PD-1 binding proteins, nucleic acids encoding same, and methods of use

Abstract

Described herein are PD-1 specific single domain agonist antibodies and binding molecules comprising such single domain agonist antibodies, as well as nucleic acids encoding the same. Also provided are nucleic acids (e.g., mRNA) encoding such single domain agonist antibodies and binding molecules and delivery vehicles (e.g., lipid nanoparticles) comprising said nucleic acids. The disclosure also features methods of using such agonist antibodies, binding molecules, nucleic acids, and delivery vehicles comprising said nucleic acids to treat various diseases such as inflammatory diseases and T cell driven autoimmune diseases.

Description

[0001] Attorney Docket No.: 45817-0163WO1 PD-1 BINDING MOLECULES, NUCLEIC ACIDS ENCODING SAME, AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of priority of U. S. Provisional Appl. No.

[0002] 63 / 744,954 filed January 14, 2025, the contents of which are incorporated by reference in their entirety herein.

[0003] SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 6, 2026, is named 45817-0163WO1_SL.xml and is 244,481 bytes in size.

[0005] BACKGROUND

[0006] 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.

[0007] Programmed Death-1 receptor (PD-1), also known as PDCD1 or CD279, is type I transmembrane protein belonging to the CD28 family of immune regulatory receptors which include CD28, CTLA-4, ICOS, and BTLA. The PD-1 pathway is a key immune checkpoint that plays an important role in maintaining peripheral T cell tolerance and regulating adaptive immune responses. The PD-1 receptor can be induced on activated T cells, while its natural ligands, PD-L1 and PD-L2, are expressed by various immune and nonimmune cells. PD-1 and its ligands constitute an inhibitory pathway that mediates immune tolerance and provides immune homeostasis. There is mounting evidence that PD-1 pathway impairment plays an important role in autoimmune disease pathogenesis.

[0008] Accordingly, as PD-1 is a promising immunotherapeutic target there is a need for new compositions that can more effectively bind to and potently agonize this receptor.Attorney Docket No.: 45817-0163WO1

[0009] SUMMARY

[0010] The present disclosure provides, among other things, binding molecules that specifically bind to PD-1 (human or human and cynomolgus), nucleic acids (e.g., mRNA) encoding same, nucleic acids formulated with delivery vehicles, and methods of using these agents in treating diseases such as inflammatory diseases. Surprisingly, the anti-PD-1 VHHs when fused to Fc had superior agonist activity than control antibodies; the smaller size of the VHH-Fc versus an IgG antibody makes it advantageous to enter the immune synapse and regulate T cell responses.

[0011] Accordingly, featured herein is a binding molecule comprising a polypeptide that specifically binds to PD-1 (human or human and cynomolgus), i.e., the polypeptide binds at essentially background levels to other proteins (i.e., proteins other than PD-1). In some cases, the PD-1 binding polypeptide is an agonist of human PD-1. The PD-1 binding polypeptide can comprise a single domain antibody. In some cases, the PD-1 binding polypeptide comprises a humanized single domain antibody. In certain cases, the PD-1 binding polypeptide comprises a humanized and affinity matured single domain antibody The binding molecule can be monovalent, bivalent, multivalent, bispecific, or multispecific. The binding molecule can also be an antibody conjugate. Also featured are nucleic acids such as mRNA that encode these binding molecules. The nucleic acids (e.g., mRNA) can be formulated in a delivery vehicle such as nanoparticles e.g., lipid nanoparticles ((LNPs) for administration to a subject. These binding molecules and the mRNA (e.g., formulated in LNPs) can be used to treat diseases that are related to PD-1 expression or overexpression (e.g., PD-1 agonists can be used to treat inflammatory diseases and T cell driven autoimmune diseases).

[0012] In one aspect, the disclosure features a polypeptide comprising a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds to PD-1 (human or human and cyno), the VHH comprising VHH complementarity determining region (CDR) 1, VHH CDR2, and VHH CDR3 of any of the VHH amino acid sequencesAttorney Docket No.: 45817-0163WO1 set forth in SEQ ID NOs.: 1 to 5 or 65 to 104. In some cases, the VHH comprises VHH complementarity determining region (CDR) 1, VHH CDR2, and VHH CDR3 of any of the VHH amino acid sequences set forth in SEQ ID NOs.: 103, 90, 71, 74, or 2. In some cases, the VHH comprises VHH complementarity determining region (CDR) 1, VHH CDR2, and VHH CDR3 of any of the VHH amino acid sequences set forth in SEQ ID NOs.: 103, 90, 71, 74, or 2, but with 1, 2, 3, 4, or 5 amino acid substitutions in one, two, or all three VHH CDRs. In certain cases, the substitutions are not made at one or more of the amino acids of the VHH identified in FIG. 13 as involved in binding to PD-1. In some instances, the polypeptide does not inhibit the interaction of PD-1 with PD-L1.

[0013] In one aspect, the disclosure features a polypeptide comprising a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds to PD-1 (human or human and cyno), wherein the VHH binds to at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the following residues of human PD-1: 38-45, 48, 49, 51, 53, 98, 107, 109, 143 (numbering according to SEQ ID NO:465). In some instances, the VHH does not inhibit the interaction of PD-1 with PD-L1.

[0014] In another aspect, the disclosure features a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds to PD-1 (human or human and cyno), the VHH comprising VHH complementarity determining region (CDR) 1, VHH CDR2, and VHH CDR3 of any of the amino acid sequences set forth in SEQ ID NOs.: 1 to 5 or 65 to 104. In some cases, the VHH comprises VHH complementarity determining region (CDR) 1, VHH CDR2, and VHH CDR3 of any of the VHH amino acid sequences set forth in SEQ ID NOs.: 103, 90, 71, 74, or 2. In some instances, the VHH does not inhibit the interaction of PD-1 with PD-L1. In one aspect, the disclosure features a VHH that specifically binds to PD-1 (human or human and cyno), wherein the VHH binds to at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the following residues of human PD-1: 38-45, 48-49, 51, 53, 98, 107, 109, 143 (numbering according to SEQ IDNO:465). In some instances, the VHH does not inhibit the interaction of PD-1 with PD-L1.Attorney Docket No.: 45817-0163WO1 In some instances, the VHH CDR1, VHH CDR2, and VHH CDR3 are based on CDR definitions according to any one of the known CDR definitions (e.g., Kabat, Chothia, enhanced Chothia, contact, IMGT, or AbM CDR definitions).

[0015] In another aspect, the disclosure features a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds to PD-1 (human or human and cyno), the VHH comprising 0, 1, 2, or 3 amino acid substitutions in any one or more (1, 2, or 3) of the VHH CDR1, CDR2, or CDR3 of a VHH set forth in SEQ ID NOs: 1 to 5 or 65 to 104 (e.g., SEQ ID NOs.: 103, 90, 71, 74, or 2). In some cases, the VHH comprises 0, 1, 2, or 3 amino acid substitutions in any one or more (1, 2, or 3) of VHH CDR1, VHH CDR2, and VHH CDR3 of any of the VHH amino acid sequences set forth in SEQ ID NOs.: 103, 90, 71, 74, or 2. In some instances, the VHH does not inhibit the interaction of PD-1 with PD-L1.

[0016] In some cases, the VHH comprises 1, 2, or 3 amino acid substitutions in any one or more (1, 2, or 3) of the VHH CDR1, CDR2, or CDR3 of a VHH set forth in any one of SEQ ID NOs.: 1-5 or 65 to 104 (e.g., SEQ ID NOs.: 103, 90, 71, 74, or 2). In some cases, the VHH comprises 1 amino acid substitution in one, two or all three of the VHH CDRs of a VHH set forth in any one of SEQ ID NOs.: 1-5 or 65 to 104 (e.g., SEQ ID NOs.: 103, 90, 71, 74, or 2). In some cases, the amino acid substitution is conservative. The CDR substitution(s) improves the binding affinity of the VHH to human PD-1 relative to the parental VHH. In some instances, the substitutions are in VHH CDR1. In some instances, the substitutions are in VHH CDR2. In some instances, the substitutions are in VHH CDR3. In some instances, the substitutions are in VHH CDR1 and / or VHH CDR2. In other instances, the substitutions are in VHH CDR1 and / or VHH CDR3. In certain instances, the substitutions are in VHH CDR2 and / or VHH CDR3. In some instances, the substitutions are in VHH CDR1, VHH CDR2, and VHH CDR3. In making the substitutions, in some instances, the substitutions are not at one or more, or not at any, of the amino acid residues of the VHH (CDR and / or framework region) identified in FIG.

[0017] 13 as involved in binding to human PD-1. In some cases, the VHH comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,Attorney Docket No.: 45817-0163WO1 at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of the sequences set forth in SEQ ID NOs.:103, 90, 71, 74, or 2. In other cases, the VHH comprises an amino acid sequence set forth in any one of SEQ ID NOs.: 103, 90, 71, 74, or 2, except having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions. In some cases, the VHH comprises the VHH CDR1 set forth in SEQ ID NO: 123. In some cases, the VHH comprises the VHH CDR2 set forth in SEQ ID NO: 124. In some cases, the VHH comprises the VHH CDR3 set forth in SEQ ID NO: 125. In some instances, the VHH does not inhibit the interaction of PD-1 with PD-L1.

[0018] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two or all of the VHH CDRs set forth in SEQ ID NO: 9, 10, and 11. In some cases, the VHH comprises the VHH CDRs set forth in SEQ ID NOs.: 123, 124, and 125.

[0019] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 23, 24, and 25.

[0020] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 35, 36, and 25.

[0021] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 44, 45, and 25.

[0022] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 54, 55, and 56.

[0023] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 9, 10, and 11.

[0024] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 109, 110, and 108.

[0025] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 23, 113, and 112.Attorney Docket No.: 45817-0163WO1 In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 116, 117, and 112.

[0026] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 54, 119, and 515.

[0027] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 122, 36, and 112.

[0028] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 106, 107, and 108.

[0029] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 23, 111, and 112.

[0030] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 114, 115, and 112.

[0031] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 54, 118, and 515.

[0032] In some cases, the VHH comprises 0, 1, or 2 amino acid substitutions in one, two, or all of the VHH CDRs set forth in SEQ ID NO: 120, 121, and 112.

