFITNGS (SEQ ID NO: 84) CLASRKTSFMMALCLS SIYEDLKMYQVE FKTMNAKLLMDPKRQIFLDQNMLAVIDE LMQALNFNSETVPQKSSLEEPDFYKTKIK LCILLHAFRIRAVTIDRVMSYLNASGGSP WGLSGRSGGSDAHKSEVAHRFKDLGEEN FKALVLIAFAQYLQQCPFEDHVKLVNEVT EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL1184920-5746-9564.2Attorney Docket No. 131986-6001QHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDE GKASSAKQRLKCASLQKFGERAFKAWA VARLSQRFPKAEFAEVSKLVTDLTKVHTE CCHGDLLECADDRADLAKYICENQDSISS KLKECCEKPLLEKSHCIAEVENDEMPADL PSLAADFVESKDVCKNYAEAKDVFLGMF LYEYARRHPDYSWLLLRLAKTYETTLE KCCAAADPHECYAKVFDEFKPLVEEPQN LIKQNCELFEQLGEYKFQNALLVRYTKK VPQVSTPTLVEVSRNLGKVGSKCCKHPE AKRMPCAEDYLSWLNQLCVLHEKTPVS DRVTKCCTESLVNRRPCFSALEVDETYVP KEFNAETFTFHADICTLSEKERQIKKQTAL VELVKHKPKATKEQLKAVMDDFAAFVE KCCKADDKETCFAEEGKKLVAASQAALGL(SEQ ID NO: 83)CRTSECCFQDPPYPDADSGSASGPRDLRC YRISSDRYECSWQYEGPTAGVSHFLRCCL SSGRCCYFAAGSATRLQFSDQAGVSVLY TVTLWVESWARNQTEKSPEVTLQLYNSV KGGGGSPWGLSGRSGGGGSIWELKKDVY WELDWYPDAPGEMWLTCDTPEEDGIT WTLDQSSEVLGSGKTLTIQVKEFGDAGQ CRTSECCFQDPPYPDAD YTCHKGGEVLSHSLLLLHKKEDGIWSTDI SGSASGPRDLRCYRISS LKDQKEPKNKTFLRCEAKNYSGRFTCW DRYECSWQYEGPTAGVro2 IL-12 Substrate-2 HSA HSA- WLTTISTDLTFSVKSSRGSSDPQGVTCGA N / A SHFLRCCLSSGRCCYFA ECD-pi fusion ATLSAERVRGDNKEYEYSVECQEDSACP AGSATRLQFSDQAGVS AAEESLPIEVMVDAVHKLKYENYTSSFFI VLYTVTLWVESWARN RDIIKPDPPKNLQLKPLKNSRQVEVSWEY QTEKSPEVTLQLYNSVK PDTWSTPHSYFSLTFCVQVQGKSKREKK (SEQ ID NO: 86) DRVFTDKTSATVICRKNASISVRAQDRYY SSSWSEWASVPCSGGGGSGGGGSGGGGS RNLPVATPDPGMFPCLHHSQNLLRAVSN MLQKARQTLEFYPCTSEEIDHEDITKDKT STVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLS SIYEDLKMYQVE1194920-5746-9564.2Attorney Docket No. 131986-6001FKTMNAKLLMDPKRQIFLDQNMLAVIDE LMQALNFNSETVPQKSSLEEPDFYKTKIK LCILLHAFRIRAVTIDRVMSYLNASGGSP WGLSGRSGGSDAHKSEVAHRFKDLGEEN FKALVLIAFAQYLQQCPFEDHVKLVNEVT EFAKTCVADESAENCDKSLHTLFGDKLC TVATLRETYGEMADCCAKQEPERNECFL QHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDE GKASSAKQRLKCASLQKFGERAFKAWA VARLSQRFPKAEFAEVSKLVTDLTKVHTE CCHGDLLECADDRADLAKYICENQDSISS KLKECCEKPLLEKSHCIAEVENDEMPADL PSLAADFVESKDVCKNYAEAKDVFLGMF LYEYARRHPDYSWLLLRLAKTYETTLE KCCAAADPHECYAKVFDEFKPLVEEPQN LIKQNCELFEQLGEYKFQNALLVRYTKK VPQVSTPTLVEVSRNLGKVGSKCCKHPE AKRMPCAEDYLSWLNQLCVLHEKTPVS DRVTKCCTESLVNRRPCFSALEVDETYVP KEFNAETFTFHADICTLSEKERQIKKQTAL VELVKHKPKATKEQLKAVMDDFAAFVE KCCKADDKETCFAEEGKKLVAASQAALGL(SEQ ID NO: 85)IWELKKDVYVVELDWYPDAPGEMWLT ESKYGPPCPPCPAPEFEGGPS CDTPEEDGITWTLDQSSEVLGSGKTLTIQ VFLFPPKPKDTLMISRTPEVT VKEFGDAGQYTCHKGGEVLSHSLLLLHK CVWDVSQEDPEVQFNWYV KEDGIWSTDILKDQKEPKNKTFLRCEAKN DGVEVHNAKTKPREEQFNS YSGRFTCWWLTTISTDLTFSVKSSRGSSD TYRWSVLTVLHQDWLNGKFc- PQGVTCGAATLSAERVRGDNKEYEYSVE EYKCKVSNKGLPSSIEKTISK unmaskedrol4 Substrate-2 fusionIL-12-Fc Fc CQEDSACPAAEESLPIEVMVDAVHKLKY AKGQPREPQVCTLPPSQEEM N / A fusion Heterod ENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQ TKNQVSLSCAVKGFYPSDIA imerVEVSWEYPDTWSTPHSYFSLTFCVQVQG VEWESNGQPENNYKTTPPV KSKREKKDRVFTDKTSATVICRKNASISV LD SDGSFFL VSRLTVDKSRW QEGNVFSCSVMHEALHNRF RAQDRYYSSSWSEWASVPCSGGGGGGSR NLPVATPDPGMFPCLHHSQNLLRAVSNM TQKSLSLSLGK LQKARQTLEFYPCTSEEIDHEDITKDKTST (SEQ ID NO: 111)1204920-5746-9564.2Attorney Docket No. 131986-6001VEACLPLELTKNESCLNSRETSFITNGSCL ASRKTSFMMALCLSSIYEDLKMYQVEFK TMNAKLLMDPKRQIFLDQNMLA VIDELM QALNFNSETVPQKSSLEEPDFYKTKIKLCI LLHAFRIRAVTIDRVMSYLNASGPWGLSG RSGGSGPPCPPCPAPEFEGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRW SVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPCQEEMTK NQVSLWCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO: 110)GTAECPKHFQETLISGKDGPPGSGHSGSIK NCQPRKLHGSSGGSGGSGGPWGLSGRSG GSIWELKKDVYWELDWYPDAPGEMW LTCDTPEEDGITWTLDQSSEVLGSGKTLTI QVKEFGDAGQYTCHKGGEVLSHSLLLLH KKEDGIWSTDILKDQKEPKNKTFLRCEAK ESKYGPPCPPCPAPEFEGGPS NYSGRFTCWWLTTISTDLTFSVKSSRGSS VFLFPPKPKDTLMISRTPEVT DPQGVTCGAATLSAERVRGDNKEYEYSV ECQEDSACPAAEESLPIEVMVDAVHKLK CVWDVSQEDPEVQFNWYV YENYTS SFFIRDIIKPDPPKNLQLKPLKNSR DGVEVHNAKTKPREEQFNS TYRWSVLTVLHQDWLNGK GTAECPKHFQETLISGK Fc- QVEVSWEYPDTWSTPHSYFSLTFCVQVQ EYKCKVSNKGLPSSIEKTISKpeptide fusion GKSKREKKDRVFTDKTSATVICRKNASIS DGPPGSGHSGSIKNCQProl5 PM1B2 Substrate-2 Fc AKGQPREPQVCTLPPSQEEMHeterod VRAQDRYYSSSWSEWASVPCSGGGGGGS RKLHimer RNLPVATPDPGMFPCLHHSQNLLRAVSN TKNQVSLSCAVKGFYPSDIA(SEQ ID NO: 114) VEWESNGQPENNYKTTPPV MLQKARQTLEFYPCTSEEIDHEDITKDKTLD SDGSFFL VSRLTVDKSRW STVEACLPLELTKNESCLNSRETSFITNGS QEGNVFSCSVMHEALHNRF CLASRKTSFMMALCLS SIYEDLKMYQVE TQKSLSLSLGK FKTMNAKLLMDPKRQIFLDQNMLAVIDE(SEQ ID NO: 113) LMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGPWG LSGRSGGSGPPCPPCPAPEFEGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTY RWSVLTVLHQDWLNGKEYKCKVSNKG1214920-5746-9564.2Attorney Docket No. 131986-6001LPS SIEKTISKAKGQPREPQVYTLPPCQEE MTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHYTQKSLS LSLG(SEQ ID NO: 112)GMYSTAECPKHFQETLISGKDGPPGSGHS GSIKNCQPRKLHGSSGGSGGSGGPWGLS GRSGGSIWELKKDVYWELDWYPDAPGE MWLTCDTPEEDGITWTLDQSSEVLGSG KTLTIQVKEFGDAGQYTCHKGGEVLSHS LLLLHKKEDGIWSTDILKDQKEPKNKTFL RCEAKNYSGRFTCWWLTTISTDLTFSVKS SRGSSDPQGVTCGAATLSAERVRGDNKE YEYSVECQEDSACPAAEESLPIEVMVDAV HKLKYENYTSSFFIRDIIKPDPPKNLQLKP ESKYGPPCPPCPAPEFEGGPS VFLFPPKPKDTLMISRTPEVT LKNSRQVEVSWEYPDTWSTPHSYFSLTFC VQVQGKSKREKKDRVFTDKTSATVICRK CVWDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNS NASISVRAQDRYYSSSWSEWASVPCSGG TYRWSVLTVLHQDWLNGKFc- GGGGSRNLPVATPDPGMFPCLHHSQNLL GMYSTAECPKHFQETLI EYKCKVSNKGLPSSIEKTISKfusion RAVSNMLQKARQTLEFYPCTSEEIDHEDI SGKDGPPGSGHSGSIKN rol6 peptide Substrate-2 Fc AKGQPREPQVCTLPPSQEEMPM2B2 Heterod TKDKTSTVEACLPLELTKNESCLNSRETSF CQPRKLH TKNQVSLSCAVKGFYPSDIAimer ITNGSCLASRKTSFMMALCLSSIYEDLKM (SEQ ID NO: 117) YQVEFKTMNAKLLMDPKRQIFLDQNML VEWESNGQPENNYKTTPPV LD SDGSFFL VSRLTVDKSRW AVIDELMQ ALNFNSETVPQKS SLEEPDFY QEGNVFSCSVMHEALHNRF KTKIKLCILLHAFRIRAVTIDRVMSYLNAS TQKSLSLSLGK GPWGLSGRSGGSGPPCPPCPAPEFEGGPS(SEQ ID NO: 116) VFLFPPKPKDTLMISRTPEVTCVWDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRWSVLTVLHQDWLNGKEYKCKV SNKGLPSSIEKTISKAKGQPREPQVYTLPP CQEEMTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLG(SEQ ID NO: 115)GMYSTAESPKHFQETLISGKDGSSGGSGG ESKYGPPCPPCPAPEFEGGPS GMYSTAESPKHFQETLIrol7 peptide Substrate-2 Fc- SGGPWGLSGRSGGSIWELKKDVYWELD VFLFPPKPKDTLMISRTPEVT SGKDPM3B2 Fc fusionWYPDAPGEMWLTCDTPEEDGITWTLDQ CVWDVSQEDPEVQFNWYV (SEQ ID NO: 120)1224920-5746-9564.2Attorney Docket No. 131986-6001Heterod SSEVLGSGKTLTIQVKEFGDAGQYTCHKG DGVEVHNAKTKPREEQFNSimer GEVLSHSLLLLHKKEDGIWSTDILKDQKE TYRWSVLTVLHQDWLNGK PKNKTFLRCEAKNYSGRFTCWWLTTISTD EYKCKVSNKGLPSSIEKTISK LTFSVKSSRGSSDPQGVTCGAATLSAERV AKGQPREPQVCTLPPSQEEM RGDNKEYEYS VECQED S ACP AAEESLPIE TKNQVSLSCAVKGFYPSDIA VMVD AVHKLKYENYTS SFFIRDIIKPDPP VEWESNGQPENNYKTTPPV KNLQLKPLKNSRQVEVSWEYPDTWSTPH LD SDGSFFL VSRLTVDKSRW SYFSLTFCVQVQGKSKREKKDRVFTDKTS QEGNVFSCSVMHEALHNRF ATVICRKNASISVRAQDRYYSSSWSEWAS TQKSLSLSLGK VPCSGGGGGGSRNLPVATPDPGMFPCLH (SEQ ID NO: 119) HSQNLLRAVSNMLQKARQTLEFYPCTSE EIDHEDITKDKTSTVEACLPLELTKNESCL NSRETSFITNGSCLASRKTSFMMALCLSSI YEDLKMYQVEFKTMNAKLLMDPKRQIFL DQNMLAVIDELMQALNFNSETVPQKSSL EEPDFYKTKIKLCILLHAFRIRAVTIDRVM SYLNASGPWGLSGRSGGSGPPCPPCPAPE FEGGPSVFLFPPKPKDTLMISRTPEVTCW VDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRWSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPCQEEMTKNQVSLWCLVKGFYPS DIAVE WESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLG(SEQ ID NO: 118)GRISSDRYEGPPGSFSDQAGVSVLYTVGP ESKYGPPCPPCPAPEFEGGPS VFLFPPKPKDTLMISRTPEVT PGSTLQLYNSVKGSSGGSGGSGGPWGLS GRSGGSIWELKKDVYWELDWYPDAPGE CVWDVSQEDPEVQFNWYV MWLTCDTPEEDGITWTLDQSSEVLGSG DGVEVHNAKTKPREEQFNS KTLTIQVKEFGDAGQYTCHKGGEVLSHS TYRWSVLTVLHQDWLNGK GRISSDRYEGPPGSFSD Fc- LLLLHKKEDGIWSTDILKDQKEPKNKTFL EYKCKVSNKGLPSSIEKTISKrol8 peptidePM4B1 Substrate-2 fusionFc AKGQPREPQV QAGVSVLYTVGPPGST Heterod RCEAKNYSGRFTCWWLTTISTDLTFSVKS CTLPPSQEEM SRGSSDPQGVTCGAATLSAERVRGDNKE TKNQVSLSCAVKGFYPSDIA LQLYNSVKimer (SEQ ID NO: 123) YEYSVECQEDS ACP AAEESLPIE VMVD AV VEWESNGQPENNYKTTPPV HKLKYENYTSSFFIRDIIKPDPPKNLQLKP LD SDGSFFL VSRLTVDKSRW QEGNVFSCSVMHEALHNRF LKNSRQVEVSWEYPDTWSTPHSYFSLTFC VQVQGKSKREKKDRVFTDKTSATVICRK TQKSLSLSLGK(SEQ ID NO: 122) NASISVRAQDRYYSSSWSEWASVPCSGG1234920-5746-9564.2Attorney Docket No. 131986-6001GGGGSRNLPVATPDPGMFPCLHHSQNLL RAVSNMLQKARQTLEFYPCTSEEIDHEDI TKDKTSTVEACLPLELTKNESCLNSRETSF ITNGSCLASRKTSFMMALCLSSIYEDLKM YQVEFKTMNAKLLMDPKRQIFLDQNML AVIDELMQ ALNFNSETVPQKS SLEEPDFY KTKIKLCILLHAFRIRAVTIDRVMSYLNAS GPWGLSGRSGGSGPPCPPCPAPEFEGGPS VFLFPPKPKDTLMISRTPEVTCVWDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRWSVLTVLHQDWLNGKEYKCKV SNKGLPSSIEKTISKAKGQPREPQVYTLPP CQEEMTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLG(SEQ ID NO: 121)GCYRISSDRYECGPPGSFSDQAGVSVLYT VGPPGSTLQLYNSVKGSSGGSGGSGGPW GLSGRSGGSIWELKKDVYVVELDWYPDA PGEMWLTCDTPEEDGITWTLDQS SEVLG SGKTLTIQVKEFGDAGQYTCHKGGEVLS HSLLLLHKKEDGIWSTDILKDQKEPKNKT ESKYGPPCPPCPAPEFEGGPS VFLFPPKPKDTLMISRTPEVT FLRCEAKNYSGRFTCWWLTTISTDLTFSV KSSRGSSDPQGVTCGAATLSAERVRGDN CVWDVSQEDPEVQFNWYV KEYEYSVECQEDSACPAAEESLPIEVMVD DGVEVHNAKTKPREEQFNS TYRWSVLTVLHQDWLNGKFc- AVHKLKYENYTSSFFIRDIIKPDPPKNLQLEYKCKVSNKGLPSSIEKTISK GCYRISSDRYECGPPGSfusion KPLKNSRQVEVSWEYPDTWSTPHSYFSLTrol9 peptide Substrate-2 AKGQPREPQVCTLPPSQEEM FSDQAGVSVLYTVGPP PM5B1 FcHeterod FCVQVQGKSKREKKDRVFTDKTSATVIC TKNQVSLSCAVKGFYPSDIA GSTLQLYNSVK imer RKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 126)GGGGGGSRNLPVATPDPGMFPCLHHSQN VEWESNGQPENNYKTTPPV LLRAVSNMLQKARQTLEFYPCTSEEIDHE LD SDGSFFL VSRLTVDKSRW QEGNVFSCSVMHEALHNRF DITKDKTSTVEACLPLELTKNESCLNSRET TQKSLSLSLGK SFITNGSCLASRKTSFMMALCLSSIYEDLK(SEQ ID NO: 125) MYQVEFKTMNAKLLMDPKRQIFLDQNM L AVIDELMQ ALNFNSETVPQKS SLEEPDF YKTKIKLCILLHAFRIRAVTIDRVMSYLNA SGPWGLSGRSGGSGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQ1244920-5746-9564.2Attorney Docket No. 131986-6001EDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRWSVLTVLHQDWLNGKEYKCKV SNKGLPSSIEKTISKAKGQPREPQVYTLPP CQEEMTKNQVSLWCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLG(SEQ ID NO: 124)GFSDQAGVSVLYTVGPPGSTLQLYNSVK GSSGGSGGSGGPWGLSGRSGGSIWELKK DVYWELDWYPDAPGEMWLTCDTPEE DGITWTLDQSSEVLGSGKTLTIQVKEFGD AGQYTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKNKTFLRCEAKNYSGRFT CWWLTTISTDLTFSVKSSRGSSDPQGVTC GAATLSAERVRGDNKEYEYSVECQEDSA CPAAEESLPffiVMVD AVHKLKYENYTS SF ESKYGPPCPPCPAPEFEGGPS FIRDIIKPDPPKNLQLKPLKNSRQVEVSWE VFLFPPKPKDTLMISRTPEVT YPDTWSTPHSYFSLTFCVQVQGKSKREK CVWDVSQEDPEVQFNWYV KDRVFTDKTSATVICRKNASISVRAQDRY DGVEVHNAKTKPREEQFNS YSSSWSEWASVPCSGGGGGGSRNLPVAT