[0033] The substitutions can be made, e.g., at a position or positions that vary in an alignment of the CDRs of the P3. B3 parental clone with the affinity matured and humanized VHHs of Table IB. In some instances, the substitutions are in VHH CDR1 and / or VHH CDR2. In other instances, the substitutions are in VHH CDR1 and / or VHH CDR3. In certain instances, the substitutions are in VHH CDR2 and / or VHH CDR3. In making the substitutions, in some instances, the substitutions are not at any one or more, or not at any of the amino acid residues of the VHH identified in FIG. 13 as involved in binding to human PD-1. In some instances, the VHH does not inhibit the interaction of PD-1 with PD-L1.Attorney Docket No.: 45817-0163WO1 In some cases, the VHH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, m at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at;east 97%, at least 98%,,at least 99%, or 100% identical to a VHH set forth in any one of SEQ ID NOs.: 1-5 or 65 to 104. In certain cases, the VHH comprises an amino acid sequence that is 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;east 97%, at least 98%,,at least 99%, or 100% identical to a VHH set forth in any one of SEQ ID NOs.: 103, 90, 71, 74, or 2.

[0034] In some instances, the polypeptide or VHH binds to human PD-1. In other instances, the polypeptide or VHH binds to both human and cynomolgus PD-1. In some instances, the VHHs in a bivalent format (e.g., when conjugated at its C-terminal to the hinge-Fc domain of IgGl) act as PD-1 agonists. In certain instances, the VHHs in a bivalent format (e.g., when conjugated at its C-terminal to the hinge-Fc domain of IgGl) have an EC50 for human PD-1 of about 5 nM to about 60 nM (e.g., about 45 nM to about 55 nM).

[0035] In some instances, the VHH is humanized. In some cases, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 5, 1 to 4, 5 to 15, 6 to 12, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) of any of the VHHs set forth in SEQ ID NOs.: 1 to 5 are substituted in the process of humanization. In certain cases, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) of the VHHs set forth in SEQ ID NOs.: 1 to 5 are substituted in the process of humanization. In some cases, humanization of the VHH is by resurfacing / veneering. In some cases, humanization of the VHH is by CDR grafting. In some cases, one or more of positions 42, 49, 50, and 52 (numbering based on IMGT numbering) are humanized. In other cases, one or more of positions 42, 49, 50, and 52 (numbering based on IMGT numbering) are not humanized. In yet other cases none of positions 42, 49, 50, and 52 (numbering based on IMGT numbering) are humanized. In certain cases, positions 42 and / or 52 (numbering based on IMGT numbering) are not humanized. In other cases, positions 49 and / or 50Attorney Docket No.: 45817-0163WO1 (numbering based on IMGT numbering) are not humanized. In certain cases, positions 49 and / or 50 (numbering based on TMGT numbering) are humanized.

[0036] In some cases, the VHH is humanized and affinity matured. In some cases, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 5, 1 to 4, 5 to 15, 6 to 12, or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) of any of the VHHs set forth in SEQ ID NOs.: 1 to 5 or 65 to 104 (e.g., SEQ ID NOs.: 103, 90, 71, 74, or 2) are substituted in the process of humanization and affinity maturation. In certain cases, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) of the VHHs set forth in SEQ ID NOs.: 1 to 5 or 65 to 104 are substituted in the process of humanization and affinity maturation. The humanized and affinity matured VHH binds to human PD-1 with about 50 fold, 6o fold, 70 fold, 80 fold, 90 fold, 100 fold or 110 fold improvement in binding relative to the parental VHH. In some instances, the substitutions for affinity maturation are in VHH CDR1 and / or VHH CDR2. In other instances, the substitutions for affinity maturation are in VHH CDR1 and / or VHH CDR3. In certain instances, the substitutions for affinity maturation are in VHH CDR2 and / or VHH CDR3. In making the substitutions for affinity maturation, in some instances, the substitutions are not at any of the amino acid residues of the VHH identified in FIG. 14 as involved in binding to human PD-1. In some instances, the VHH does not inhibit the interaction of PD-1 with PD-L1.

[0037] In some cases, the polypeptide or the VHH is conjugated, fused, or linked to an agent selected from the group consisting of a purification tag, a fluorophore, a drug, a photosensitizer, a nanoparticle, a toxic agent, a radionuclide, another VHH, an Fab, a scFv, a multimerization module, a moiety that facilitates the polypeptide crossing the blood brain barrier, and a half-life extension moiety. In some instances, the agent is conjugated, attached, or linked to the C-terminus of the VHH. In certain instances, the agent is another VHH. In some instances, the agent is an Fab. In yet other instances, the agent is a scFv.Attorney Docket No.: 45817-0163WO1 In some cases, the polypeptide or VHH is conjugated, fused, or linked to a human Ig Fc domain, optionally, wherein the human Ig Fc domain is conjugated, fused, or linked to the polypeptide or VHH at the C-terminus of the polypeptide or VHH. In some cases, the human Ig Fc domain further comprises a human Ig hinge domain, wherein the hinge domain is attached or linked at the N-terminus of the human Ig Fc domain. In some cases, the human Ig is a human IgGl, human IgG2, human IgG3, or human IgG4. In one instance, the human Ig is human IgGl. In some cases, the polypeptide or VHH is conjugated, fused, or linked to an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to the human IgGl hinge and Fc domain sequence set forth in SEQ ID NO:468. In certain cases, the polypeptide or VHH is conjugated, fused, or linked to an amino acid sequence that is identical to a human IgGl hinge and Fc domain sequence. In some cases, the Fc domain includes mutations that increase or improve FcyR binding. In some cases, the Fc domain includes mutations that promote heterodimerization (e.g., knob-into hole mutations, electrostatic steering mutations).

[0038] In one aspect, the disclosure features a polypeptide comprising a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds to PD-1 (human or human and cyno), the VHH comprising VHH complementarity determining region (CDR) 1, VHH CDR2, and VHH CDR3 of any of the amino acid sequences set forth in SEQ ID NOs.: 1 to 5 or 65 to 104 (e.g., SEQ ID NOs.:103, 90, 71, 74, or 2), wherein 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or wherein 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids in the framework regions of the VHH are substituted (e.g., in the humanization or affinity maturation process). In certain cases, provided herein is a polypeptide comprising a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds to PD-1 (human or human and cyno), the VHH comprising VHH complementarity determining region (CDR) 1, VHH CDR2, and VHH CDR3 of any of the amino acid sequences set forth in SEQ ID NOs.: 1 to 5 or 65 to 104 (e.g., SEQ ID NOs.:103, 90, 71, 74, or 2), wherein 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6,Attorney Docket No.: 45817-0163WO1 1 to 5, 1 to 4, 1 to 3, 1 to 2, or wherein 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids in one, two or three CDRs of the VHH are substituted (e.g., in the affinity maturation process). In some cases, one or two amino acids of a VHH-CDR are substituted. In certain cases, the polypeptide is conjugated, fused, or linked to an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identical to the human IgGl hinge and Fc domain sequence set forth in SEQ ID NO:468. In certain instances, the polypeptide specifically binds to human PD-1. In some cases, the polypeptide binds to both human and cynomolgus PD-1.

[0039] In another aspect, provided herein is a bispecific binder comprising a polypeptide or VHH that specifically binds to PD-1 (human or human and cyno) disclosed herein and a second VHH that specifically binds to an antigen that is expressed on a T cell. In some instances, the bispecific binder comprises a second VHH that specifically binds to an antigen that is expressed on aNK cell. In other instances, the bispecific binder comprises a second VHH that specifically binds to an antigen that is expressed on a tumor cell. In some instances, the bispecific binder comprises a second VHH that specifically binds to any one of VEGF, TIM-3, CTLA-4, or PD-L1. In certain instances, the polypeptide specifically binds to human PD-1. In some cases, the polypeptide binds to both human and cynomolgus PD-1.

[0040] In some instances, the VHH specifically binds to PD-1 (human or human and cyno) and the VHH CDR1, VHH CDR2, and VHH CDR3 comprises the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of any one of the clones set forth in Tables 3, 4, 5, 6, 7. In some instances, the VHH binds to human PD-1. In some instances, the VHH binds to human and cynomolgus PD-1.

[0041] In certain instances, the VHH specifically binds to PD-1 (human or human and cyno) and comprises:Attorney Docket No.: 45817-0163WO1 (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:6, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:8;

[0042] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:9, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11;

[0043] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14;

[0044] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:9, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16;

[0045] (e) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19;

[0046] (f) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:20, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:21, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:22;

[0047] (g) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:24, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25;

[0048] (h) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:26, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27;Attorney Docket No.: 45817-0163WO1 (i) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:28, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:29;

[0049] (j) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:30, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:31, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:32;

[0050] (k) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:33, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:34, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:22;

[0051] (l) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:35, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:36, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25;

[0052] (m) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:37, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:38, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27;

[0053] (n) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:35, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:39, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:29;

[0054] (o) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:40, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:41, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:32;

[0055] (p) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:42, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:43, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:22;Attorney Docket No.: 45817-0163WO1 (q) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:44, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:45, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25;

[0056] (r) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:46, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:47, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27;

[0057] (s) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:44, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:48, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:29;

[0058] (t) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:49, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:32;

[0059] (u) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:51, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53;

[0060] (v) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:54, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 55, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 56;

[0061] (w) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:57, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 58, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 59;

[0062] (x) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:54, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:60, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:61; orAttorney Docket No.: 45817-0163WO1 (y) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:62, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:63, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:64.

[0063] In some instances, the VHH specifically binds to PD-1 (human or human and cyno) and the VHH CDR1, VHH CDR2, and VHH CDR3 comprises the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of any one of the clones set forth in Table IB (e.g., clone v14 or v27). The CDRs can be based on any CDR definition (e.g., IMGT, Kabat, Chothia, enhanced Chothia, Contact).

[0064] In some instances, the VHH specifically binds to PD-1 (human or human and cyno) and the VHH CDR1, VHH CDR2, and VHH CDR3 comprises the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of any one of the clones set forth in any one of Tables 2A, 2B, 2C, 2D, or 2E.