TYRWSVLTVLHQDWLNGKFc- PDPGMFPCLHHSQNLLRAVSNMLQKARQ EYKCKVSNKGLPSSIEKTISK GFSDQAGVSVLYTVGP ro20 peptide Substrate-2 fusionPM6B1 Fc TLEFYPCTSEEIDHEDITKDKTSTVEACLP AKGQPREPQVCTLPPSQEEM PGSTLQLYNSVK Heterod LELTKNESCLNSRETSFITNGSCLASRKTS TKNQVSLSCAVKGFYPSDIA (SEQ ID NO: 129) imer FMMALCLSSIYEDLKMYQVEFKTMNAKL VEWESNGQPENNYKTTPPV LMDPKRQIFLDQNMLAVIDELMQALNFN LD SDGSFFL VSRLTVDKSRW SETVPQKSSLEEPDFYKTKIKLCILLHAFRI QEGNVFSCSVMHEALHNRF TQKSLSLSLGK RAVTIDRVMSYLNASGPWGLSGRSGGSG(SEQ ID NO: 128) PPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRWSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPCQEEMTKNQVSLW CLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLG(SEQ ID NO: 127)1254920-5746-9564.2Attorney Docket No. 131986-6001GFSDQAGVSVLYTVGPPGSEVTLQLYNS VKGSSGGSGGSGGPWGLSGRSGGSIWEL KKDVYWELDWYPDAPGEMWLTCDTP EEDGITWTLDQSSEVLGSGKTLTIQVKEF GDAGQYTCHKGGEVLSHSLLLLHKKEDG IWSTDILKDQKEPKNKTFLRCEAKNYSGR FTCWWLTTISTDLTFSVKSSRGSSDPQGV TCGAATLSAERVRGDNKEYEYSVECQED SACPAAEESLPIEVMVDAVHKLKYENYTS ESKYGPPCPPCPAPEFEGGPS SFFIRDIIKPDPPKNLQLKPLKNSRQVEVS VFLFPPKPKDTLMISRTPEVT WEYPDTWSTPHSYFSLTFCVQVQGKSKR CVWDVSQEDPEVQFNWYV EKKDRVFTDKTSATVICRKNASISVRAQD DGVEVHNAKTKPREEQFNS TYRWSVLTVLHQDWLNGKFc- RYYSSSWSEWASVPCSGGGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKA EYKCKVSNKGLPSSIEKTISK GFSDQAGVSVLYTVGPro21 peptide Substrate-2 fusionPM7B1 Fc RQTLEFYPCTSEEIDHEDITKDKTSTVEAC AKGQPREPQVCTLPPSQEEM PGSEVTLQLYNSVK Heterodimer LPLELTKNESCLNSRETSFITNGSCLASRK TKNQVSLSCAVKGFYPSDIA (SEQ ID NO: 132) TSFMMALCLSSIYEDLKMYQVEFKTMNA VEWESNGQPENNYKTTPPV KLLMDPKRQIFLDQNMLAVIDELMQALN LD SDGSFFL VSRLTVDKSRW FNSETVPQKSSLEEPDFYKTKIKLCILLHA QEGNVFSCSVMHEALHNRF FRIRAVTIDRVMSYLNASGPWGLSGRSGG TQKSLSLSLGK SGPPCPPCPAPEFEGGPSVFLFPPKPKDTL (SEQ ID NO: 131) MISRTPEVTCVWDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRWSVLT VLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPCQEEMTKNQVS LWCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO: 130)IWELKKDVYVVELDWYPDAPGEMWLT ESKYGPPCPPCPAPEFEGGPS CDTPEEDGITWTLDQSSEVLGSGKTLTIQ VFLFPPKPKDTLMISRTPEVT VKEFGDAGQYTCHKGGEVLSHSLLLLHK CVWDVSQEDPEVQFNWYV KEDGIWSTDILKDQKEPKNKTFLRCEAKNunmasked Fc- DGVEVHNAKTKPREEQFNSro3 fusion YSGRFTCWWLTTISTDLTFSVKSSRGSSD TYRWSVLTVLHQDWLNGKIL-12-Fc NSUB Fc PQGVTCGAATLSAERVRGDNKEYEYSVE EYKCKVSNKGLPSSIEKTISK N / Afusion Heterodimer CQEDSACPAAEESLPIEVMVDAVHKLKY AKGQPREPQVCTLPPSQEEM ENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQ TKNQVSLSCAVKGFYPSDIA VEVSWEYPDTWSTPHSYFSLTFCVQVQG VEWESNGQPENNYKTTPPVKSKREKKDRVFTDKTSATVICRKNASISV LD SDGSFFL VSRLTVDKSRW1264920-5746-9564.2Attorney Docket No. 131986-6001RAQDRYYSSSWSEWASVPCSGGGGSGGG QEGNVFSCSVMHEALHNRF GSGGGGSRVIPVSGPARCLSQSRNLLKTT TQKSLSLSLGK DDMVKTAREKLKHYSCTAEDIDHEDITR (SEQ ID NO: 134) DQTSTLKTCLPLELHKNESCLATRETSSTT RGSCLPPQKTSLMMTLCLGSIYEDLKMY QTEFQAINAALQNHNHQQIILDKGMLVAI DELMQSLNHNGETLRQKPPVGEADPYRV KMKLCILLHAFSTRVVTINRVMGYLSSAG GGGSGGSGGGGSESKYGPPCPPCPAPEFE GGPSVFLFPPKPKDTLMISRTPEVTCVW DVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRWSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQV YTLPPCQEEMTKNQVSLWCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLG(SEQ ID NO: 133)IWELKKDVYVVELDWYPDAPGEMWLT CDTPEEDGITWTLDQSSEVLGSGKTLTIQ VKEFGDAGQYTCHKGGEVLSHSLLLLHK KEDGIWSTDILKDQKEPKNKTFLRCEAKN QSVLTQPPSVSGAPGQR VTISCSGSRSNIGSNTVK YSGRFTCWWLTTISTDLTFSVKSSRGSSD ESKYGPPCPPCPAPEFEGGPS WYQQLPGTAPKLLIYY PQGVTCGAATLSAERVRGDNKEYEYSVE VFLFPPKPKDTLMISRTPEVT CQEDSACPAAEESLPIEVMVDAVHKLKY NDQRPSGVPDRFSGSKS CVWDVSQEDPEVQFNWYV GTSASLAITGLQAEDEA ENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQ DGVEVHNAKTKPREEQFNS DYYCQSYDRYTHPALL VEVSWEYPDTWSTPHSYFSLTFCVQVQG TYRWSVLTVLHQDWLNGKFc- FGTGTKVTVLGGGGSG Bria KSKREKKDRVFTDKTSATVICRKNASISV EYKCKVSNKGLPSSIEKTISKkinum fusionro4 NSUB AKGQPREPQVCTLPPSQEEM SGGSGGGGSQVQLVES Fc RAQDRYYSSSWSEWASVPCSGGGGSGGGab scFv Heterod GGGVVQPGRSLRLSCA GSGGGGSRVIPVSGPARCLSQSRNLLKTT TKNQVSLSCAVKGFYPSDIAimer DDMVKTAREKLKHYSCTAEDIDHEDITR ASGFTFSSYGMHWVRQ VEWESNGQPENNYKTTPPV DQTSTLKTCLPLELHKNESCLATRETSSTT APGKGLEWVAFIRYDG LD SDGSFFL VSRLTVDKSRW RGSCLPPQKTSLMMTLCLGSIYEDLKMY QEGNVFSCSVMHEALHNRF SNKYYADSVKGRFTISR DNSKNTLYLQMNSLRA QTEFQAINAALQNHNHQQIILDKGMLVAI TQKSLSLSLGK EDTAVYYCKTHGSHDN DELMQSLNHNGETLRQKPPVGEADPYRV (SEQ ID NO: 136)WGQGTMVTVSS KMKLCILLHAFSTRVVTINRVMGYLSSAG (SEQ ID NO: 137) GGGSGGGGSGGSGSGGGGSGGGGSGGSG SGGGGSGGGSQSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLI1274920-5746-9564.2Attorney Docket No. 131986-6001YYNDQRPSGVPDRFSGSKSGTSASLAITG LQAEDEADYYCQSYDRYTHPALLFGTGT KVTVLGGGGSGSGGSGGGGSQVQLVESG GGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAFIRYDGSNKYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCKTHGSHDNWGQGTMVTVSSGGGG SESKYGPPCPPCPAPEFEGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRW SVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPCQEEMTK NQVSLWCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO: 135)IWELKKDVYVVELDWYPDAPGEMWLT CDTPEEDGITWTLDQSSEVLGSGKTLTIQ VKEFGDAGQYTCHKGGEVLSHSLLLLHK KEDGIWSTDILKDQKEPKNKTFLRCEAKN QSVLTQPPSVSGAPGQR YSGRFTCWWLTTISTDLTFSVKSSRGSSD VTISCSGSRSNIGSNTVK PQGVTCGAATLSAERVRGDNKEYEYSVE ESKYGPPCPPCPAPEFEGGPS WYQQLPGTAPKLLIYY VFLFPPKPKDTLMISRTPEVT CQEDSACPAAEESLPIEVMVDAVHKLKY NDQRPSGVPDRFSGSKS CVWDVSQEDPEVQFNWYV ENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQ GTSASLAITGLQAEDEA DGVEVHNAKTKPREEQFNS VEVSWEYPDTWSTPHSYFSLTFCVQVQG DYYCQSYDRYTHPALL TYRWSVLTVLHQDWLNGKFc- KSKREKKDRVFTDKTSATVICRKNASISV EYKCKVSNKGLPSSIEKTISK FGTGTKVTVLGGGGSG Briakinum fusionro5 Substrate-2 RAQDRYYSSSWSEWASVPCSGGGGSGGGFc AKGQPREPQVCTLPPSQEEM SGGSGGGGSQVQLVES ab scFv Heterod GSGGGGSRVIPVSGPARCLSQSRNLLKTT TKNQVSLSCAVKGFYPSDIA GGGVVQPGRSLRLSCA imer DDMVKTAREKLKHYSCTAEDIDHEDITR ASGFTFSSYGMHWVRQ DQTSTLKTCLPLELHKNESCLATRETSSTT VEWESNGQPENNYKTTPPV APGKGLEWVAFIRYDG RGSCLPPQKTSLMMTLCLGSIYEDLKMY LD SDGSFFL VSRLTVDKSRW QEGNVFSCSVMHEALHNRF SNKYYADSVKGRFTISR QTEFQAINAALQNHNHQQIILDKGMLVAI DNSKNTLYLQMNSLRA TQKSLSLSLGK DELMQSLNHNGETLRQKPPVGEADPYRV EDTAVYYCKTHGSHDN(SEQ ID NO: 139) KMKLCILLHAFSTRVVTINRVMGYLSSAG WGQGTMVTVSS GGGSGGGGSGGSGSGGGGSGGGGSGGSP (SEQ ID NO: 137) WGLSGRSGGSQSVLTQPPSVSGAPGQRV TISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAIT1284920-5746-9564.2Attorney Docket No. 131986-6001GLQAEDEADYYCQSYDRYTHPALLFGTG TKVTVLGGGGSGSGGSGGGGSQVQLVES GGGVVQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAFIRYDGSNKYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCKTHGSHDNWGQGTMVTVSSGGG GSESKYGPPCPPCPAPEFEGGPSVFLFPPK PKDTLMISRTPEVTCVWDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPCQEEMTK NQVSLWCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO: 138)IWELKKDVYVVELDWYPDAPGEMWLT CDTPEEDGITWTLDQSSEVLGSGKTLTIQ VKEFGDAGQYTCHKGGEVLSHSLLLLHK KEDGIWSTDILKDQKEPKNKTFLRCEAKN QSVLTQPPSVSGAPGQR YSGRFTCWWLTTISTDLTFSVKSSRGSSD PQGVTCGAATLSAERVRGDNKEYEYSVE VTISCSGSRSNIGSNTVK ESKYGPPCPPCPAPEFEGGPS CQEDSACPAAEESLPIEVMVDAVHKLKY VFLFPPKPKDTLMISRTPEVT WYQQLPGTAPKLLIYY ENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQ CVWDVSQEDPEVQFNWYV NDQRPSGVPDRFSGSKS GTSASLAITGLQAEDEA VEVSWEYPDTWSTPHSYFSLTFCVQVQG DGVEVHNAKTKPREEQFNS DYYCQSYDRYTHPALLFc- KSKREKKDRVFTDKTSATVICRKNASISV TYRWSVLTVLHQDWLNGKFGBriakinum fusion RAQDRYYSSSWSEWASVPCSGGGGSGGG EYKCKVSNKGLPSSIEKTISK TGTKVTVLGGGGSG ro24 Substrate-2 GSGGGGSRVIPVSGPARCLSQSRNLLKTT AKGQPREPQVCTLPPSQEEM SGGSGGGGSQVQLVES ab scFv Fc Heterod DDMVKTAREKLKHYSCTAEDIDHEDITR TKNQVSLSCAVKGFYPSDIA GGGVVQPGRSLRLSCA imer ASGFTFSSYGMHWVRQ DQTSTLKTCLPLELHKNESCLATRETSSTT VEWESNGQPENNYKTTPPV APGKGLEWVAFIRYDG RGSCLPPQKTSLMMTLCLGSIYEDLKMY LD SDGSFFL VSRLTVDKSRW SNKYYADSVKGRFTISR QTEFQAINAALQNHNHQQIILDKGMLVAI QEGNVFSCSVMHEALHNRF DNSKNTLYLQMNSLRA TQKSLSLSLGK DELMQSLNHNGETLRQKPPVGEADPYRV EDTAVYYCKTHGSHDN (SEQ ID NO: 141) KMKLCILLHAFSTRVVTINRVMGYLSSAG WGQGTMVTVSS GGGSGGGGSGGSPWGLSGRSGGSQSVLT (SEQ ID NO: 137) QPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVPDRFS GSKSGTSASLAITGLQAEDEADYYCQSYD RYTHPALLFGTGTKVTVLGGGGSGSGGS1294920-5746-9564.2Attorney Docket No. 131986-6001GGGGSQVQLVESGGGWQPGRSLRLSCA ASGFTFSSYGMHWVRQAPGKGLEWVAFI RYDGSNKYYADSVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCKTHGSHDNWG QGTMVTVSSGGSPWGLSGRSGGSGPPCPP CPAPEFEGGPSVFLFPPKPKDTLMISRTPE VTCVWDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRWSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQVYTLPPCQEEMTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG(SEQ ID NO: 140)IWELKKDVYVVELDWYPDAPGEMWLT CDTPEEDGITWTLDQSSEVLGSGKTLTIQ VKEFGDAGQYTCHKGGEVLSHSLLLLHK KEDGIWSTDILKDQKEPKNKTFLRCEAKN YSGRFTCWWLTTISTDLTFSVKSSRGSSD QSVLTQPPSVSGAPGQR PQGVTCGAATLSAERVRGDNKEYEYSVE VTISCSGSRSNIGSNTVK CQEDSACPAAEESLPIEVMVDAVHKLKY ESKYGPPCPPCPAPEFEGGPS WYQQLPGTAPKLLIYY ENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQ VFLFPPKPKDTLMISRTPEVT NDQRPSGVPDRFSGSKS VEVSWEYPDTWSTPHSYFSLTFCVQVQG CVWDVSQEDPEVQFNWYV GTSASLAITGLQAEDEA KSKREKKDRVFTDKTSATVICRKNASISV DGVEVHNAKTKPREEQFNS DYYCQSYDRYTHPALL TYRWSVLTVLHQDWLNGK RAQDRYYSSSWSEWASVPCSGGGGSGGGFc- GSGGGGSRVIPVSGPARCLSQSRNLLKTT EYKCKVSNKGLPSSIEKTISK FGTGTKVTVLGGGGSG Briakinum fusionro22 Substrate-3 SGGSGGGGSQVQLVES Fc DDMVKTAREKLKHYSCTAEDIDHEDITR AKGQPREPQVCTLPPSQEEMab scFv Heterod GGGVVQPGRSLRLSCA DQTSTLKTCLPLELHKNESCLATRETSSTT TKNQVSLSCAVKGFYPSDIAimer ASGFTFSSYGMHWVRQ RGSCLPPQKTSLMMTLCLGSIYEDLKMY VEWESNGQPENNYKTTPPV APGKGLEWVAFIRYDG QTEFQAINAALQNHNHQQIILDKGMLVAI LD SDGSFFL VSRLTVDKSRW SNKYYADSVKGRFTISR QEGNVFSCSVMHEALHNRF DELMQSLNHNGETLRQKPPVGEADPYRV DNSKNTLYLQMNSLRA TQKSLSLSLGK KMKLCILLHAFSTRVVTINRVMGYLSSAG EDTAVYYCKTHGSHDN(SEQ ID NO: 143) GGGSGGGGSGGSGSGGGGSGGGGSGGSQWGQGTMVTVSS NQALRSAGGSQSVLTQPPSVSGAPGQRVT(SEQ ID NO: 137) ISCSGSRSNIGSNTVKWYQQLPGTAPKLLI YYNDQRPSGVPDRFSGSKSGTSASLAITG LQAEDEADYYCQSYDRYTHPALLFGTGT KVTVLGGGGSGSGGSGGGGSQVQLVESG GGWQPGRSLRLSCAASGFTFSSYGMHW1304920-5746-9564.2Attorney Docket No. 131986-6001VRQAPGKGLEWVAFIRYDGSNKYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCKTHGSHDNWGQGTMVTVSSGGSQ NQALRSAGGSGPPCPPCPAPEFEGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNST YRWSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPCQE EMTKNQVSLWCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLG(SEQ ID NO: 142)IWELKKDVYVVELDWYPDAPGEMWLT CDTPEEDGITWTLDQSSEVLGSGKTLTIQ VKEFGDAGQYTCHKGGEVLSHSLLLLHK KEDGIWSTDILKDQKEPKNKTFLRCEAKN YSGRFTCWWLTTISTDLTFSVKSSRGSSD PQGVTCGAATLSAERVRGDNKEYEYSVE QSVLTQPPSVSGAPGQR