[0065] The VHHs bind human PD-1. In some instances, the VHHs when linked to an Ig hinge-Fc domain (e.g., from human IgGl) act as PD-1 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 5 nM to about 60 nM.

[0066] In some instances, the VHH specifically binds to PD-1 (human or human and cyno) and comprises a sequence that is 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 VHH amino acid sequence set forth in Table 1, Table 1A, or Table IB. In certain cases, the VHH comprises a sequence that is set forth in any one of SEQ ID NOs.: 1 to 5 or 65 to 104. In certain cases, the VHH is a PD-1 agonist and comprises a sequence that is set forth in SEQ ID NO.: 2. In certain cases, the VHH is a PD-1 agonist and comprises a sequence that is set forth in any one of SEQ ID NOs.: 103, 90, 71, or 74. The VHHs bind to human PD-1. In some instances, the VHHs when linked to an Ig hinge-Fc domain (e.g., from human IgGl) act as PD-1 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 5 nM to about 60 nM.Attorney Docket No.: 45817-0163WO1 In some instances, the VHH specifically binds to PD-1 (human or human and cyno) and comprises a sequence that is identical to a sequence that is set forth in any one of SEQ ID NOs.: 1 to 5 (e.g., SEQ ID NO: 2) or 65 to 104 (e.g., SEQ ID NO: 71, 74, 90, or 103), except for having 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions in the framework regions. In some instances, the VHH specifically binds to human PD-1. In other instances, the VHH specifically binds to both human PD-1 and cynomolgus PD-1.

[0067] In some instances, the VHH binds to PD-1 expressed on a cell with an EC50of about 5 nM to about 60 nM. In other instances, the VHH binds to PD-1 expressed on a cell with an EC50 of about 5 nM to about 55 nM. In certain instances, the VHH binds to PD-1 expressed on a cell with an EC50 of about 5 nM to about 50 nM. In some instances, the VHH binds to PD-1 expressed on a cell with an EC50 of about 5 nM to about 40 nM. In other instances, the VHH binds to PD-1 expressed on a cell with an EC50 of about 5 nM to about 35 nM. In yet other instances, the VHH binds to PD-1 expressed on a cell with an EC50 of about 5 nM to about 30 nM. In yet other instances, the VHH binds to PD-1 expressed on a cell with an EC50 of about 5 nM to about 25 nM. In other instances, the VHH binds to PD-1 expressed on a cell with an EC50 of about 5 nM to about 20 nM. In other instances, the VHH binds to PD-1 expressed on a cell with an EC50 of about 5 nM to about 15 nM. In some other instances, the VHH binds to PD-1 expressed on a cell with an EC50 of about 5 nM to about 10 nM. In some other instances, the VHH binds to PD-1 expressed on a cell with an EC50 of about 8 nM to about 12 nM. In some instances, the above binding assays are done with the VHH in a bivalent format (e.g., the C-terminal of the VHH is linked to the N-terminus of a sequence that is identical to the sequence set forth in SEQ ID NO:468 except for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 substitutions). In certain cases, the PD-1 is human PD-1.

[0068] In one aspect, the disclosure features a polypeptide comprising a VHH that specifically binds to PD-1 (human or human and cyno) and comprises a sequence that is 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%Attorney Docket No.: 45817-0163WO1 identical to a sequence that is set forth in any one of SEQ ID NOs.: 65 to 104 (e.g., SEQ ID NO: 71, 74, 90, or 103), and wherein the VHH comprises the three CDRs of the P3. B3 (see e.g.,, Tables 3 to 7). In some cases, provided herein is a polypeptide comprising a VHH that specifically binds to PD-1 (human or human and cyno) and comprises a sequence that is set forth in any one of SEQ ID NOs.: 65 to 104 (e.g., SEQ ID NO: 71, 74, 90, or 103), except for having 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions in the framework regions, and wherein the VHH comprises the three CDRs of the P3.B3 (see e.g.,, Tables 3 to 7). In some instances, the VHH specifically binds to human PD-1. In some instances, the VHH specifically binds to human PD-1 and cynomolgus PD-1.

[0069] In another aspect, the disclosure features a bispecific antibody comprising the polypeptide or VHH described above that specifically binds to PD-1; and a binding molecule that binds to a different epitope of PD-1 than the polypeptide or VHH described above, or binds to a different antigen (e.g., as T cell antigen, aNK cell antigen, a tumor associated antigen).

[0070] In a different aspect, the disclosure features a multispecific binding molecule comprising the polypeptide or VHH described above that specifically binds to PD-1; and a binding molecule that binds to a different epitope(s) of PD-1 than the polypeptide or VHH described above or binds to a different antigen(s) (e.g., as T cell antigen, a NK cell antigen, a tumor associated antigen).

[0071] In a further aspect, the disclosure relates to a binding polypeptide that binds to two, or at least two, different epitopes or antigens. The binding polypeptide comprises a means for binding PD-1 (human or human and cyno PD-1). The binding polypeptide also comprises a polypeptide that binds to a different epitope or antigen (e.g., a T cell antigen, a NK cell antigen, a tumor associated antigen). In some cases, the polypeptide is a VHH. In other cases, the polypeptide is a scFv. In yet other cases, the polypeptide is a Fab. In some cases, the means for binding PD-1 (human or human and cyno PD-1) is linked to the polypeptide via a peptide linker (e.g., glycine linker, serine linker, glycine-serineAttorney Docket No.: 45817-0163WO1 linker). In some cases, the linker is (G4S)nwhere n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO:473) In one case, the linker is (G4S)3(SEQ ID NO:474).

[0072] In some instances, the binding molecule is a VHH. In other instances, the binding molecule is a scFv.

[0073] In some instances, a human Ig hinge domain and Fc domain are directly or indirectly linked to the C-terminus of the VHH that specifically binds to PD-1 (human or human and cyno). In some cases, the N-terminus of the human Ig hinge domain-Fc domain are directly or indirectly linked to the C-terminus of the VHH. In certain cases, the human Ig hinge domain -Fc domain are from a human IgGl, human IgG2, human IgG3, or human IgG4, or variants thereof. In some instances, the variant differs from the native human IgG hinge and / or Fc sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids. In some cases, the human Ig hinge domain -Fc domain is a sequence comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:468. In some cases, the human Ig Fc domain include one or more mutations that alter (e.g., increase) binding to FcyR. In other cases, the human Ig Fc domain include one or more mutations that promote heterodimerization (e.g., knob into hole or electrostatic steering mutations).

[0074] In yet another aspect, the disclosure features a nucleic acid or nucleic acids encoding the polypeptide or VHH described above, or the bispecific or multispecific antibody described above.

[0075] In another aspect, provided are a vector or vectors comprising the nucleic acid or nucleic acids described above.

[0076] Also provided is a host cell comprising the vector or vectors described above. In yet another aspect, the disclosure features a method of making a polypeptide or bispecific antibody. The method comprises culturing a host cell described above under conditions that facilitate expression of the polypeptide, or the bispecific antibody. In some cases, the method further involves isolating the polypeptide or the bispecificAttorney Docket No.: 45817-0163WO1 antibody. In certain cases, the method further comprises formulating the polypeptide or the bispecific antibody as a sterile pharmaceutical composition.

[0077] In another aspect, provided herein is a pharmaceutical composition comprising the polypeptide or the bispecific antibody described herein, and a pharmaceutically acceptable carrier.

[0078] In yet another aspect, the disclosure relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a means for binding PD-1 (human or human and cyno PD-1).

[0079] In yet another aspect, the disclosure relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a means for agonizing human PD-1.

[0080] In a different aspect, the disclosure features a method of treating an inflammatory disorder or disease or an autoimmune disease (e.g., a T cell drive autoimmune disease) in a human subject in need thereof. The method comprises administering to the human subject a therapeutically effective amount of a polypeptide (e.g., a polypeptide comprising a VHH comprising the three VHH CDRs of any one of SEQ ID NOs.: 1, 2, 3, 4, or 5), a bispecific antibody, a multispecific antibody, or a pharmaceutical composition described herein. In one instance, the human subject in need thereof has or is at risk of developing rheumatoid arthritis, ulcerative colitis, Type I diabetes, or multiple sclerosis. In some instances, administration is performed intravenously. In other instances, administration is performed subcutaneously. In yet other instances, administration is performed intramuscularly.

[0081] In certain aspects, the disclosure features a nucleic acid molecule, e.g., an mRNA comprising an open reading frame encoding a VHH, a polypeptide, a bispecific antibody, or multispecific binding molecule described herein.

[0082] In another aspect, the disclosure features a polynucleotide comprising an mRNA comprising: (i) a 5' UTR; (ii) an open reading frame (ORF) encoding a VHH, aAttorney Docket No.: 45817-0163WO1 polypeptide, a bispecific antibody, or multispecific binding molecule described herein; (iii) a stop codon; and (iv) a 3' UTR. Tn some cases, the polynucleotide comprises a nucleic acid encoding a signal sequence that is positioned between the 3’ end of the 5 ’UTR and the 5’ end of the ORF. In some cases, a nucleic acid encoding a signal sequence is included between the 3’ end of the 5’ UTR and the 5’ end of the ORF.

[0083] In some cases, the mRNA comprises a microRNA (miR) binding site. In some cases, the microRNA is expressed in an immune cell of hematopoietic lineage or a cell that expresses TLR7 and / or TLR8 and secretes pro-inflammatory cytokines and / or chemokines. In certain cases, the microRNA binding site is for a microRNA selected from miR-122, miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27, miR-26a, or any combination thereof. In some cases, the microRNA binding site is for a microRNA selected from miR122-5p, miR126-3p, miR-142-3p, miR-142-5p, miR-155, or any combination thereof. In certain cases, the microRNA binding site is located in the 3' UTR of the mRNA.

[0084] In some instances, the 5' UTR comprises a nucleic acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NO: 479 to 482 or 486 to 501.

[0085] In some instances, the 3' UTR comprises a nucleic acid sequence at least 90%, 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: 502 to 514.

[0086] In some instances, wherein the mRNA comprises a 5' terminal cap. In some cases, the 5' terminal cap comprises a CapO, Capl, ARC A, inosine, N1 -methylguanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof. In some instances, the 5’terminal cap is or comprises m7GpppGm.

[0087] In certain instances, the mRNA comprises a poly A region. In some cases, the poly A region is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90Attorney Docket No.: 45817-0163WO1 nucleotides in length, or at least about 100 nucleotides in length. In some cases, the poly A region is about 10 to about 200, about 20 to about 180, about 50 to about 160, about 70 to about 140, or about 80 to about 120 nucleotides in length.

[0088] In some instances, the mRNA comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof. In certain cases, the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil (ψ), N1-methylpseudouracil (m1ψ), 1 -ethylpseudouracil, 2-thiouracil (s2U), 4’-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof. In some cases, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uracils are

[0089] N1-methylpseudouracils. In some instances, 100% of the uracils of the mRNA are N1-methylpseudouracils. In some instances, 100% of the uracils of the mRNA are 5-methoxyuracils.

[0090] In some instances, the open reading frame consists of nucleosides selected from the group consisting of (i) uridine or a modified uridine, (ii) cytidine or a modified cytidine, (iii) adenosine or a modified adenosine, and (iv) guanosine or a modified guanosine. In some cases, the modified uridine is 1 -methylpseudouridine.

[0091] In some instances, the mRNA comprises a 5’terminal cap comprising m7GpppGm and a poly A region 100 nucleotides in length.

[0092] In some instances, all uracils of the polynucleotide are N1 -methylpseudouracils. In certain aspects, the disclosure features a polynucleotide comprising an mRNA nucleotide sequence encoding a polypeptide, a bispecific antibody, or a multispecific binding molecule described herein, comprising from 5’ to 3’end: (i) a 5’cap, optionally which comprises m7GpppGm; (ii) a 5’UTR, (iii) an ORF encoding a polypeptide, a bispecific antibody, or a multispecific binding molecule described herein; (iv) a 3’UTR; and (v) a poly A tail, optionally which is about 100 nucleotides in length. In someAttorney Docket No.: 45817-0163WO1 instances, the polynucleotide comprises a nucleic acid encoding a signal sequence positioned between the 3’ end of the 5’UTR and the 5’ end of the ORF.

[0093] In another aspect, the disclosure features a delivery agent (e.g., LNP) comprising a polynucleotide (e.g., mRNA) described herein. The mRNA is formulated in the delivery agent (e.g., a nanoparticle such as a LNP).

[0094] In another aspect, the disclosure features a pharmaceutical composition comprising a polynucleotide described herein formulated with a delivery agent (e.g., a LNP).

[0095] In some cases, the delivery agent comprises a nanoparticle such as a lipid nanoparticle. In some cases, the lipid nanoparticle has a mean particle size of from 80 nm to 160 nm. In certain cases, the lipid nanoparticle has a poly dispersity index (PDI) of from 0.02 to 0.2 and / or a lipid:nucleic acid ratio of from 10 to 20.

[0096] In some cases, the lipid nanoparticle comprises an ionizable amino lipid, a structural lipid, a phospholipid, and a polyethyleneglycol (PEG)-modified lipid.

[0097] In certain cases, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0098] In some cases, the ionizable amino lipid is a compound of Formula (I). In one case, the ionizable amino lipid is Compound 1-1.

[0099] In certain cases, the PEG-modified lipid is PEG 2000 dimyristoyl glycerol (PEG-DMG) or 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90, 93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate.

[0100] In some cases, the structural lipid is cholesterol, adosterol, agosterol A, atheronals, avenasterol, azacosterol, blazein, cerevisterol, colestolone, cycloartenol, daucosterol, 7-dehydrocholesterol, 5 -dehydroepi sterol, 7-dehydrositosterol, 20a,22R-Attorney Docket No.: 45817-0163WO1 dihydroxycholesterol, dinosterol, epibrassicasterol, episterol, ergosterol, ergosterol, fecosterol, fucosterol, fungisterol, ganoderenic acid, ganoderic acid, ganoderiol, ganodermadiol, 7a-hydroxycholesterol, 22R-hydroxycholesterol, 27-hydroxycholesterol, inotodiol, lanosterol, lathosterol, lichesterol, lucidadiol, lumisterol, oxycholesterol, oxysterol, parkeol, saringosterol, spinasterol, sterol ester, trametenolic acid, zhankuic acid, or zymosterol. In some cases, the structural lipid is cholesterol.

[0101] In some instances, the delivery vehicle is a lipid nanoparticle that comprises an ionizable amino lipid, a structural lipid, a phospholipid, and a polyethylene glycol (PEG)-modified lipid. In some cases, the ionizable amino lipid is present at about 40 to 50 mole ratio%, the structural lipid is present at 35 to 45 mole ratio%, the phospholipid is present at 5 to 15 mole ratio%, and the PEG-modified lipid is present at 1.5 to 5 mole ratio%. In certain cases, the ionizable amino lipid is present at about 47.5 mole ratio%, the structural lipid is present at about 39 mole ratio%, the phospholipid is present at about 10.5 mole ratio%, and the PEG-modified lipid is present at about 3 mole ratio%. In certain cases, the ionizable amino lipid is present at 47.5 mole ratio%, the structural lipid is present at 39 mole ratio%, the phospholipid is present at 10.5 mole ratio%, and the PEG-modified lipid is present at 3 mole ratio%.

[0102] In certain instances, the ionizable amino lipid is Compound 1-1 or a salt thereof. In some instances, the structural lipid is cholesterol. In other instances, the phospholipid is DSPC. In yet other instances, the PEG-modified lipid is Compound A. In certain instances, the LNP comprises Compound 1-1 or a salt thereof, cholesterol, DSPC or a salt thereof, and Compound A or a salt thereof. In other instances, the LNP comprises Compound 1-1, cholesterol, DSPC, and Compound A. In some cases, the ionizable amino lipid is present at about 40 to 50 mole ratio%, the structural lipid is present at 35 to 45 mole ratio%, the phospholipid is present at 5 to 15 mole ratio%, and the PEG-modified lipid is present at 1.5 to 5 mole ratio%. In certain cases, the ionizable amino lipid is present at about 47.5 mole ratio%, the structural lipid is present at about 39 mole ratio%, the phospholipid is present at about 10.5 mole ratio%, and the PEG-modified lipid is present at about 3 mole ratio%.Attorney Docket No.: 45817-0163WO1 In other instances, the ionizable amino lipid is Compound 1-1 and is present at about 47.5 mole ratio%, the structural lipid is cholesterol and is present at about 39 mole ratio%, the phospholipid is DSPC and is present at about 10.5 mole ratio%, and the PEG-modified lipid is Compound A and is present at about 3 mole ratio%.

[0103] In some instances, the ionizable amino lipid is Compound 1-1 and is present at 47.5 mole ratio%, the structural lipid is cholesterol and is present at 39 mole ratio%, the phospholipid is DSPC and is present at 10.5 mole ratio%, and the PEG-modified lipid is Compound A and is present at 3 mole ratio%.

[0104] In one instance, the lipid nanoparticle comprises an ionizable amino lipid that is Compound I- 1, a phospholipid that is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), a PEG-modified lipid that is 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90, 93,96,99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132-tetratetracontaoxatetratriacontahectyl stearate, and a structural lipid that is cholesterol. In some cases, the ionizable amino lipid is present at about 47.5 mole ratio%, the phospholipid is present at about 10.5 mole ratio%, the PEG-modified lipid is present at about 3 mole ratio%, and the structural lipid is present at about 39 mole ratio%. In certain cases, the ionizable amino lipid is present at 47.5 mole ratio%, the phospholipid is present at 10.5 mole ratio%, the PEG-modified lipid is present at 3 mole ratio%, and the structural lipid is present at 39 mole ratio%.

[0105] In a different aspect, provided is a method of treating an inflammatory disease or an autoimmune disease (e.g., a T cell drive autoimmune disease) in a human subject in need thereof. In one instance, the human subject has or is at risk of developing rheumatoid arthritis, ulcerative colitis, psoriasis, Type I diabetes, or multiple sclerosis. The method comprises administering to the human subject a therapeutically effective amount of a nucleic acid (e.g., mRNA) that encodes a VHH (e.g., a polypeptide comprising a VHH comprising the three VHH CDRs of any one of SEQ ID NOs.: 1, 2, 3, 4, or 5), a polypeptide, a bispecific antibody, or a multispecific binding moleculeAttorney Docket No.: 45817-0163WO1 described herein, wherein the mRNA is formulated in a delivery vehicle (e.g., LNP). In some instances, the delivery vehicle is a LNP that comprises an ionizable amino lipid that is Compound 1-1 and is present at 47.5 mole ratio%, a structural lipid that is cholesterol and is present at 39 mole ratio%, a phospholipid that is DSPC and is present at 10.5 mole ratio%, and a PEG-modified lipid that is Compound A and is present at 3 mole ratio%. In some instances, administration is performed intravenously. In other instances, administration is performed subcutaneously. In some instances, administration is performed intramuscularly.

[0106] In another aspect, the disclosure features a kit comprising (i) the VHH, the polypeptide; the bispecific antibody; the multispecific binding molecule; the pharmaceutical composition; or the polynucleotide formulated in an LNP as described herein, and (ii) a package insert instructing a user of the kit to administer the polypeptide, bispecific antibody, the multispecific binding molecule, pharmaceutical composition, or polynucleotide formulated in an LNP to a human subject in need thereof. In some cases, the human subject in need thereof has an inflammatory disease or disorder or an autoimmune disease (e.g., rheumatoid arthritis, ulcerative colitis, psoriasis).

[0107] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 provides graphical depictions of the binding of anti PD-1 VHH to PD-1+ Jurkat cells.

[0108] FIG. 2 depicts data showing that anti PD-1 VHH inhibit Staphylococcal Enterotoxin B (SEB)-driven CD4+T cell activation.

[0109] FIG. 3 is a graph that shows that PD-1 agonists suppress disease in collagen induced arthritis model.

[0110] FIG. 4 is a table summarizing the properties of the anti-PD-1 VHHs of this disclosure.