CQEDSACPAAEESLPIEVMVDAVHKLKY VTISCSGSRSNIGSNTVK ESKYGPPCPPCPAPEFEGGPS WYQQLPGTAPKLLIYY ENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQ VFLFPPKPKDTLMISRTPEVT VEVSWEYPDTWSTPHSYFSLTFCVQVQG NDQRPSGVPDRFSGSKS CVWDVSQEDPEVQFNWYV KSKREKKDRVFTDKTSATVICRKNASISV GTSASLAITGLQAEDEA DGVEVHNAKTKPREEQFNS RAQDRYYSSSWSEWASVPCSGGGGSGGG DYYCQSYDRYTHPALL TYRWSVLTVLHQDWLNGK GSGGGGSRVIPVSGPARCLSQSRNLLKTT Fc- EYKCKVSNKGLPSSIEKTISK FGTGTKVTVLGGGGSG Briakinum fusion DDMVKTAREKLKHYSCTAEDIDHEDITRro23 SGGSGGGGSQVQLVES Substrate- 1 Fc AKGQPREPQVCTLPPSQEEM DQTSTLKTCLPLELHKNESCLATRETSSTT GGGVVQPGRSLRLSCA ab scFv Heterod TKNQVSLSCAVKGFYPSDIAimer RGSCLPPQKTSLMMTLCLGSIYEDLKMY ASGFTFSSYGMHWVRQ VEWESNGQPENNYKTTPPV QTEFQAINAALQNHNHQQIILDKGMLVAI APGKGLEWVAFIRYDG LD SDGSFFL VSRLTVDKSRW DELMQSLNHNGETLRQKPPVGEADPYRV SNKYYADSVKGRFTISR QEGNVFSCSVMHEALHNRF DNSKNTLYLQMNSLRA KMKLCILLHAFSTRVVTINRVMGYLSSAG TQKSLSLSLGK GGGSGGGGSGGSGSGGGGSGGGGSGGSD EDTAVYYCKTHGSHDN(SEQ ID NO: 145) HQSRSGPWGLLGGSQSVLTQPPSVSGAPG WGQGTMVTVSS QRVTISCSGSRSNIGSNTVKWYQQLPGTA (SEQ ID NO: 137) PKLLIYYNDQRPSGVPDRFSGSKSGTSASL AITGLQAEDEADYYCQSYDRYTHPALLF GTGTKVTVLGGGGSGSGGSGGGGSQVQL VESGGGWQPGRSLRLSCAASGFTFSSYG MHWVRQAPGKGLEWVAFIRYDGSNKYY1314920-5746-9564.2Attorney Docket No. 131986-6001ADSVKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCKTHGSHDNWGQGTMVTVSSG GSDHQSRSGPWGLLGGSGPPCPPCPAPEF EGGPSVFLFPPKPKDTLMISRTPEVTCVW DVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRWSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQV YTLPPCQEEMTKNQVSLWCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLG(SEQ ID NO: 144)IWELKKDVYVVELDWYPDAPGEMWLT CDTPEEDGITWTLDQSSEVLGSGKTLTIQ VKEFGDAGQYTCHKGGEVLSHSLLLLHK KEDGIWSTDILKDQKEPKNKTFLRCEAKN YSGRFTCWWLTTISTDLTFSVKSSRGSSD PQGVTCGAATLSAERVRGDNKEYEYSVE QSVLTQPPSVSGAPGQR CQEDSACPAAEESLPIEVMVDAVHKLKY VTISCSGSRSNIGSNTVK ESKYGPPCPPCPAPEFEGGPS ENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQ WYQQLPGTAPKLLIYY VEVSWEYPDTWSTPHSYFSLTFCVQVQG VFLFPPKPKDTLMISRTPEVT NDQRPSGVPDRFSGSKS KSKREKKDRVFTDKTSATVICRKNASISV CVWDVSQEDPEVQFNWYV GTSASLAITGLQAEDEA RAQDRYYSSSWSEWASVPCSGGGGSGGG DGVEVHNAKTKPREEQFNS DYYCQSYDRYTHPALL GSGGGGSRVIPVSGPARCLSQSRNLLKTT TYRWSVLTVLHQDWLNGKFc- FGTGTKVTVLGGGGSG DDMVKTAREKLKHYSCTAEDIDHEDITR EYKCKVSNKGLPSSIEKTISKBriakinum fusionro29 Substrate-2 SGGSGGGGSQVQLVES Fc DQTSTLKTCLPLELHKNESCLATRETSSTT AKGQPREPQVCTLPPSQEEMab scFv Het GGGVVQPGRSLRLSCAerod RGSCLPPQKTSLMMTLCLGSIYEDLKMY TKNQVSLSCAVKGFYPSDIAimer ASGFTFSSYGMHWVRQ QTEFQAINAALQNHNHQQIILDKGMLVAI VEWESNGQPENNYKTTPPV APGKGLEWVAFIRYDG DELMQSLNHNGETLRQKPPVGEADPYRV LD SDGSFFL VSRLTVDKSRW SNKYYADSVKGRFTISR QEGNVFSCSVMHEALHNRF KMKLCILLHAFSTRVVTINRVMGYLSSAG DNSKNTLYLQMNSLRA TQKSLSLSLGK GGGSGGGGSGGSGSGGGGSGGGGSGGSP EDTAVYYCKTHGSHDN(SEQ ID NO: 147) WGLSGRSGGSQSVLTQPPSVSGAPGQRVWGQGTMVTVSS TISCSGSRSNIGSNTVKWYQQLPGTAPKL(SEQ ID NO: 137) LIYYNDQRPSGVPDRFSGSKSGTSASLAIT GLQAEDEADYYCQSYDRYTHPALLFGTG TKVTVLGGGGSGSGGSGGGGSQVQLVES GGGVVQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAFIRYDGSNKYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDT1324920-5746-9564.2Attorney Docket No. 131986-6001AVYYCKTHGSHDNWGQGTMVTVSSGGS PWGLSGRSGGSGPPCPPCPAPEFEGGPSVF LFPPKPKDTLMISRTPEVTCVWDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNS TYRWSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPCQ EEMTKNQVSLWCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLG(SEQ ID NO: 146)IWELKKDVYVVELDWYPDAPGEMWLT CDTPEEDGITWTLDQSSEVLGSGKTLTIQ VKEFGDAGQYTCHKGGEVLSHSLLLLHK KEDGIWSTDILKDQKEPKNKTFLRCEAKN YSGRFTCWWLTTISTDLTFSVKSSRGSSD PQGVTCGAATLSAERVRGDNKEYEYSVE CQEDSACPAAEESLPIEVMVDAVHKLKY QSVLTQPPSVSGAPGQR ENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQ VTISCSGSRSNIGSNTVK VEVSWEYPDTWSTPHSYFSLTFCVQVQG WYQQLPGTAPKLLIYY KSKREKKDRVFTDKTSATVICRKNASISV NDQRPSGVPDRFSGSKS RAQDRYYSSSWSEWASVPCSGGGGSGGG GTSASLAITGLQAEDEA GSGGGGSRVIPVSGPARCLSQSRNLLKTT DYYCQSYDRYTHPALL DDMVKTAREKLKHYSCTAEDIDHEDITR FGTGTKVTVLGGGGSGBriakinum Anti- DQTSTLKTCLPLELHKNESCLATRETSSTTro25 Substrate-2 anti-HSA HSA- N / A SGGSGGGGSQVQLVES ab scFv RGSCLPPQKTSLMMTLCLGSIYEDLKMY GGGVVQPGRSLRLSCA fusionQTEFQAINAALQNHNHQQIILDKGMLVAI ASGFTFSSYGMHWVRQ DELMQSLNHNGETLRQKPPVGEADPYRV APGKGLEWVAFIRYDG KMKLCILLHAFSTRVVTINRVMGYLSSAG SNKYYADSVKGRFTISR GGGSGGGGSGGGGSPWGLSGRSGGGGS DNSKNTLYLQMNSLRA GGGGSGGGGSQSVLTQPPSVSGAPGQRV EDTAVYYCKTHGSHDN TISCSGSRSNIGSNTVKWYQQLPGTAPKL WGQGTMVTVSS LIYYNDQRPSGVPDRFSGSKSGTSASLAIT (SEQ ID NO: 137) GLQAEDEADYYCQSYDRYTHPALLFGTG TKVTVLGGGGSGSGGSGGGGSQVQLVES GGGVVQPGRSLRLSCAASGFTFSSYGMH WVRQAPGKGLEWVAFIRYDGSNKYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSSGGS1334920-5746-9564.2Attorney Docket No. 131986-6001PWGLSGRSGGSEVQLVESGGGLVQPGNS LRLSCAASGFTFSSFGMSWVRQAPGKGL EWVSSISGSGSDTLYADSVKGRFTISRDN AKTTLYLQMNSLRPEDTAVYYCTIGGSLS RSSQGTLVTVSS(SEQ ID NO: 148)RNA Sequencesnce Name SEQ ID ORF SequenceNO:149 AUGGAGACAGACACCCUCCUGCUGUGGGUUCUGCUCCUCUGGGUGCCUGGGAGCACGGGUAUCUGGGAG CUGAAGAAAGACGUGUACGUGGUUGAGCUGGACUGGUACCCUGACGCCCCAGGCGAGAUGGUGGUGCU GACCUGCGACACCCCGGAAGAAGACGGCAUCACCUGGACUCUGGACCAGAGCAGCGAGGUCCUUGGGAG CGGCAAGACCCUGACCAUCCAGGUAAAGGAGUUCGGUGACGCCGGCCAGUACACCUGCCACAAGGGCGG CGAAGUGCUGAGCCACAGCCUGUUGCUACUCCACAAGAAGGAGGACGGCAUUUGGAGCACCGACAUCCU GAAGGACCAGAAGGAGCCCAAGAAUAAGACCUUCCUGCGGUGCGAGGCCAAGAAUUACAGCGGCCGGUU CACUUGCUGGUGGCUGACCACCAUUAGUACCGACCUGACCUUUAGUGUGAAGAGCAGCCGUGGCUCCAG CGACCCACAGGGCGUGACCUGUGGCGCAGCCACACUCUCUGCUGAGCGUGUCCGGGGCGACAACAAGGA AUACGAGUACAGCGUGGAGUGCCAGGAAGACAGCGCUUGUCCAGCCGCCGAGGAAAGCCUGCCCAUCGA GGUGAUGGUAGACGCCGUGCACAAGCUGAAGUACGAGAACUACACCAGCAGCUUCUUCAUACGGGACAU-2 CAUCAAGCCCGACCCUCCGAAGAACUUGCAACUGAAGCCCCUGAAGAACAGCCGGCAGGUCGAAGUGAG CUGGGAGUACCCCGACACCUGGAGCACUCCCCACAGCUACUUCUCACUGACCUUCUGCGUGCAGGUGCA AGGCAAGAGCAAGCGGGAGAAGAAGGAUCGGGUGUUCACCGACAAGACCUCUGCCACCGUGAUCUGCCG UAAGAACGCCAGCAUCAGCGUGCGGGCCCAGGACAGAUACUACAGCAGCAGCUGGAGCGAGUGGGCAUC GGUUCCGUGUAGCGGAGGCGGUGGAAGUGGCGGUGGCGGAAGUGGAGGCGGAGGUUCUAGGGUGAUCC CCGUGAGCGGUCCAGCUCGGUGCCUAAGCCAGAGCCGCAACCUGCUGAAGACUACCGACGACAUGGUGA AGACCGCCCGGGAGAAGCUGAAGCACUACAGCUGUACCGCAGAGGACAUCGACCACGAGGACAUCACAC GGGACCAGACGAGCACCCUCAAGACCUGCCUGCCCCUGGAGCUGCACAAGAACGAAAGCUGCCUGGCAA CACGGGAGACUAGCAGCACGACGAGAGGCAGCUGUCUGCCGCCGCAGAAGACCAGCCUGAUGAUGACCC UGUGCCUGGGUAGUAUCUACGAGGACCUGAAGAUGUACCAGACCGAGUUCCAGGCCAUCAACGCUGCCCUUCAGAACCACAAUCACCAGCAGAUUAUCCUGGAUAAGGGCAUGCUAGUGGCCAUCGACGAGCUGAUGC1344920-5746-9564.2Attorney Docket No. 131986-6001AGAGUCUGAACCAUAACGGCGAGACUCUGCGACAGAAGCCUCCUGUAGGAGAGGCCGACCCCUAUCGUG UGAAGAUGAAGCUGUGCAUCCUACUGCACGCCUUCAGCACACGGGUCGUGACGAUCAAUCGGGUGAUGG GCUACCUUUCCUCCGCAGGUGGAGGCGGAAGUGGUGGUGGUGGCUCGGGUGGUUCAGGUUCUGGUGGC GGUGGAUCAGGAGGCGGUGGUAGUGGAGGAUCUGGAGGUUCCGGUGGCGGAGGGUCAGGAGGUAGCCA AAGCGUGUUGACCCAGCCUCCAAGCGUGAGCGGCGCUCCUGGACAGAGGGUGACCAUUAGCUGUAGCGG CAGCCGGAGCAACAUAGGCAGCAACACCGUGAAGUGGUACCAGCAACUCCCUGGCACCGCCCCAAAGUU GCUGAUCUACUACAACGACCAACGCCCAUCUGGCGUGCCCGACCGGUUCAGCGGAAGCAAGAGUGGCAC CAGCGCUAGCCUGGCCAUCACCGGCUUGCAGGCCGAAGACGAAGCCGACUAUUACUGCCAGAGCUACGA CCGAUACACACACCCCGCCCUACUGUUCGGCACCGGCACCAAGGUGACAGUGUUAGGAGGUGGCGGCAG UGGUAGCGGCGGUUCAGGCGGUGGUGGAAGCCAGGUGCAGCUGGUUGAAAGCGGAGGAGGAGUGGUCC AACCUGGGCGAUCCCUACGGCUGAGCUGCGCUGCCAGCGGCUUCACCUUUAGCAGCUACGGCAUGCACU GGGUGAGACAAGCUCCGGGUAAGGGUCUGGAGUGGGUGGCCUUCAUCCGGUACGACGGCAGCAAUAAG UACUACGCCGACAGCGUGAAGGGGCGGUUUACCAUCUCACGGGACAACAGCAAGAACACCCUGUACCUG CAGAUGAACAGCCUGAGAGCCGAGGACACCGCCGUGUACUACUGUAAGACCCACGGCAGCCACGACAAC UGGGGCCAGGGCACUAUGGUGACCGUGAGUUCCGGAGGCUCCGGUGGAUCCGGAGGAGGUGGUUCAGG CGGCAGUGGCCCACCUUGUCCUCCCUGUCCCGCUCCUGAGUUUGAGGGCGGACCCUCAGUGUUCCUGUU CCCUCCUAAGCCCAAAGACACCCUUAUGAUCAGCCGGACCCCUGAGGUUACCUGCGUGGUGGUGGACGU GAGCCAGGAGGAUCCCGAGGUGCAAUUCAAUUGGUACGUGGACGGCGUGGAAGUGCAUAACGCCAAGA CCAAGCCCCGGGAGGAACAGUUCAAUAGCACCUACCGGGUGGUGAGCGUACUGACGGUGCUGCACCAGG ACUGGCUGAACGGCAAGGAGUACAAGUGCAAGGUGAGCAACAAGGGCCUCCCCAGCAGCAUCGAGAAGA CCAUCAGCAAGGCCAAGGGCCAACCACGAGAGCCCCAGGUGUAUACCCUGCCUCCGUGUCAGGAAGAGA UGACAAAGAACCAGGUGAGCCUGUGGUGCCUGGUGAAGGGCUUCUACCCCAGCGACAUCGCCGUUGAGU GGGAGAGCAACGGCCAGCCCGAGAACAACUACAAGACCACCCCUCCCGUGCUGGAUAGCGACGGCUCGU UCUUCCUGUACAGCCGCCUUACCGUGGACAAGAGCCGGUGGCAAGAGGGCAACGUGUUCAGCUGCAGCG UGAUGCACGAGGCCCUGCACAACCACUACACCCAGAAGAGCCUGUCACUGAGCUUGGGC150 AUGGAGACAGACACCCUCCUGCUGUGGGUUCUGCUCCUCUGGGUUCCCGGAAGCACGGGCAUCUGGGAG CUGAAGAAAGACGUGUACGUGGUUGAGCUGGACUGGUACCCUGACGCCCCAGGCGAGAUGGUGGUGCU GACCUGCGACACCCCGGAAGAAGACGGCAUCACCUGGACUCUGGACCAGAGCAGCGAAGUGCUCGGCAG-5 CGGCAAGACCCUGACCAUCCAGGUAAAGGAGUUUGGGGACGCCGGCCAGUACACCUGCCACAAGGGCGG CGAAGUUCUGAGCCACAGCCUGUUGCUAUUGCAUAAGAAGGAGGACGGCAUUUGGAGCACCGACAUCCU GAAGGACCAGAAGGAGCCCAAGAAUAAGACCUUCCUGCGGUGCGAGGCCAAGAAUUACAGCGGCCGGUUCACUUGCUGGUGGCUGACCACCAUUAGUACCGACCUGACCUUUAGUGUGAAGAGCAGCAGAGGCUCAAG1354920-5746-9564.2Attorney Docket No. 131986-6001CGACCCACAGGGCGUGACCUGUGGCGCAGCCACACUGAGCGCUGAGAGAGUGCGGGGCGACAACAAGGA AUACGAGUACAGCGUGGAGUGCCAGGAAGACAGCGCUUGUCCAGCCGCCGAGGAAAGCCUGCCCAUCGA GGUGAUGGUAGACGCCGUGCACAAGCUGAAGUACGAGAACUACACCAGCAGCUUCUUCAUACGGGACAU CAUCAAGCCCGACCCUCCGAAGAACUUGCAACUGAAGCCCCUGAAGAACAGCCGGCAGGUGGAAGUGAG CUGGGAGUACCCCGACACCUGGAGCACUCCCCACAGCUACUUCUCACUGACCUUCUGCGUGCAGGUGCA AGGCAAGAGCAAGCGGGAGAAGAAGGAUCGGGUGUUCACCGACAAGACCUCUGCCACCGUGAUCUGCCG UAAGAACGCCAGCAUCAGCGUGCGGGCCCAGGACAGAUACUACAGCAGCAGCUGGAGCGAGUGGGCCAG CGUGCCUUGUAGCGGCGGUGGUGGAUCUGGAGGAGGUGGAUCAGGAGGCGGCGGUUCUAGGGUGAUCC CCGUGAGCGGUCCAGCUCGGUGCCUAAGCCAAAGCCGCAACCUGCUGAAGACUACCGACGACAUGGUGA AGACCGCCCGGGAGAAGCUGAAGCACUACAGCUGUACCGCAGAGGACAUCGACCACGAGGACAUCACAC GGGACCAGACGAGCACCCUCAAGACCUGCCUGCCCCUGGAGCUGCACAAGAACGAAAGCUGCCUGGCAA CACGGGAGACUAGCAGCACAACCCGGGGUUCUUGCCUGCCGCCACAGAAGACCAGCCUGAUGAUGACCC UGUGCCUGGGUAGUAUCUACGAGGACCUGAAGAUGUACCAGACCGAGUUCCAGGCCAUCAACGCUGCCC UUCAGAACCACAAUCACCAGCAGAUUAUCCUGGAUAAGGGCAUGCUAGUGGCCAUCGACGAGCUGAUGC AGAGUCUGAACCAUAACGGCGAGACUCUGCGACAGAAACCUCCUGUAGGAGAGGCCGACCCCUAUCGUG UGAAGAUGAAGCUGUGCAUUCUGCUGCACGCUUUCAGCACACGGGUCGUGACCAUCAAUCGGGUGAUG GGCUACCUGUCAAGCGCAGGAGGAGGUGGUAGCGGAGGAGGAGGUUCCGGUGGAUCAGGAUCUGGUGG AGGAGGAAGUGGUGGAGGCGGUAGUGGCGGAUCUCCCUGGGGCCUGAGCGGAAGGUCUGGAGGAAGCC AGAGCGUGUUGACCCAGCCUCCAAGCGUGUCUGGAGCUCCAGGACAGCGGGUGACCAUUAGCUGUAGCG GCAGCCGGAGCAACAUAGGCAGCAACACCGUGAAGUGGUACCAGCAACUGCCUGGGACCGCCCCAAAGU UGCUGAUCUACUACAACGACCAACGCCCAUCUGGCGUGCCCGACCGGUUCAGCGGAAGCAAGAGUGGCA CCAGCGCAAGCCUGGCCAUCACCGGCUUGCAGGCGGAAGACGAGGCUGACUAUUACUGCCAAAGCUACG AUCGGUACACUCACCCCGCCCUACUGUUCGGCACCGGCACCAAAGUGACUGUGCUGGGCGGAGGAGGAU CCGGGAGCGGAGGUUCUGGAGGUGGAGGAUCACAAGUCCAGCUAGUUGAGUCCGGAGGCGGAGUGGUU CAACCUGGGCGAUCCCUACGGCUGAGCUGCGCUGCCAGCGGCUUCACCUUCAGCAGUUACGGUAUGCAC UGGGUGAGACAAGCACCAGGAAAGGGCCUCGAGUGGGUGGCCUUCAUCCGGUACGACGGCAGCAAUAA GUACUACGCCGACAGCGUGAAGGGGCGGUUUACCAUCUCACGGGACAACAGCAAGAACACCCUGUACCU GCAGAUGAACAGCCUGAGAGCCGAGGACACCGCCGUGUACUACUGUAAGACCCACGGCAGCCACGACAA CUGGGGCCAGGGCACUAUGGUGACCGUUAGCUCAGGAGGCUCCCCUUGGGGUCUGAGUGGUCGGUCAGG CGGGUCUGGGCCACCUUGUCCUCCCUGUCCCGCUCCUGAAUUUGAGGGCGGACCAUCAGUAUUCCUGUU CCCUCCUAAGCCCAAGGAUACCCUCAUGAUCAGCCGGACCCCUGAGGUUACCUGCGUGGUGGUCGACGUGAGCCAGGAGGACCCCGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAGGUCCACAACGCUAAGAC1364920-5746-9564.2Attorney Docket No. 131986-6001CAAGCCUCGCGAGGAACAGUUCAAUAGCACCUACCGGGUGGUGAGCGUACUGACGGUGCUGCAUCAGGA CUGGCUGAACGGCAAGGAGUACAAGUGCAAGGUGAGCAACAAGGGCCUCCCCAGCAGCAUCGAGAAGAC CAUCAGCAAGGCCAAGGGCCAACCUCGAGAGCCCCAGGUGUAUACCCUGCCUCCGUGUCAGGAAGAGAU GACAAAGAACCAGGUGAGCCUGUGGUGCCUGGUGAAGGGCUUCUACCCCAGCGACAUCGCCGUUGAGUG GGAGAGCAACGGCCAGCCCGAGAACAACUACAAGACCACCCCUCCCGUGCUGGAUAGCGACGGCUCGUU CUUCCUGUACAGCCGCCUUACCGUGGACAAGAGCCGGUGGCAAGAGGGCAACGUGUUCAGCUGCAGCGU GAUGCACGAGGCCCUGCACAACCACUACACCCAGAAGAGCCUGUCACUGAGCUUGGGC151 AUGGAGACAGACACCCUCCUGCUGUGGGUUCUGCUCCUCUGGGUGCCCGGUAGCACAGGAAUCUGGGAG CUGAAGAAAGACGUGUACGUGGUUGAGCUAGACUGGUACCCUGACGCCCCAGGCGAGAUGGUGGUGCU GACCUGCGACACCCCGGAAGAAGACGGCAUCACCUGGACUCUGGACCAGAGCAGCGAGGUGUUGGGCAG CGGCAAGACCCUGACCAUCCAGGUAAAGGAGUUCGGUGACGCCGGCCAGUACACGUGCCACAAGGGCGG CGAAGUGCUGAGCCACAGCCUGUUGCUAUUGCAUAAGAAGGAGGACGGCAUUUGGAGCACCGACAUCCU GAAGGACCAGAAGGAGCCCAAGAAUAAGACCUUCCUGCGGUGCGAGGCCAAGAAUUACAGCGGCCGGUU CACUUGCUGGUGGCUGACCACCAUUAGUACCGACCUGACCUUUAGUGUAAAGAGCAGCAGAGGGUCCAG CGACCCACAGGGCGUGACCUGUGGCGCAGCCACACUGAGCGCAGAAAGAGUGCGCGGGGACAACAAGGA AUACGAGUACAGCGUGGAGUGCCAGGAAGACAGCGCUUGUCCAGCGGCUGAGGAGAGCCUGCCCAUCGA GGUGAUGGUAGACGCCGUGCACAAGCUGAAGUACGAGAACUACACAAGCAGCUUCUUCAUACGGGACA UCAUCAAGCCCGACCCUCCGAAGAACUUGCAACUUAAGCCCCUGAAGAACAGCCGGCAGGUGGAAGUGA GCUGGGAGUACCCCGACACCUGGAGCACUCCCCACAGCUACUUCUCACUGACCUUCUGCGUGCAGGUGC-3 AAGGCAAGAGCAAGCGGGAGAAGAAGGAUCGGGUGUUCACCGACAAGACCUCUGCCACCGUGAUCUGCC GUAAGAACGCCAGCAUCAGCGUGCGGGCCCAGGACAGAUAUUACAGCAGCAGCUGGAGCGAGUGGGCUA GCGUGCCCUGUUCUGGCGGUGGCGGAUCUGGUGGAGGAGGUAGCGGAGGCGGUGGUUCUAGGGUGAUC CCCGUGAGCGGUCCAGCUCGGUGCCUAAGCCAGAGCCGCAACCUGCUGAAGACUACCGACGACAUGGUG AAGACCGCCCGGGAGAAACUGAAGCACUAUAGCUGUACCGCAGAGGAUAUCGACCACGAGGACAUCACA CGGGACCAGACGAGCACCCUCAAGACCUGCCUGCCCCUGGAGCUGCACAAGAACGAAAGCUGCCUGGCA ACACGGGAGACUAGCAGCACUACCCGUGGGAGUUGCCUGCCGCCGCAGAAGACCUCACUGAUGAUGACC CUGUGCCUGGGUAGUAUCUACGAGGACCUGAAGAUGUACCAGACCGAGUUCCAGGCCAUCAACGCUGCC CUUCAGAACCACAAUCACCAGCAGAUUAUCCUGGAUAAGGGCAUGCUAGUGGCCAUCGACGAGCUGAUG CAAAGUCUGAACCAUAACGGCGAGACUCUGCGACAGAAGCCUCCUGUAGGAGAGGCCGACCCCUAUCGU GUGAAGAUGAAGCUGUGCAUCCUGCUGCACGCCUUCAGCACACGGGUCGUGACCAUCAACCGGGUGAUG GGCUACCUGUCAAGCGCCGGAGGAUCAGGCGGUUCAGGAGGUGGUGGCAGUGGAGGCUCCGGACCACCUUGUCCUCCCUGUCCAGCCCCGGAGUUUGAGGGCGGACCCAGCGUGUUCCUGUUCCCUCCUAAGCCCAAG1374920-5746-9564.2Attorney Docket No. 131986-6001GACACCCUGAUGAUCAGCCGGACCCCUGAGGUUACCUGCGUGGUGGUGGACGUGAGCCAGGAGGAUCCC GAGGUGCAGUUCAACUGGUACGUGGACGGCGUGGAGGUGCACAACGCCAAGACCAAGCCCCGGGAGGAA CAGUUCAAUAGCACCUACCGGGUGGUGAGCGUACUGACGGUGCUGCACCAGGACUGGCUGAACGGCAAG GAGUACAAGUGCAAGGUGAGCAACAAGGGCCUCCCCAGCAGCAUCGAGAAGACCAUCAGCAAGGCCAAG GGCCAACCGCGAGAACCGCAGGUGUAUACCCUGCCUCCGUGUCAGGAAGAGAUGACAAAGAACCAGGUG AGCCUGUGGUGCCUGGUGAAGGGCUUCUACCCCAGCGACAUCGCCGUUGAGUGGGAGAGCAACGGCCAG CCCGAGAACAACUACAAGACCACCCCUCCCGUGCUGGAUAGCGACGGCUCGUUCUUCCUGUACAGCCGC CUUACCGUGGACAAGAGCCGGUGGCAAGAGGGCAACGUGUUCAGCUGCAGCGUGAUGCACGAGGCCCUG CACAACCACUACACCCAGAAGAGCCUGUCACUGAGCUUGGGA152 AUGGAGACAGACACCCUCCUGCUGUGGGUUCUGCUCCUCUGGGUUCCUGGGAGCACGGGCAUCUGGGAG CUGAAGAAAGACGUGUACGUGGUUGAGCUGGACUGGUACCCUGACGCCCCAGGCGAGAUGGUGGUGCU GACCUGCGACACCCCGGAAGAAGACGGCAUCACCUGGACUCUGGACCAGAGCAGCGAGGUUCUGGGCAG CGGCAAGACCCUGACCAUCCAGGUAAAGGAGUUCGGUGACGCCGGCCAGUACACCUGCCACAAGGGCGG CGAAGUGCUGAGCCACAGCCUGUUGCUAUUGCACAAGAAGGAGGACGGCAUUUGGAGCACCGACAUCCU GAAGGACCAGAAGGAGCCCAAGAAUAAGACCUUCCUGCGGUGCGAGGCCAAGAAUUACAGCGGCCGGUU CACUUGCUGGUGGCUGACCACCAUUAGUACCGACCUGACCUUUAGUGUAAAGAGCAGCCGGGGUUCCAG CGACCCACAGGGCGUGACCUGUGGCGCAGCCACACUGUCAGCAGAGAGAGUGCGGGGCGACAACAAGGA AUACGAGUACAGCGUGGAGUGCCAGGAAGACAGCGCUUGUCCAGCCGCCGAGGAAAGCCUGCCCAUCGA GGUGAUGGUAGACGCCGUGCACAAGCUGAAGUACGAGAACUACACUUCUAGCUUCUUCAUACGGGACA UCAUAAAGCCCGACCCUCCGAAGAACUUGCAACUCAAGCCCCUGAAGAACAGCCGGCAGGUGGAAGUGA-1 GCUGGGAGUACCCCGACACCUGGAGCACUCCCCACAGCUACUUCUCACUGACCUUCUGCGUGCAAGUAC AAGGCAAGAGCAAGCGGGAGAAGAAGGAUCGGGUGUUCACCGACAAGACCUCUGCCACCGUGAUCUGCC GUAAGAACGCCAGCAUCAGCGUGCGGGCCCAGGACAGAUAUUACAGCAGCAGCUGGAGCGAGUGGGCCA GCGUGCCUUGCUCUGGAGGAGGUGGUUCCGGCGGUGGAGGGAGUGGAGGCGGUGGUUCUAGGGUGAUC CCCGUCUCAGGACCAGCUCGGUGCCUAAGCCAGAGCCGCAACCUGCUGAAGACUACCGACGACAUGGUG AAGACCGCCCGGGAGAAACUGAAGCACUACUCCUGUACCGCAGAGGAUAUCGACCACGAGGACAUCACA CGGGACCAGACGAGCACCCUCAAGACCUGCCUGCCCCUGGAGUUACACAAGAACGAAAGCUGCCUGGCA ACACGGGAGACUAGCAGCACAACCCGGGGAUCUUGUCUGCCGCCGCAGAAGACCUCCUUGAUGAUGACC CUGUGCCUGGGUAGUAUCUACGAGGACCUGAAGAUGUACCAGACCGAGUUCCAGGCCAUCAACGCUGCC CUUCAGAACCACAAUCACCAGCAGAUUAUCCUGGAUAAGGGCAUGCUAGUGGCCAUCGACGAGCUGAUG CAAAGUCUGAACCAUAACGGCGAGACUCUGCGACAGAAGCCUCCUGUAGGAGAGGCCGACCCCUAUCGUGUGAAGAUGAAGCUGUGCAUCCUGCUGCACGCCUUCAGCACACGGGUCGUGACCAUCAACCGGGUGAUG1384920-5746-9564.2Attorney Docket No. 131986-6001GGCUACCUGUCAAGCGCCGGUGGUGGCGGAUCUGGCGGAGGUGGGAGUGGUGGGUCAGGAUCAGGUGG CGGCGGUAGUGGUGGAGGCUCGGACCACCAAUCUCGGUCAGGGCCUUGGGGCCUGUUGGGAGGAAGCCA AAGCGUGUUGACCCAGCCUCCAAGCGUGAGUGGCGCUCCUGGACAGCGGGUGACCAUUAGCUGUAGCGG CAGCCGGAGCAACAUAGGCAGCAACACCGUGAAGUGGUACCAGCAACUGCCGGGAACGGCCCCAAAGUU GCUGAUCUACUACAACGACCAACGCCCAUCUGGCGUGCCCGACCGGUUCAGCGGAAGUAAGAGUGGCAC CAGCGCUAGCCUGGCUAUCACCGGCUUGCAGGCGGAGGACGAGGCAGACUACUACUGCCAGAGCUACGA CCGGUACACUCACCCCGCCCUACUGUUCGGCACCGGCACCAAGGUGACAGUGCUGGGAGGUGGAGGUUC GGGGAGUGGCGGUUCAGGAGGAGGCGGAAGCCAGGUGCAGCUGGUGGAGAGCGGAGGAGGAGUGGUCC AACCUGGGCGAUCCCUACGGCUGAGCUGCGCUGCCAGCGGCUUCACCUUUAGCAGCUACGGCAUGCACU GGGUGAGGCAGGCUCCGGGUAAGGGCCUGGAGUGGGUGGCCUUCAUCCGGUACGACGGCAGCAAUAAG UACUACGCCGACAGCGUGAAGGGGCGGUUUACCAUCUCACGGGACAACAGCAAGAACACCCUGUACCUG CAGAUGAACAGCCUGAGAGCCGAGGACACCGCCGUGUACUACUGUAAGACCCACGGCAGCCACGACAAC UGGGGCCAGGGCACUAUGGUGACCGUGAGCAGCGGAGGUUCCGAUCACCAAAGCAGGAGCGGUCCCUGG GGUCUACUGGGCGGAUCAGGCCCACCUUGCCCUCCCUGUCCAGCACCGGAGUUUGAGGGCGGACCCAGC GUGUUCCUGUUCCCUCCUAAGCCCAAGGACACCCUGAUGAUCAGCCGGACCCCUGAGGUUACCUGCGUG GUGGUGGACGUGAGCCAGGAGGAUCCCGAGGUGCAGUUCAACUGGUACGUGGACGGCGUGGAGGUGCA CAACGCCAAGACCAAGCCCCGGGAGGAACAGUUCAAUAGCACCUACCGGGUGGUGAGCGUACUGACGGU GCUGCACCAGGACUGGCUGAACGGCAAGGAGUACAAGUGCAAGGUGAGCAACAAGGGCCUCCCCAGCAG CAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAACCCAGGGAACCCCAGGUGUAUACCCUGCCUCCGUG UCAGGAAGAGAUGACAAAGAACCAGGUGAGCCUGUGGUGCCUGGUGAAGGGCUUCUACCCCAGCGACA UCGCCGUUGAGUGGGAGAGCAACGGCCAGCCCGAGAACAACUACAAGACCACCCCUCCCGUGCUGGAUA GCGACGGCUCGUUCUUCCUGUACAGCCGCCUUACCGUGGACAAGAGCCGGUGGCAAGAGGGCAACGUGU UCAGCUGCAGCGUGAUGCACGAGGCCCUGCACAACCACUACACCCAGAAGAGCCUGUCACUGAGCUUGG GA153 AUGGAGACAGACACCCUCCUGCUGUGGGUUCUGCUCCUCUGGGUGCCAGGGAGCACGGGUAUCUGGGAG CUGAAGAAAGACGUGUACGUGGUUGAGCUGGACUGGUACCCUGACGCCCCAGGCGAGAUGGUGGUGCU GACCUGCGACACCCCGGAAGAAGACGGCAUCACCUGGACUCUGGACCAGAGCAGCGAAGUGCUCGGCAG CGGCAAGACCCUGACCAUCCAGGUAAAGGAAUUCGGGGACGCCGGCCAGUACACCUGCCACAAGGGCGG-4CGAAGUCCUGAGCCACAGCCUGUUGCUAUUGCAUAAGAAGGAGGACGGCAUUUGGAGCACCGACAUCCU GAAGGACCAGAAGGAGCCCAAGAAUAAGACCUUCCUGCGGUGCGAGGCCAAGAAUUACAGCGGCCGGUU CACUUGCUGGUGGCUGACCACCAUUAGUACCGACCUGACCUUUAGUGUGAAGAGCAGCCGCGGCUCCAGUGACCCACAGGGCGUGACCUGUGGCGCAGCCACUCUGAGCGCCGAGAGGGUCAGAGGGGACAACAAGGA1394920-5746-9564.2Attorney Docket No. 131986-6001AUACGAGUACAGCGUGGAGUGCCAGGAAGACAGCGCUUGCCCGGCUGCUGAGGAGAGCCUGCCCAUCGA GGUGAUGGUAGACGCCGUGCACAAGCUGAAGUACGAGAACUACACCAGCAGCUUCUUCAUACGGGACAU CAUCAAGCCCGACCCUCCGAAGAACUUGCAACUGAAGCCCCUGAAGAACAGCCGGCAGGUGGAAGUGAG CUGGGAGUACCCCGACACCUGGAGCACUCCCCACAGCUACUUCUCACUGACCUUCUGCGUGCAGGUGCA AGGCAAGAGCAAGCGGGAGAAGAAGGAUCGGGUGUUCACCGACAAGACCUCUGCCACCGUGAUCUGCCG UAAGAACGCCAGCAUCAGCGUGCGGGCCCAGGACAGAUACUACAGCAGCAGCUGGAGCGAGUGGGCCAG CGUGCCUUGUAGCGGUGGCGGAGGAUCUGGUGGUGGCGGUUCUGGAGGAGGUGGUUCUAGGGUGAUCC CCGUGAGCGGUCCAGCUCGGUGCCUAAGCCAGAGCCGCAACCUGCUGAAGACUACCGACGACAUGGUGA AGACCGCCCGGGAGAAGCUGAAGCACUACAGCUGUACCGCAGAGGACAUCGACCACGAGGACAUCACAC GGGACCAGACGAGCACCCUCAAGACCUGCCUGCCCCUGGAGCUGCACAAGAACGAAAGCUGCCUGGCAA CACGGGAGACUAGCAGCACAACCAGAGGCUCCUGUCUGCCGCCGCAGAAGACCAGCCUGAUGAUGACCC UGUGCCUGGGUAGUAUCUACGAGGACCUGAAGAUGUACCAGACCGAGUUCCAGGCCAUCAACGCUGCCC UUCAGAACCACAAUCACCAGCAGAUUAUCCUGGAUAAGGGCAUGCUAGUGGCCAUCGACGAGCUGAUGC AGAGUCUGAACCAUAACGGCGAGACUCUGCGACAGAAGCCUCCUGUAGGAGAGGCCGACCCCUAUCGUG UGAAGAUGAAGCUGUGCAUCCUGCUGCACGCCUUCAGCACACGGGUCGUGACCAUCAACCGGGUGAUGG GCUACCUGUCAAGCGCUGGCGGUGGUGGCUCUGGAGGUGGAGGUUCCGGCGGGUCAGGAUCAGGUGGA GGAGGAAGUGGUGGAGGCGGUAGUGGCGGAUCUCAGAAUCAAGCAUUACGGAGCGCGGGCGGAAGUCA GAGCGUGUUGACCCAGCCUCCAAGCGUCUCUGGGGCACCUGGACAGCGGGUGACCAUUAGCUGUAGCGG CAGCCGGAGCAACAUAGGCAGCAACACCGUGAAGUGGUACCAGCAACUGCCGGGCACUGCCCCAAAGUU GCUGAUCUACUACAACGACCAACGCCCAUCUGGCGUGCCCGACCGGUUCAGCGGAAGCAAGAGUGGCAC CAGCGCUAGCCUGGCCAUCACCGGCUUGCAGGCGGAAGACGAGGCCGACUACUACUGCCAGAGCUACGA CCGGUACACUCACCCCGCCCUACUGUUCGGCACCGGCACCAAGGUGACAGUGUUAGGCGGCGGAGGAAG CGGAUCUGGCGGUUCAGGAGGAGGCGGAAGCCAGGUGCAGCUGGUGGAGAGCGGAGGAGGAGUGGUCC AACCUGGGCGAUCCCUACGGCUGAGCUGCGCUGCCAGCGGCUUCACCUUCAGCAGCUACGGCAUGCAUU GGGUAAGGCAAGCGCCAGGGAAGGGCCUGGAGUGGGUGGCCUUCAUCCGGUACGACGGCAGCAAUAAG UACUACGCCGACAGCGUGAAGGGGCGGUUUACCAUCUCACGGGACAACAGCAAGAACACCCUGUACCUG CAGAUGAACAGCCUGAGAGCCGAGGACACCGCCGUGUACUACUGUAAGACCCACGGCAGCCACGACAAC UGGGGCCAGGGCACUAUGGUGACCGUGAGCUCAGGCGGGUCCCAGAACCAAGCCCUGAGAAGCGCAGGC GGAUCAGGCCCACCUUGUCCUCCUUGCCCAGCCCCGGAGUUUGAGGGCGGACCCAGCGUGUUCCUGUUC CCUCCUAAGCCCAAGGACACCCUGAUGAUCAGCCGGACCCCUGAGGUUACCUGCGUGGUGGUGGACGUG AGCCAGGAGGAUCCCGAGGUGCAGUUCAACUGGUACGUGGACGGCGUGGAGGUGCACAACGCCAAGACCAAGCCCCGGGAGGAACAGUUCAAUAGCACCUACCGGGUGGUGAGCGUACUGACGGUGCUGCACCAGGAC1404920-5746-9564.2Attorney Docket No. 131986-6001UGGCUGAACGGCAAGGAGUACAAGUGCAAGGUGAGCAACAAGGGCCUCCCCAGCAGCAUCGAGAAGACC AUCAGCAAGGCCAAGGGCCAACCACGAGAGCCCCAGGUGUAUACCCUGCCUCCGUGUCAGGAAGAGAUG ACAAAGAACCAGGUGAGCCUGUGGUGCCUGGUGAAGGGCUUCUACCCCAGCGACAUCGCCGUUGAGUGG GAGAGCAACGGCCAGCCCGAGAACAACUACAAGACCACCCCUCCCGUGCUGGAUAGCGACGGCUCGUUC UUCCUGUACAGCCGCCUUACCGUGGACAAGAGCCGGUGGCAAGAGGGCAACGUGUUCAGCUGCAGCGUGAUGCACGAGGCCCUGCACAACCACUACACCCAGAAGAGCCUGUCACUGAGCUUGGGA1414920-5746-9564.2Attorney Docket No. 131986-6001IX. EXAMPLES