[0111] FIG. 5 is a graph showing the binding of monovalent P3. B3 humanized constructs to Jurkat-PD1+ cells.Attorney Docket No.: 45817-0163WO1 FIG. 6 is a graph showing CD4+T cell activation inhibition potential in SEB stimulated human PBMCs

[0112] FIG. 7 is a graph showing Tm measurements of additional sequence variants of humanized P3. B3 intended to optimize VHH stability

[0113] FIG. 8 is a graph showing monovalent binding affinity measurements of P3. B3 progeny to human and cyno PD-1 from affinity maturation process

[0114] FIG. 9 is a graph showing melting temperature and polyreactivity of select affinity maturation progeny of P3. B3

[0115] FIG. 10 is a graph showing inhibition of CD4+T cell proliferation in the SEB stimulation assay with human PBMCs.

[0116] FIG. 11 is a table that summarizes data on the most improved affinity matured antibodies.

[0117] FIG. 12A shows the overall complex, with the human PD-1 ectodomain displayed as a gray surface and the anti-PDl VHH as ribbons, binding near the C-terminal region of PD-1.

[0118] FIG. 12B shows an open-book view of the epitope-paratope interface. Epitope residues are labeled on PD-1, and paratope residues are labeled on the VHH using linear residue numbering (for simplicity, instead of conventional Kabat numbering). Paratope residues within CDRs are labeled in bold, and CDR2 and CDR3 are indicated. The residues on PD-1 that form the epitope are residues for the anti-PD-1 VHH2 are amino acids 38-45, 48-49, 51, 53, 98, 107, 109, and 143 of human PD-1 as set forth in SEQ ID NO:465.

[0119] FIG. 13 shows the VHH2 PD-1 epitope residues on the PD-1 ectodomain sequence (SEQ ID NO:525) in enlarged underlined font. It also shows the VHH2 sequence with IMGT-defined CDRs highlighted by larger font size with grey background and paratope residues indicated by bold font with underlining. Figure discloses SEQ IDAttorney Docket No.: 45817-0163WO1 NOs: 525 and 2, respectively, in order of appearance. The PD-1 ectodomain includes a C62S mutation (underlined but not in bold) to improve recombinant expression and enable structure determination.

[0120] FIG. 14. Shows the cell killing activity driven by VHH2 and its affinity matured progeny P3. B3_v.27 in an in vitro NK cell - T cell co-culture assay; the effect on life PD-1 CD4+and CD8+T cells is shown

[0121] DETAILED DESCRIPTION

[0122] The compositions and methods of the disclosure feature PD-1 specific single domain antibodies (anti -PD-1 VHHs) and complementarity determining regions (CDRs) thereof, as well as binding proteins that comprise the disclosed VHHs or the three VHH-CDRs of a VHH that specifically binds to PD-1. The VHHs bind human PD-1 or human and cynomolgus PD-1. In some instances, the VHHs when linked to an Ig Fc domain (e.g., human IgGl) act as PD-1 agonists. In certain instances, the VHHs have an EC50 of about 5 nM to about 60 nM. The disclosure also provides nucleic acids (e.g., mRNA) encoding the disclosed proteins, and such nucleic acids formulated in delivery vehicles (e.g., nanoparticles such as lipid nanoparticles). Also featured are methods of using such VHHs, binding proteins, nucleic acids, and delivery vehicles that are formulated with such nucleic acids in the treatment of inflammatory diseases and autoimmune diseases (e.g., T cell driven autoimmune diseases). In some cases, the VHHs, binding proteins, nucleic acids, and delivery vehicles that are formulated with such nucleic acids of this disclosure are used to treat rheumatoid arthritis, ulcerative colitis, or psoriasis.

[0123] Definitions

[0124] “VHH” means a variable domain of the heavy chain of heavy chain-only antibodies that are produced by camelids. They are monovalent and do not include a light chain. The VHH can be used as is or after humanization. The VHH can also be made bivalent or multivalent by e.g., fusing to sequences that facilitate dimerization, trimerization, tetramerization, pentamerization, or by being linked to a whole antibody orAttorney Docket No.: 45817-0163WO1 an antigen-binding fragment. VHHs are also referred to as single domain antibodies and nanobodies.

[0125] “Monovalent” means a binding molecule with a single antigen binding domain (e.g., a single VHH).

[0126] “Bivalent” means a binding molecule with two antigen binding domains (e.g., two VHHs linked together or brought together by a dimerization domain such as a polypeptide comprising a human Ig Fc).

[0127] “Multivalent” means a binding molecule with more than two antigen binding domains.

[0128] “% identity” is the percentage of identical residues between two or more sequences and is determined over the full length of the sequences being compared. It is a function of the number of amino acids or nucleotide residues that are identical in the sequences being compared. Identical residues are defined as residues that are the same in the two sequences in a given position of the alignment of the sequences. The percentage of sequence identity is calculated from the optimal alignment by taking the number of residues identical between two sequences dividing it by the total number of residues in the shortest sequence and multiplying by 100. The optimal alignment is the alignment in which the percentage of identity is the highest possible. Gaps may be introduced into one or both sequences in one or more positions of the alignment to obtain the optimal alignment. These gaps are then taken into account as non-identical residues for calculation of the percentage of sequence identity. Alignment for purposes of determining the percentage of amino acid or nucleic acid sequence identity can be achieved in various ways using computer programs. For example, the BLAST program (Tatiana t al., FEMS Microbiol. Lett., 174:247-250 (1999)) set to the default parameters, available from the National Center for Biotechnology Information (NCBI) at ncbi.nlm.nih.gov / BLAST / bl2seq / wblast2.cgi, can be used to obtain an optimal alignment of amino acid or nucleic acid sequences to calculate the percentage of sequence identity.Attorney Docket No.: 45817-0163WO1 “about” means + or - 10% of the recited value, so “about 250 nM” means 225 nM to 27 nM.

[0129] PD-1

[0130] Programmed cell death 1 protein (PD-1) belongs to the CD28 superfamily and is a type I transmembrane protein of 288 amino acid residues PD-1 is about 15% identical to the amino acid sequence of CD28, about 20% identical to CTLA-4, and about 13% identical to inducible co-stimulatory molecule (ICOS). The 50-55 kDaPD-1 protein includes an immunoglobulin (Ig) superfamily domain, a 20 amino acid stalk, a transmembrane domain, an intracellular domain of approximately 95 residues containing an immunoreceptor tyrosine-based inhibitory motif (ITIM), and an immunoreceptor tyrosine-based switch motif (ITSM). PD-1 is an immunosuppressive checkpoint and is mainly expressed in activated T lymphocytes, B lymphocytes, natural killer cells, macrophages, dendritic cells, monocytes, and myeloid cells. PD-1 expression is wider than the restricted expression of other CD28 family members on T cells, resulting in a broader spectrum of immune responses.

[0131] The PD-1 receptor binds to two ligands namely, PD-L l and PD-L2, leading to inhibition of T cell proliferation, inhibition of T cell activation, inhibition of cytokine production, inhibition of cytotoxic T lymphocyte killer functions, and eventual death of activated T cells. The inhibitory function of PD-1 depends on its interaction with the phosphatase SHP-2. PD-1 contains inhibitory motifs, including ITIM and ITSM, and the interaction of the SHP-2 SH2 domains with PD-1 ITSM induces PD-1 dimerization and SHP-2 activation. After interacting with its ligands, PD-1 is activated and recruits the phosphatase SHP-2 in proximity to TCRs, which dephosphorylates critical protein molecules for TCR signaling and affects downstream signaling pathways, including the phosphoinositide 3-kinase (PI3K)-phosphoinositide-dependent kinase 1 (PDK1)-AKT-mammalian target of rapamycin (mTOR) pathway, RAS-RAF-MEK (mitogen-activated protein kinase kinase or MAPKK)-extracellular-signal-regulated kinase (ERK) pathway.Attorney Docket No.: 45817-0163WO1 and Janus kinases (JAKs)-signal transducers and activators of transcription (STAT) pathways.

[0132] PD-1, is an immune checkpoint receptor that downregulates T cell activation, proliferation, and cytokine production, rendering T cells dysfunctional. Thus, agonizing PD-1 has utility in treating autoimmune and inflammatory disorders.

[0133] The amino acid sequence ofhumanPD-1 (Uniprot: QI 5116) is provided below: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFT

[0134] MSVVTTARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAFIPSPSPRP AGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAV PVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGP RSAQPLRPEDGHCSWPL (SEQ ID NO:465)

[0135] The ectodomain of human PD-1 used for X-ray crystallography is provided below (the amino acid residues involved in binding to anti-PD-1 VHHs described herein are shown in bold:

[0136] WNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPED RSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR AELRVTER (SEQ ID NO: 126)

[0137] The amino acid sequence of Macaca fascicularis PD-1 (Uniprot B0LAJ3) is provided below:

[0138] MQIPQAPWPVVWAVLQLGWRPGWFLESPDRPWNAPTFSPALLLVTEG

[0139] GQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPAPCVPEQTEYATIVFPSGLGTSS PARRGSADGPRSPRPLRPEDGHCSWPL (SEQ ID NO:466)

[0140]

[0141] Attorney Docket No.: 45817-0163WO1 The amino acid sequence of a fragment of cynomolgus PD-1 used for binding studies is provided below:

[0142] LESPDRPWNAPTF SP ALLL VTEGDNATFTC SF SNASE SF VLNW YRMSP SN QTDKLAAFPEDRSQPGQDCRFRVTRLPNGRDFHMSVVRARRNDSGTYLCGAISL APKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQ (SEQ ID NO: 105)

[0143] The amino acid sequence of murine PD-1 (Uniprot: Q02242) is provided below: MWVRQVPWSFTWAVLQLSWQSGWLLEVPNGPWRSLTFYPAWLTVSEGANATF TCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDF FIMNII DTRRNDSGIYLCG AISLHPKAKIEESPGAELVVTERILETSTRYPSPSPKPE GRFQGMVIGIMSALVGIPVLLLLAWALAVFCSTSMSEARGAGSKDDTLKEEPSA APVPSVAYEELDFQGREKTPELPTACVHTEYATIVFTEGLGASAMGRRGSADGL QGPRPPRHEDGHCSWPL (SEQ ID NO:467)

[0144] VHHs that Specifically Bind PD-1

[0145] Provided herein are novel VHHs that specifically bind to PD-1 (human or human and cynomolgus); i.e., the VHHs bind to a protein or proteins other than PD-1 at essentially background levels. The VHHs bind to human PD-1. In some instances, the VHHs bind to human and cynomolgus PD-1. In some instances, the VHH do not bind to murine PD-1. In some instances, the VHHs bind to human and cynomolgus PD-1 but not to murine PD-1. Table 1 provides the amino acid sequences of several examples of novel VHHs that bind to PD-1.

[0146] This disclosure features a binding molecule or polypeptide that specifically binds to PD-1 (human or human and cyno) and comprises the VHH amino acid sequence of any of the clones listed in Table 1. In some instances, the VHH do not bind to murine PD-1. In some instances, the binding molecule or polypeptide comprises a VHH that specifically binds to PD-1 (human or human and cyno) and comprises a sequence that is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at leastAttorney Docket No.: 45817-0163WO1 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 an amino acid sequence set forth in any one of SEQ ID NOs.: 1 to 5. In some instances, the binding molecule or polypeptide comprises a VHH that specifically binds to PD-1 (human, or human and cyno) and comprises an amino acid sequence set forth in any one of SEQ ID NO:1 to 5 except for having 1, 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 acid substitutions. In certain instances, the binding molecule or polypeptide comprises a VHH that specifically binds to PD-1 (human, or human and cyno) and comprises an amino acid sequence set forth in any one of SEQ ID NO:1 to 5 except for having 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions. The amino acid substitutions may be conservative or non-conservative amino acid substitutions. The variability of the above sequences from an amino acid sequence set forth in any one of SEQ ID NO: 1 to 5 may be due to humanization of the VHH. The humanized VHHs still specifically bind to PD-1 (human, or human and cyno). In certain cases, variability of the above sequences from an amino acid sequence set forth in any one of SEQ ID NO: 1 to 5 may be due to humanization and affinity maturation of the VHH. In some cases, the process of affinity maturation leads to substitutions of 1 to 3 amino acids in one, two or all three VHH CDRs. In some cases, the process of affinity maturation leads to 1 amino acid substitution in one, two or all three VHH CDRs. The humanized and affinity matured VHHs specifically bind to PD-1 (human, or human and cyno) with improved affinity relative to the parental clone. In some instances, the abovedescribed binding molecule or polypeptide specifically binds to human PD-1. In some instances, the above-described binding molecule or polypeptide binds to both human PD-1 and cynomolgus PD-1. In some instances, the VHHs act as PD-1 agonists when the VHH is connected to the hinge and Fc domain of a human IgG (e.g., human IgGl). In certain instances, the VHHs have an EC50 of about 5 nM to about 60 nM. In certain instances, the VHHs have an EC50 of about 5 nM to about 35 nM. In some instances, the VHHs have an EC50 of about 5 nM to about 25 nM. In other instances, the VHHs haveAttorney Docket No.: 45817-0163WO1 an EC50 of about 5 nM to about 15 nM. In certain instances, the VHHs have an EC50 of about 45 nM to about 55 nM.

[0147] In some instances, the binding molecule or polypeptide that binds PD-1 (human, or human and cyno) is in a monovalent format. In some instances, the binding molecule or polypeptide that binds PD-1 (human, or human and cyno) comprises the VHH in a bivalent format. In some cases, a bivalent format of the VHH is produced by linking the VHH to the same or another VHH. In some cases, a bivalent format of the VHH is arrived at by linking the C-terminus of the VHH of the binding molecule or polypeptide to the N-terminus of a hinge and Fc domain of a human immunoglobulin (e.g., IgA, IgGl, IgG2, IgG3, IgG4). In one case, the hinge and Fc domain is from human IgGl. In some cases, the hinge and Fc domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of human IgGl (SEQ ID NO:468) The amino acid sequence of the hinge and Fc domain of human IgGl is provided below (the hinge is shown in bold; the CH2 domain is italicized; and the CH3 domain is underlined; the CH2 and CH3 domains together form the Fc):

[0148] EPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO:468).

[0149] The Fc gamma receptor interactions of the Fc are important for PD-1 VHH-Fc agonism. Thus, when PD-1 VHH is formatted with an IgG4-Fc, it loses its agonistic activity.

[0150] In some cases, the hinge and Fc domain comprises an amino acid sequence set forth in SEQ ID NO:468 except for 1, 2, 3 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. These substitutions can be made to e.g., alter effector function (e.g., increasing or decreasing effector function), or to promote heterodimerization. Such substitutions made as part of Fc domain engineering are well known in the art. See e.g., Liu H, Saxena A,Attorney Docket No.: 45817-0163WO1 Sidhu SS and Wu D (2017) Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds Front. Immunol. 8:38 doi: 10.3389 / firnmu.2017.00038; Wilkinson I, Anderson S, Fry J, Julien LA, Neville D, Qureshi O, et al. (2021) Fc- engineered antibodies with immune effector functions completely abolished. PLoS ONE 16(12): e0260954. doi org / 10 1371 / journal.pone.0260954; Delidakis et al., Wilkinson I, Anderson S, Fry J, Julien LA, Neville D, Qureshi O, et al. (2021) Fc-engineered antibodies with immune effector functions completely abolished. PLoS ONE 16(12): e0260954. / / doi. org / 10.1371 / journal.pone.0260954, all of which are incorporated by reference herein,

[0151] In some cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format (e.g., wherein the C-terminus of the VHH is linked to the N-terminus of the hinge-Fc domain of a human IgG such as human IgGl) binds a cell expressing human PD-1 with an EC50 of about 5 nM to about 60 nM. In some other cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human PD-1 with an EC50 of about 5 nM to about 50 nM. In certain cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human PD-1 with an EC50 of about 5 nM to about 40 nM. In other cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human PD-1 with an EC50 of about 5 nM to about 30 nM. In some cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human PD-1 with an EC50 of about 5 nM to about 20 nM. In other cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human PD-1 with an EC50 of about 5 nM to about 15 nM. In certain cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human PD-1 with an EC50 of about 5 nM to about 10 nM. In some cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human PD-1 with an EC50 of about 8 nM to about 15 nM. In other cases, the binding molecule or polypeptide comprising aAttorney Docket No.: 45817-0163WO1 VHH described herein in bivalent format binds a cell expressing human PD-1 with an EC50 of about 8 nM to about 10 nM.

[0152] Table 1 provides the sequences of exemplary VHHs that specifically bind to PD-1 (human PD-1, or human and cynomolgus PD-1).

[0153] 5 Table 1: Amino Acid Sequences of VHHs that Bind PD-1

[0154] VHH VHH Amino Acid Sequence

[0155] ID VHH1 / QVQLVESGGGLVQPGGSLRLSCGASGFSLEHFDIGWFRQAPGKEREGVSCINSSDGRTWYTDSVK P1. C5_V2 GRFTISRDSGKNTVYLQMNNLKPEDTAVYYCSTDVVCYSSTSSYPAYWGQGTQVTVSS (SEQ ID NO: 1)

[0156] VHH2 / QVQLVESGGGFVQAGGSLRLSCSASGRTFSSYAMGWFRQGPGKEREFVAAITWSSGSSGYSDPVK P3. B3 S3 DRFTVSRDNAKNTVYLQMNDLKPEDTATYYCAARRNRVGFESVISVFGDDYDYWGQGTQVTVSS (SEQ ID NO 2)

[0157] VHH3 / QVQLVESGGGLVQAGGSLRLSCSASGRSFGSYAMGWFRQGPGKDREFVAAITWSSSSSGYSDPVK P2. D8 S3 DRFTISRDNAKNTVYLQMNSLKPEDTATYYCAARRNREGFESVISVFFDDYDYWGQGTQVTVSS (SEQ ID NO 3)

[0158] VHH4 / QVQLVESGGGLVQAGGSLRLSCSASGRTFSSYAMGWFRQGPGKEREFVAAITSSAGSSGYAGSVK P2 E8_S3 DRFTISRDNAKNTVYLQMNSLKPEDTAAYYCAARRNRVGFESVISVFLDDYDYWGQGTQVTVSS (SEQ ID NO:4)

[0159] VHH5 / QVQLVESGGGLVQPGGSLRLSCVASGFILDDYDIGWFRQAPGKEREGVSCISTSNRRTWYADFVK YF-12 GRFTISRDNAKNTVYLQMNSLKPEDAAVYYCSTDVICYSGSPKGRGYLKVWGQGTLVTVSS

[0160]

[0161] (SEQ ID NO:5)

[0162] This disclosure features a polypeptide comprising a VHH that specifically binds to PD-1 (human, or human and cyno) and comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 5. In some cases, the VHH that specifically binds to PD-1 (human, w or human and cyno) comprises the amino acid sequence of any one of SEQ ID NOs: 1 to

[0163] 5 except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions within the VHH. In some cases, the substitutions are in the framework region(s). In other cases, the substitutions are in one or more (e.g., 1, 2, 3) of the three VHH CDRs. In some cases, one amino acid of one, two, or all three VHH CDRs are substituted. In yet other

[0164] 15 instances, the substitutions are in the framework region(s) and in one or more (e.g., 1, 2,

[0165] 3) of the three VHH CDRs. In some cases, the substitutions are conservative amino acid substitutions. In other instances, the substitutions are not conservative amino acid substitutions. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl)Attorney Docket No.: 45817-0163WO1 act as PD-1 agonists. In certain instances, the VHHs have an EC50 of about 5 nM to

[0166] about 60 nM. In certain instances, the VHHs have an EC50 of about 5 nM to about 35

[0167] nM. In other instances, the VHHs have an EC50 of about 5 nM to about 15 nM.

[0168] Also encompassed by this disclosure are binding molecules or polypeptides that 5 comprise a VHH that specifically binds to PD-1 (human, or human and cyno), wherein

[0169] the VHH comprises the VHH CDR1, VHH CDR2, and VHH CDR3 of the VHH of any one of the clones listed in Table 1. The VHH CDRs of the VHH can be based on any

[0170] CDR definition. Exemplary CDR numbering systems are provided below as Table 2 (see, e.g., Lafrance et al,. Dev. Comp. Immunol., 27(l):55-77 (2003)).