[0568] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.Example 1: Assessment of IL-12-Based Masked Proteins

[0569] IL- 12 was selected as a molecule for showing proof-of-concept of systemic mRNA delivery of a masked protein that is expressed in the liver or spleen, and activated in a different tissue. While IL-12 is well-characterized and has potential as an immunotherapy, and systemic delivery of IL-12 alone is dose limited due to associated toxicities even at low concentration. In particular, high doses of IL- 12 were necessary for treatment of the various tested cancers, but these doses of IL- 12 resulted in potentially severe side effects (e.g., neutropenia, thrombocytopenia, hyperbilirubinemia, and hypoalbuminemia) and, in some cases, death. Accordingly, the following examples more broadly illustrate that the disclosed mRNA-based approach for systemic administration of masked proteins is applicable to a range of therapeutic proteins, even when high doses of protein are needed and toxicity is a concern.

[0570] This example describes the composition and characterization of illustrative masked proteins that employ IL- 12 as the therapeutic protein together with a variety of different mask types (e.g., a scFv mask, an extracellular domain (ECD) mask, a peptide mask, and a steric mask), and substrates, and a half-life extending moiety: either a Fc domain dimer, an anti-HSA domain, or human serum albumin (HSA). The Fc domain comprised a “knob and hole” mutation, with the first Fc domain of the dimer comprising a hole mutation and the second Fc domain of the dimer comprising a knob mutation.