[0171] w Table 2: Exemplary CDR Definitions

[0172] Kabat + IMGT Kabat AbM Chothia Contact Chothia

[0173] VHH CDR 1 26-35 27-38 31-35 26-35 26-32 30-35 VHH CDR2 50-65 56-65 50-65 50-58 52-56 47-58 VHHH 95-102 105-117 95-102 95-102 95-102 93-101

[0174] CDR3

[0175]

[0176] Provided in Table 3 are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the clones of Table 1 according to the IMGT definition. The disclosure encompasses polypeptides, single domain antibodies, and binding molecules that

[0177] 15 specifically bind to PD-1 (human, or human and cyno) and which comprise the three

[0178] VHH CDRs of any one of the VHHs listed in Table 3.

[0179] Table 3: Anti-PD-1 VHH CDRs Based on IMGT CDR Definition

[0180] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH1 / P1. C5 V2 GFSLEHFD INSSDGRT STDVVCYSSTSSYPAY

[0181] (SEQ ID NO:6) (SEQ ID NO: 7) (SEQ ID NO: 8) VHH2 / P3. B3 S3 GRTFSSYA ITWSSGSS AARRNRVGFESVISVFGDDYDY

[0182]

[0183] (SEQ ID NO:9) (SEQ ID NO: 10) (SEQ ID NO: 11)Attorney Docket No.: 45817-0163WO1 VHH3 / P2. D8 S3 GRSFGSYA ITWSSSSS AARRNREGFESVISVFFDDYDY (SEQ ID NO: 12) (SEQ ID NO: 13) (SEQ ID NO: 14) VHH4 / P2. E8 S3 GRTFSSYA ITSSAGSS AARRNRVGFESVISVFLDDYDY (SEQ ID NO:9) (SEQ ID NO: 15) (SEQ ID NO: 16)

[0184] VHH5 / YF-12 GFILDDYD ISTSNRRT STDVICYSGSPKGRGYLKV

[0185]

[0186] (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19)

[0187] In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to PD-1 (human, or human and cyno), wherein the VHH comprises the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid

[0188] sequences of any one clone listed in Table 3. Thus, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds PD-1 (human, or

[0189] human and cyno), wherein the VHH comprises:

[0190] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:6, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:8;

[0191] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:9, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a VHH

[0192] CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11;

[0193] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a

[0194] VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14;

[0195] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:9, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:15, and a VHH

[0196] CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; or

[0197] (e) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a

[0198] VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19.Attorney Docket No.: 45817-0163WO1 The VHHs bind human PD-1. In some instances, the VHHs when linked to an Ig Fc domain (e g., IgGl) act as PD-1 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for binding human PD-1 of about 5 nM to about 60 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for binding human PD-1 of about 5 nM to about 35 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 45 nM to about 55 nM.

[0199] In some instances, featured herein are single domain antibodies that specifically bind to PD-1 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to PD-1 (human, or human and cyno), which comprise: a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:9, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11. In certain cases, the single domain antibodies or binding molecules comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:2. In other cases, the single domain antibodies or binding molecules comprise an amino acid sequence set forth in SEQ ID NO:2 except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. In some cases, the substitutions are in the framework regions. In other cases, the substitutions are in one or more of the three VHH CDRs. In yet other instances, the substitutions are in the framework regions and one or more of the three VHH CDRs. In some cases, the VHHs bind to human PD-1. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as PD-1 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 40 nM to about 60 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 45 nM to about 55 nM.

[0200] Provided in Table 4 are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the clones of Table 1 according to the Kabat CDR definition. TheAttorney Docket No.: 45817-0163WO1 disclosure encompasses single domain antibodies that specifically bind to PD-1 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to PD-1 (human, or human and cyno) and which comprise the three VHH CDRs of any one of the VHHs listed in Table 4.Attorney Docket No.: 45817-0163WO1

[0201] Table 4. Anti-PD-1 VHH CDRs Based on Kabat CDR Definition

[0202] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0203] VHH1 / P1. C5_V2 HFDIG CINSSDGRTWYTDSVKG DVVCYSSTSSYPAY

[0204] (SEQ ID NO:20) (SEQ ID NO:21) (SEQ ID NO:22)

[0205] VHH2 / P3. B3 S3 SYAMG AITWSSGSSGYSDPVKD RRNRVGFESVISVFGDDYDY

[0206] (SEQ ID NO:23) (SEQ ID NO:24) (SEQ ID NO:25)

[0207] VHH3 / P2. D8 S3 SYAMG AITWSSSSSGYSDPVKD RRNREGFESVISVFFDDYDY

[0208] (SEQ ID NO:23) (SEQ ID NO:26) (SEQ ID NO:27)

[0209] VHH4 / P2. E8_S3 SYAMG AITSSAGSSGYAGSVKD RRNRVGFESVISVFLDDYDY

[0210] (SEQ ID NO:23) (SEQ ID NO:28) (SEQ ID NO:29)

[0211] VHH5 / YF-12 DYDIG CISTSNRRTWYADFVKG DVICYSGSPKGRGYLKV

[0212]

[0213] (SEQ ID NO:30) (SEQ ID NO:31) (SEQ ID NO:32)Attorney Docket No.: 45817-0163WO1 In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to PD-1 (human, or human and cyno PD-1), wherein the VHH comprises the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of any one clone listed in Table 4. Thus, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds PD-1 (human or human and cyno), wherein the VHH comprises:

[0214] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:20, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:21, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:22;

[0215] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:24, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25;

[0216] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:26, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27;

[0217] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:28, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:29; or

[0218] (e) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:30, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:31, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:32.

[0219] In some cases, the VHHs bind to human PD-1. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as PD-1 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 5 nM to about 60 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 5 nM to about 35 nM. In other instances, theAttorney Docket No.: 45817-0163WO1 VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 45 nM to about 55 nM.

[0220] In some instances, featured herein are single domain antibodies that specifically bind to PD-1 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to PD-1 (human, or human and cyno), which comprise: a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:24, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25. In certain cases, the single domain antibodies or binding molecules comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:2. In other cases, the single domain antibodies or binding molecules comprise an amino acid sequence set forth in SEQ ID NO:2 except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. In some cases, the substitutions are in the framework regions. In other cases, the substitutions are in one or more of the three VHH CDRs. In yet other instances, the substitutions are in the framework regions and one or more of the three VHH CDRs. In some cases, the VHHs bind to human PD-1. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as PD-1 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for human PD-1 of about 40 nM to about 60 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for human PD-1 of about 45 nM to about 55 nM.

[0221] Provided in Table 5 are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the clones of Table 1 according to the Chothia CDR definition. The disclosure encompasses single domain antibodies that specifically bind to PD-1 (human, or human and cyno) or binding molecules or polypeptides comprising single domain antibodies that specifically bind to PD-1 (human, or human and cyno) and which comprise the three VHH CDRs of any one of the VHHs listed in Table 5.Attorney Docket No.: 45817-0163WO1 Table 5. Anti-PD-1 VHH CDRs Based on Chothia CDR Definition

[0222] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH1 / P1. C5 V2 GFSLEHF NSSDGR DVVCYSSTSSYPAY (SEQ ID NO:33) (SEQ ID NO:34) (SEQ ID NO:22) VHH2 / P3. B3 S3 GRTFSSY TWSSGS RRNRVGFESVISVFGDDYDY (SEQ ID NO:35) (SEQ ID NO:36) (SEQ ID NO:25) VHH3 / P2. D8 S3 GRSFGSY TWSSSS RRNREGFESVISVFFDDYDY (SEQ ID NO:37) (SEQ ID NO:38) (SEQ ID NO:27) VHH4 / P2. E8 S3 GRTFSSY TSSAGS RRNRVGFESVISVFLDDYDY (SEQ ID NO:35) (SEQ ID NO:39) (SEQ ID NO:29) VHH5 / YF-12 GFILDDY STSNRR DVICYSGSPKGRGYLKV

[0223]

[0224] (SEQ ID NO:40) (SEQ ID NO:41) (SEQ ID NO:32)

[0225] In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to PD-1 (human, or human and cyno PD-1), wherein the VHH comprises the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of any one clone listed in Table 5. Thus, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds PD-1 (human, or human and cyno), wherein the VHH comprises:

[0226] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:33, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:34, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:22;

[0227] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:35, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:36, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25;

[0228] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:37, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:38, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27;

[0229] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:35, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:39, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:29; orAttorney Docket No.: 45817-0163WO1 (e) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:40, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:41, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:32.

[0230] In some cases, the VHHs bind to human PD-1. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as PD-1 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 5 nM to about 60 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 5 nM to about 35 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 45 nM to about 55 nM.

[0231] In some instances, featured herein are single domain antibodies that specifically bind to PD-1 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to PD-1 (human, or human and cyno), which comprise: a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:35, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:36, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25. In certain cases, the single domain antibodies or binding molecules comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:2. In other cases, the single domain antibodies or binding molecules comprise an amino acid sequence set forth in SEQ ID NO:2 except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. In some cases, the substitutions are in the framework regions. In other cases, the substitutions are in one or more of the three VHH CDRs. In yet other instances, the substitutions are in the framework regions and one or more of the three VHH CDRs. In some cases, the VHHs bind to human PD-1. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as PD-1 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for human PD-1 of about 40 nM to about 60 nM. In other instances, the VHHs linked to an Ig Fc domainAttorney Docket No.: 45817-0163WO1 (or in another bivalent format) have an EC50 for human PD-1 of about 45 nM to about 55 nM,

[0232] Provided in Table 6 are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the clones of Table 1 according to the enhanced Chothia CDR definition. The disclosure encompasses single domain antibodies that specifically bind to PD-1 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to PD-1 (human, or human and cyno) and which comprise the three VHH CDRs of any one of the VHHs listed in Table 6.