[0571] A description of illustrative masked IL-12 proteins and control proteins is provided in Table 4.Table 4 - Description of illustrative masked IL-12 proteins and control proteinsHalf-LifeCompound Masking Moiety Cleavable Substrate Extending FormatMoietyMaskPro 1 IL-12 ECD-pi Substrate-2 HSA HSA-fusion MaskPro2 IL-12 ECD-pi Substrate-2 HSA HSA-fusion MaskPro6 unmasked IL-12-Fc fusion Substrate-7 Fc Fc-fusion HeterodimerMaskPro7 peptide PM1 Substrate-7 Fc Fc-fusion Heterodimer1424920-5746-9564.2Attorney Docket No. 131986-6001MaskPro8 peptide PM2 Substrate-7 Fc Fc-fusion Heterodimer MaskPro9 peptide PM3 Substrate-7 Fc Fc-fusion Heterodimer MaskProlO peptide PM4 Substrate-7 Fc Fc-fusion Heterodimer MaskPro 11 peptide PM5 Substrate-7 Fc Fc-fusion Heterodimer MaskProl2 peptide 3HMXPM6 Substrate-7 Fc Fc-fusion Heterodimer MaskPro 13 peptide 3HMXPM7 Substrate-7 Fc Fc-fusion Heterodimer MaskPro 14 unmasked IL-12-Fc fusion Substrate-2 Fc Fc-fusion Heterodimer MaskPro 15 peptide PM1B2 Substrate-2 Fc Fc-fusion Heterodimer MaskPro 16 peptide PM2B2 Substrate-2 Fc Fc-fusion Heterodimer MaskPro 17 peptide PM3B2 Substrate-2 Fc Fc-fusion Heterodimer MaskPro 18 peptide PM4B1 Substrate-2 Fc Fc-fusion Heterodimer MaskPro 19 peptide PM5B1 Substrate-2 Fc Fc-fusion Heterodimer MaskPro20 peptide PM6B 1 Substrate-2 Fc Fc-fusion Heterodimer MaskPro21 peptide PM7B1 Substrate-2 Fc Fc-fusion Heterodimer MaskPro26 Briakinumab scFv Substrate-5 Fc Fc-fusion Heterodimer MaskPro27 Briakinumab scFv Substrate-5 / 6 Fc Fc-fusion Heterodimer MaskPro28 Briakinumab scFv Substrate-5 / 6 Fc Fc-fusion Heterodimer MaskPro3 unmasked IL-12-Fc fusion NSUB Fc Fc-fusion Heterodimer MaskPro4 Briakinumab scFv NSUB Fc Fc-fusion Heterodimer MaskPro5 Briakinumab scFv Substrate-2 Fc Fc-fusion Heterodimer MaskPro24 Briakinumab scFv Substrate-2 Fc Fc-fusion Heterodimer MaskPro22 Briakinumab scFv Substrate-3 Fc Fc-fusion Heterodimer MaskPro23 Briakinumab scFv Substrate- 1 Fc Fc-fusion Heterodimer MaskPro29 Briakinumab scFv Substrate-2 Fc Fc-fusion HeterodimerMaskPro25 Briakinumab scFv Substrate-2 anti-HSA Anti-HSA-fusionA. Characterization of Masked Proteins in Cell-Based IL-12 Reporter Assay

[0572] Exemplary masked proteins were tested in a cell-based IL- 12 reporter assay that utilizes IL-12-responsive HEK293 cells. The cells were generated by stable transfection with the human IL-12Rbl and IL-12Rb2 genes, along with the genes of the IL- 12 signaling pathway. The cells also feature an STAT4-inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. The addition of IL-12 to these cells activates the STAT4 and subsequently induces the production of SEAP which can be readily assessed in the supernatant using QUANTI-Blue solution, a colorimetric detection for alkaline phosphatase activity1434920-5746-9564.2Attorney Docket No. 131986-6001

[0573] Masked proteins MaskProl and MaskPro2 were constructed as described above. Each employs a different ECD masking moiety derived from the ECD pi subunit of the IL-12R. MaskProl utilizes a masking moiety corresponding to amino acids 24-236 of IL-12RP1. MaskPro2 utilizes a masking moiety corresponding to amino acids 24-138 of IL-12RP1. MaskProl and MaskPro2 have the structure depicted in Figure 2A. (see FIG. 2A). Inclusion of the cleavable substrate Substrate-2 was sufficient to render the two exemplary masked proteins cleavable by MMP2 (FIG. 2B). The assay was performed on each of the masked proteins, a control (i.e., recombinant huIL-12 (rhIL-12)), and, in addition, digestion product of masked protein + MMP2 (i.e., activated protein). Assay results for MaskProl and MaskPro2 are depicted in FIG. 2C (rhIL-12, MaskProl, and MaskProl +MMP2) and FIG. 2D (rhIL-12, MaskPro2, respectively. ECso calculations are provided in Table 5.Table 5 - Reporter Activity for IL-12 Masked Proteins Having ECD-Based Masking MoietiesEC50 in IL-12 Reporter AssayIL- 12 Control 20.3(rhIL-12)MaskProl 73.1Activated 15.5MaskProlIL- 12 Control 20.1(rhIL-12)MaskPro2 82.2Activated 13.9MaskPro2

[0574] These results indicate that IL- 12 receptor binding activity was attenuated for both MaskProl and MaskPro2.

[0575] Masked proteins MaskPro7, MaskPro8, MaskPro9, MaskProlO, MaskProl 1, MaskProl2, MaskProl4, MaskProl5, MaskProl6, MaskProl7, MaskProl8, MaskProl9, MaskPro20, and MaskPro21 were constructed as described in Table 4, above. Each employed a peptide mask in the format depicted in FIG. 3 A. Each masked protein was tested in the above-described cell-base IL-12 receptor binding assay, along with control rhIL-12. Results are depicted in FIG. 3B (rhIL-12, MaskPro6, MaskPro7, MaskPro8, and MaskPro9), FIG. 3C (rhIL-12, MaskPro6, MaskProlO, and MaskProl 1), FIG. 3D (rhIL-12, MaskPro6, MaskProl2, and MaskProl3), and FIG. 3E (rhIL-12, MaskProl4, MaskProl5, MaskProl6,1444920-5746-9564.2Attorney Docket No. 131986-6001MaskProl7, MaskProl8, MaskProl9, MaskPro20, and MaskPro21). Calculated ECso values and Masking Efficiency values (ME) are provided in Tables 6 and 7, below.Table 6 - Reporter Activity and Masking Efficiencies for First-Generation Peptide MasksMask EC50 in IL-12 Masking Efficiency vs Reporter Assay Unmasked IL-12-Fc ProteinIL- 12 Control (rhlL- 25.8112)MaskPro6 Unmasked 21.73 1.0MaskPro7 PM1 44.5 2.0MaskPro8 PM2 37.5 1.7MaskPro9 PM3 23.47 1.1MaskProlO PM4 35.25 1.6MaskProl 1 PM5 34.88 1.6MaskProl2 PM6 32.11 1.5MaskProl 3 PM6 14.3 0.7Table 7 - Reporter Activity for Second-Generation Peptide MasksMask EC50In IL-12 Masking Efficiency vs Reporter Assay Unmasked IL-12-Fc ProteinIL- 12 Control 14.91(rhIL-12)MaskProl 4 Unmasked 12.57 1.1 MaskProl 5 PM1B2 13.34 1.2 MaskProl 6 PM2B2 15.39 1.4 MaskProl 7 PM3B2 17.49 0.9 MaskProl 8 PM4B1 10.29 0.8 MaskProl 9 PM5B1 18.81 1.5 MaskPro20 PM6B1 16.37 1.3MaskPro21 PM7B1 10.34 0.8

[0576] The steric mask strategy, which utilized a structure-guided steric masking technique, was also assessed (data not shown), and other proteins of interest may utilize this construction.

[0577] IL-12p40 is human and mouse cross-reactive, but the IL-12p35 subunit is speciesspecific between mice and humans. Various IL-12 masked proteins having the mouse IL- 12p35 subunit were prepared for in vivo studies. The first had a Briakinumab masking moiety and a cleavable substate (MaskPro5), the second had a Briakinumab masking moiety without1454920-5746-9564.2Attorney Docket No. 131986-6001the cleavable moiety (MaskPro4), and the third did not have a masking moiety (MaskPro3) (FIG. 4A). The mouse forms of masked proteins (MaskPro5 and MaskPro6) and unmasked protein (MaskPro3) were tested in the IL-12 reporter assay (FIG. 4B). MaskPro5, the mouse IL- 12 masked protein with a cleavable moiety, was cleaved to generate a free mouse IL- 12 (FIG. 4C). MaskPro4, the mouse IL- 12 masked protein without the cleavable moiety, could not be activated to release a free mouse IL-12 upon protease treatment (FIG. 4C). The masked proteins MaskPro5 and MaskPro4 exhibited low activity in IL- 12 reporter assay; and the activated form of MaskPro5 exhibited similar activity as the unmasked MaskPro3 and rhIL-12 in IL-12 reporter assay (FIG. 4B). Taken together, these data indicate that mouse IL-12 masked protein can be processed as desired to produce an active IL- 12 protein following cleavage.

[0578] Various half-life extending moieties were also tested with the IL-12 masked protein, including the Fc-based half-life extending moiety used in the previous experiments and an anti-HSA antibody (MaskPro25; see FIG. 5A). The masked protein with the anti-HSA antibody was efficiently cleaved by MMP2, indicating that the anti-HSA antibody did not interfere with cleavage (FIG. 5B). Further, the IL-12 reporter assay showed the anti-HAS antibody did not interfere with scFv masking, and the addition of recombinant HSA proteins did not significantly affect overall masking (FIG. 5C).B. Assessment of Cleavability

[0579] The liver and the spleen are the primary location of mRNA translation when mRNA-based therapeutics are systemically administered to patients. Accordingly, the goal of this study was to identify cleavable moieties that were resistant to cleavage in these tissues but cleavable in cells in a tumor microenvironment. Over 200 validated cleavable moiety sequences were screened to identify 8 candidates for further analysis via tissue zymography. Substrate-1, Substrate-2, Subtrate-3, and Substrate-4 were evaluated for cleavability in normal human liver and spleen tissue in a tissue zymography assay. The results are shown in FIGs. 6A-6D as Percent (%) Cleavability. Substrate-1, Substrate-2, and Substrate-3 exhibited much lower cleavability in the human spleen samples and similarly low levels of cleavage in the human liver samples (FIGs. 6C-6D). Overall, these substrates displayed protection against activation in spleen and liver tissues.1464920-5746-9564.2Attorney Docket No. 131986-6001C. In Vivo Characterization of Masked Proteins

[0580] The tolerability and antitumor activity of exemplary masked proteins was evaluated. Masked proteins MaskPro5, MaskPro24, MaskPro24, MaskPro22, MaskPro22, MaskPro23, and MaskPro4 were prepared having the components described in Table 4. The format of the exemplary constructs is shown in FIGs. 7A-7D. A low dose (0.3 mg / kg) and a high dose 3.0 mg / kg) of the masked proteins were administered to mice at day 1, day 4, day 8, and day 11 of the time course and tumor volume was evaluated every five days on day 0, day 5, day 10, day 15, and day 20 (Table 8). The masked proteins having two Substrate-1 substrates and two Substrate-3 substrates induced complete tumor regression when administered at the 3.0 mg / kg dose (FIG. 8). The masked protein having the two Substrate-1 substrates exhibited similar anti-tumor activity as the unmasked protein at the 0.3mg / kg dose (FIG. 8).Table 8 - Study Design for Tolerability and Anti-Tumor Activity Assays for IL-12 Masked ProteinsGroup Protein Administered Cleavable Count Dose Dosing moiety Schedule 1 Vehicle Only Control N / A 8 N / A Day 1, 4, 8, 11 2 MaskPro3 (unmasked) N / A 8 0.03 Day 1, 4, 8, 11 3 MaskPro5 (masked; Substrate-2 8 3 Day 1, 4, 8, 11 single substrate)4 MaskPro24 (masked; Substrate- 8 3 Day 1, 4, 8, 11 dual substrate) 2 / Substrate-25 MaskPro24 (masked; Substrate- 8 0.3 Day 1, 4, 8, 11 dual substrate) 2 / Substrate-26 MaskPro22 (masked; Substrate- 8 3 Day 1, 4, 8, 11 dual substrate) 3 / Substrate-37 MaskPro22 (masked; Substrate- 8 0.3 Day 1, 4, 8, 11 dual substrate) 3 / Substrate-38 MaskPro23 (masked; Substrate- 8 3 Day 1, 4, 8, 11 dual substrate) 1 / Substrate-l9 MaskPro23 (masked; Substrate- 8 0.3 Day 1, 4, 8, 11 dual substrate) 1 / Substrate-l10 MaskPro4 (masked; no No substrates 8 0.3 Day 1, 4, 8, 11substrate)

[0581] To examine whether the masked proteins had protease-dependent anti -tumor activity, two masked proteins (MaskPro22 and MaskPro23) were tested alongside a control masked protein that lacked a cleavable moiety (MaskPro4) and an unmasked control protein (MaskPro3) in mice. Tumor volume was measured for each group every five days. The mean tumor volume for all conditions is shown in FIG. 9A, and the effect of treatment on tumor volume for each individual member of the various treatment groups is shown in FIGs. 9B-9F. The masked proteins with the two cleavable moieties were effective in reducing mean tumor1474920-5746-9564.2Attorney Docket No. 131986-6001size as compared to the vehicle control for all times during the tested time course (FIGs. 9A-9B and 9D-9E). The control masked protein lacking a cleavable moiety had much higher tumor volumes measured as compared to the unmasked control (MaskPro3) and the cleavable forms of the masked protein. Thus, the reduction in tumor size is not due to the presence of the masked protein in the target cells, but rather, it is dependent on the cleavage of the masked protein (FIGs. 9C-9F).

[0582] To determine whether the masked proteins had any negative side effects, body weight and liver damage follow...

Claims

Attorney Docket No. 131986-6001CLAIMSWhat is claimed is:

1. An mRNA encoding a masked protein, the masked protein comprising:(a) a therapeutic protein;(b) a masking moiety;(c) at least one cleavable moiety; and(d) a half-life extending moiety;wherein the at least one cleavable moiety is inert in mammalian liver and spleen, and wherein the therapeutic protein is activated upon cleavage of the at least one cleavable moiety.

2. The mRNA of claim 1, wherein the therapeutic protein is a cytokine.

3. The mRNA of claim 2, wherein the cytokine is a pro-inflammatory cytokine, optionally selected from IL-ip, IL-6, IL-8, IL-12, IL-17, IL-18, IFN- a, IFN- y, and TNF-a.

4. The mRNA of claim 2, wherein the cytokine is an anti-inflammatory cytokine, optionally selected from IL-IRA, IL-4, IL-6, IL-10, IL-11, IL-13, IL-35, and TGF-p.

5. The mRNA of any one of claims 1-4, wherein the masking moiety is selected from a single-chain antibody, an extracellular domain-based masking moiety, a protein aptamer-based masking moiety, and a steric masking moiety.

6. The mRNA of claim 5, wherein the single-chain antibody is an scFv comprising a heavy chain variable sequence and a light chain variable sequence of briakinumab, infliximab, etanercept, adalimumab, certolizumab, golimumab, tocilizumab, siltuximab, sarilumab, olokizumab, sirukumab, ustekinumab, tildrakizumab, guselkumab, BL655066, LY3074828, secukinumab, ixekizumab, brodalumab, CNTO6785, bimekkizumab, or SCH-900117.

7. The mRNA of any one of claims 1-6, wherein the half-life extending moiety comprises an immunoglobulin (IgG) or an Fc domain thereof, a serum albumin, a single-chain antibody that binds to a serum albumin, a single-chain antibody, a hexahat glutathione S-transferase (GST), a glutathione affinity moiety, a calmodulin-1544920-5746-9564.2Attorney Docket No. 131986-6001binding peptide (CGP), a strep-tag, a cellulose binding domain, a maltose binding protein, an s-peptide tag, a chitin binding tag, an immune-reactive epitope, or an epitope tag.

8. The mRNA of any one of claims 1-7, wherein the at least one cleavable moiety is cleavable by proteases found in a tumor or in an infection site.

9. The mRNA of any one of claims 1-8, wherein the at least one cleavable moiety can be cleaved by MMP2, MMP9, MP-ST1, uPA, or any combination thereof.

10. The mRNA of any one of claims 1-9, wherein the at least one cleavable moiety comprises 3-25 amino acids, 3-20 amino acids, 3-15 amino acids, 3-12 amino acids, 4-25 amino acids, 4-20 amino acids, 4-15 amino acids, 4-12 amino acids, 5-25 amino acids, 5-20 amino acids, 5-15 amino acids, 5-12 amino acids, 6-25 amino acids, 6-20 amino acids, 6-15 amino acids, 6-12 amino acids, 7-25 amino acids, 7-20 amino acids, 7-15 amino acids, 7-12 amino acids, 8-25 amino acids, 8-20 amino acids, 8-15 amino acids, or 8-12 amino acids.

11. The mRNA of any one of claims 1-10, wherein the at least one cleavable moiety comprises a consensus sequence comprising (i) a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid; (ii) a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid; or (ii) both a special amino acid — a hydrophobic amino acid — a special amino acid — a hydrophobic amino acid and a polar amino acid — a special or polar amino acid — a positive amino acid — a polar amino acid.

12. The mRNA of any one of claims 1-11, wherein the at least one cleavable moiety comprises 3 or fewer negatively charged amino acids; 2 or more special amino acids; 2 or more hydrophobic amino acids; 2 or more polar amino acids; 1 or more positive amino acids; or any combination thereof.

13. The mRNA of any one of claims 1-12, wherein the at least one cleavable moiety comprises or consists of an amino acid sequence selected from PWGLSGRS (SEQ ID NO: 7), DHQSRSGPWGLL (SEQ ID NO: 8), and QNQALRSA (SEQ ID NO: 9).1554920-5746-9564.2Attorney Docket No. 131986-600114. An mRNA encoding a first peptide chain of a masked protein and a second peptide chain of the masked protein;the first peptide chain of the masked peptide comprising, in the following order:(a) a therapeutic protein,(b) a first cleavable moiety,(c) a masking moiety,(d) a second cleavable moiety, and(e) a first IgG Fc domain; andthe second peptide chain of the masked peptide comprising a second IgGFc domain.

15. The mRNA of claim 14, wherein the therapeutic protein is a cytokine, optionally selected from IL-ip, IL-6, IL-8, IL-12, IL-17, IL-18, IFN- a, IFN- y, TNF-a, IL-IRA, IL-4, IL-10, IL-11, IL-13, IL-35, and TGF-p.

16. The mRNA of claim 15 or 16, wherein the masking moiety is selected from a singlechain antibody, an extracellular domain-based masking moiety, a protein aptamerbased masking moiety, and a steric masking moiety, optionally wherein the singlechain antibody is an scFv comprising a heavy chain variable sequence and a light chain variable sequence of briakinumab, infliximab, etanercept, adalimumab, certolizumab, golimumab, tocilizumab, siltuximab, sarilumab, olokizumab, sirukumab, ustekinumab, tildrakizumab, guselkumab, BL655066, LY3074828, secukinumab, ixekizumab, brodalumab, CNTO6785, bimekkizumab, or SCH-900117.

17. The mRNA of any one of claims 14-16, wherein the first cleavable moiety and the second cleavable moiety each comprise or consist of the same amino acid sequence, or wherein the first cleavable moiety and the second cleavable moiety each independently comprise or consist of a different amino acid sequence.

18. The mRNA of any one of claims 14-17, wherein the first cleavable moiety and the second cleavable moiety are each independently cleavable by MMP2, MMP9, MP- ST1, uPA, or any combination thereof.

19. The mRNA of any one of claims 14-18, wherein the first cleavable moiety and the second cleavable moiety each independently comprise or consist of an amino acid sequence selected from PWGLSGRS (SEQ ID NO: 7), DHQSRSGPWGLL (SEQ ID NO: 8), and QNQALRSA (SEQ ID NO: 9).1564920-5746-9564.2Attorney Docket No. 131986-600120. The mRNA of any one of claims 14-19, wherein the first cleavable moiety and the second cleavable moiety are each independently inert in mammalian liver and spleen.

21. The mRNA of any one of claims 14-20, wherein the first IgG Fc domain comprises a knob mutation and the second IgG Fc domain comprises a hole mutation; or wherein the first IgG Fc domain comprises a hole mutation and the second IgG Fc domain comprises a knob mutation.

22. An mRNA comprising a nucleic acid sequence comprising any one of SEQ ID NOs:151-153 or encoding a masked protein comprising an amino acid sequence of any one of SEQ ID NOs: 135, 136, or 138-148.

23. The mRNA of any one of claims 1-22, wherein the mRNA comprises at least one chemical modification.

24. A composition comprising the mRNA of any one of claims 1-23 and a lipid nanoparticle.

25. The composition of claim 24, wherein the lipid nanoparticle comprises an ionizable amino lipid, a neutral lipid, a sterol, and a PEG-modified lipid.

26. A method of delivering a therapeutic protein to a tissue of interest, comprising administering to a subject an mRNA according to any one of claims 1-23 or the composition according to claim 24 or 25.

27. A method of stimulating an immune response, comprising administering to a subject an mRNA according to any one of claims 1-23 or the composition according to claim 24 or 25.

28. A method of treating cancer, comprising administering to a subject with cancer an mRNA according to any one of claims 1-23 or the composition according to claim 24 or 25.

29. The mRNA according to any one of claims 1-23 or the composition according to claim 24 or 25 for use in the manufacture of a medicament for delivering a therapeutic protein to a tissue of interest.1574920-5746-9564.2Attorney Docket No. 131986-600130. The mRNA according to any one of claims 1-23 or the composition according to claim 24 or 25 for use in the manufacture of a medicament for stimulating an immune response in a subject.

31. The mRNA according to any one of claims 1-23 or the composition according to claim 24 or 25 for use in the manufacture of a medicament for treating cancer in a subject.

32. Use of the mRNA according to any one of claims 1-23 or the composition according to claim 24 or 25 for delivering a therapeutic protein to a tissue of interest.

33. Use of the mRNA according to any one of claims 1-23 or the composition according to claim 24 or 25 for stimulating an immune response in a subject.

34. Use of the mRNA according to any one of claims 1-23 or the composition according to claim 24 or 25 for treating cancer in a subject.1584920-5746-9564.2

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