[0233] Table 6. Anti -PD-1 VHH CDRs Based on Enhanced Chothia CDR Definition

[0234] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH1 / P1. C5 V2 GFSLEHFDIG CINSSDGRTW DVVCYSSTSSYPAY (SEQ ID NO:42) (SEQ ID NO:43) (SEQ ID NO:22) VHH2 / P3. B3 S3 GRTFSSYAMG AITWSSGSSG RRNRVGFESVISVFGDDYDY (SEQ ID NO:44) (SEQ ID NO:45) (SEQ ID NO:25) VHH3 / P2. D8 S3 GRSFGSYAMG AITWSSSSSG RRNREGFESVISVFFDDYDY (SEQ ID NO:46) (SEQ ID NO: 47) (SEQ ID NO:27) VHH4 / P2. E8 S3 GRTFSSYAMG AITSSAGSSG RRNRVGFESVISVFLDDYDY (SEQ ID NO:44) (SEQ ID NO:48) (SEQ ID NO:29) VHH5 / YF-12 GFILDDYDIG CISTSNRRTW DVICYSGSPKGRGYLKV

[0235]

[0236] (SEQ ID NO:49) (SEQ ID NO:50) (SEQ ID NO:32)

[0237] In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to PD-1 (human, or human and cyno PD-1), wherein the VHH comprises the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of any one clone listed in Table 6. Thus, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds PD-1 (human, or human and cyno), wherein the VHH comprises:

[0238] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:42, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:43, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:22;Attorney Docket No.: 45817-0163WO1 (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:44, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:45, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25;

[0239] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:46, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:47, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27;

[0240] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:44, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:48, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:29; or

[0241] (e) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:49, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 50, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:32.

[0242] In some cases, the VHHs bind to human PD-1. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as PD-1 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 5 nM to about 60 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 5 nM to about 35 nM. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 45 nM to about 55 nM.

[0243] In some instances, featured herein are single domain antibodies that specifically bind to PD-1 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to PD-1 (human, or human and cyno), which comprise: a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:44, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:45, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25. In certain cases, the single domain antibodies or binding molecules comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atAttorney Docket No.: 45817-0163WO1 least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:2. In other cases, the

[0244] single domain antibodies or binding molecules comprise an amino acid sequence set forth in SEQ ID NO:2 except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. In some cases, the substitutions are in the framework regions. In other

[0245] cases, the substitutions are in one or more of the three VHH CDRs. In yet other instances, the substitutions are in the framework regions and one or more of the three VHH CDRs.

[0246] In some cases, the VHHs bind to human PD-1. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as PD-1 agonists. In certain instances, the VHHs

[0247] linked to an Ig Fc domain (or in another bivalent format) have an EC50 for human PD-1 of about 40 nM to about 60 nM. In other instances, the VHHs linked to an Ig Fc domain

[0248] (or in another bivalent format) have an EC50 for human PD-1 of about 45 nM to about 55 nM.

[0249] Provided in Table 7 are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the clones of Table 1 according to the Contact CDR definition. The disclosure encompasses single domain antibodies that specifically bind to PD-1 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to PD-1 (human, or human and cyno) and which comprise the three

[0250] VHH CDRs of any one of the VHHs listed in Table 7.

[0251] Table 7. Anti -PD-1 VHH CDRs Based on Contact CDR Definition

[0252] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3 VHH1 / P1. C5 V2 EHFDIG GVSCINSSDGRTW STDVVCYSSTSSYPA (SEQ ID NO:51) (SEQ ID NO: 52) (SEQ ID NO:53) VHH2 / P3. B3 S3 SSYAMG FVAAITWSSGSSG AARRNRVGFESVISVFGDDYD (SEQ ID NO:54) (SEQ ID NO:55) (SEQ ID NO:56) VHH3 / P2. D8 S3 GSYAMG FVAAITWSSSSSG AARRNREGFESVISVFFDDYD (SEQ ID NO:57) (SEQ ID NO:58) (SEQ ID NO:59) VHH4 / P2. E8 S3 SSYAMG FVAAITSSAGSSG AARRNRVGFESVISVFLDDYD (SEQ ID NO: 54) (SEQ ID NO:60) (SEQ ID NO:61) VHH5 / YF-12 DDYDIG GVSCISTSNRRTW STDVICYSGSPKGRGYLK

[0253]

[0254] (SEQ ID NO:62) (SEQ ID NO:63) (SEQ ID NO: 64)Attorney Docket No.: 45817-0163WO1 In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to PD-1 (human, or human and cyno), wherein the VHH comprises the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of any one clone listed in Table 7. Thus, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds PD-1 (human, or human and cyno), wherein the VHH comprises:

[0255] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:51, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53;

[0256] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:54, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 55, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 56;

[0257] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:57, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 58, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:59;

[0258] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:54, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:60, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:61; or

[0259] (e) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:62, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:63, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:64.

[0260] In some cases, the VHHs bind to human PD-1. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as PD-1 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 5 nM to about 60 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 5 nM to about 35 nM. In other instances, theAttorney Docket No.: 45817-0163WO1 VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 45 nM to about 55 nM.

[0261] In some instances, featured herein are single domain antibodies that specifically bind to PD-1 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to PD-1 (human, or human and cyno), which comprise: a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 54, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:55, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 56. In certain cases, the single domain antibodies or binding molecules comprise an amino acid sequence that is 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 the sequence set forth in SEQ ID NO:2. In other cases, the single domain antibodies or binding molecules comprise an amino acid sequence set forth in SEQ ID NO:2 except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. In some cases, the substitutions are in the framework regions. In other cases, the substitutions are in one or more of the three VHH CDRs. In yet other instances, the substitutions are in the framework regions and one or more of the three VHH CDRs. In some cases, the VHHs bind to human PD-1. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as PD-1 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for human PD-1 of about 40 nM to about 60 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for human PD-1 of about 45 nM to about 55 nM.

[0262] In some instances, the VHHs of this disclosure are not humanized. In some instances, the VHHs are humanized. Humanization may be by, e.g. resurfacing / veneering or CDR grafting. Given the high sequence identity of most VHHs with human IGHV3 family a comparison between the human IGHV3 framework sequence (e.g., from IGHV3-23) and the VHH can be used to make amino acid substitutions to replace the camelid amino acid with the amino acid in the corresponding human framework.Attorney Docket No.: 45817-0163WO1 AlphaFold2 and / or NanoNet can, for example, be used as tools to predict a structural model of the VHH from its sequence. See e.g., Vishwakarma et al., Inti. J. Mol. Sci., 2022, 23, 3721 (incorporated by reference herein). iCAN, Llamanade, and / or INDI can also be for assisting with VHH humanization. See, e.g., Zuo et al., BMC Genom., 2017, 18, 797; Sang et al., Structure, 2022, 30, 418-429. e3; Deszynski et al., Nucleic Acids Res., 2022, 50, D1273-D1281 (all of which are incorporated by reference herein). In some instances, 1, 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 framework amino acids of the VHH are substituted as a result of humanization. In some instances, full humanization is avoided. In some cases, less than 20, 15, 10, 9, 8, 7, 6, 5, 4, or 3 amino acids, or 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, Ito 3, 1 to 2, or 1 amino acid in the VHH framework regions are substituted for humanization. In some cases, 1 to 15 amino acids in the VHH framework regions of the sequences in Table 1 are substituted for humanization. In some cases, 1 to 10 amino acids in the VHH framework regions of the sequences in Table 1 are substituted for humanization. In some cases, 1 to 5 amino acids in the VHH framework regions of the sequences in Table 1 are substituted for humanization. In some instances, one or more of IMGT positions 42, 49, 50, and 52 of the VHH are not humanized. In some instances, one or more of IMGT positions 42 and 52 of the VHH are not humanized. In some instances, one or more of IMGT positions 42, 49, 50, and 52 of the VHH are humanized. In some instances, one or more of IMGT positions 49 and 50 of the VHH are humanized. In some instances, none of IMGT positions 42, 49, 50, and 52 of the VHH are humanized. In some instances, the VHH CDR1, VHH CDR2, and VHH CDR3 are grafted onto a universal humanized VHH scaffold, h-NbBCII10FGLA. See, e.g., Vincke et al., J. Biol. Chem., 284, 3273-3284 (2009), which is incorporated by reference herein.

[0263] Table 1 A provides the sequences of exemplary humanized VHHs that specifically bind to PD-1 (human PD-1, or human and cynomolgus PD-1). Each of these humanized VHHs are encompassed by this disclosure.Attorney Docket No.: 45817-0163WO1

[0264] Table 1A. Exemplary Humanized Antibodies

[0265] VHH ID Amino Acid Sequence of Humanized Anti-PD-1 VHH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWFRQAPGKEREFVSAISWSGGSSYYADSVKGRFTISR

[0266] P3B3 hl DNSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGDDYDYWGQGTLVTVSS (SEQ ID NO:65)

[0267] EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMSWFRQAPGKEREFVSAITWSGGSSGYADSVKDRFTISR

[0268] P3B3 h2 DNSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGDDYDYWGQGTLVTVSS (SEQ ID NO:66)

[0269] EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVSAITWSSGSSGYSDPVKDRFTISR

[0270] P3B3 h3 DNSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGDDYDYWGQGTLVTVSS (SEQ ID NO:67)

[0271] EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVSAITWSSGSSGYSDPVKDRFTISR

[0272] P3B3 h4 DNSKNTVYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGDDYDYWGQGTLVTVSS (SEQ ID NO:68)

[0273] EVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSDPVKDRFTISR

[0274] P3B3 h5 DNSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGDDYDYWGQGTLVTVSS (SEQ ID NO:69)

[0275] EVQLVESGGGLVQPGGSLRLSCSASGRTFSSYAMGWFRQAPGKEREFVSAITWSSGSSGYSDPVKDRFTISR

[0276] P3B3 h6 DNSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGDDYDYWGQGTLVTVSS (SEQ ID NO:70)

[0277] QVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVSAITWSSGSSGYSDPVKDRFTISR

[0278] P3B3 h7 DNSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGDDYDYWGQGTLVTVSS (SEQ ID NO:71)

[0279] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWFRQAPGKEREFVSAITWSSGSSYYADSVKDRFTISR

[0280] P3B3 h8 DNSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGDDYDYWGQGTLVTVSS (SEQ ID NO:72)

[0281] QVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSDPVKDRFTISR

[0282] P3B3_h7_A DNSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGDDYDYWGQGTLVTVSS (SEQ ID NO: 73)

[0283] P3B3_h7_B QVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSDPVKDRFTISR DNSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGDDYDYWGQGTQVTVSS (SEQ ID NO: 74) QVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSSGSSGYSDPVKDRFTISR

[0284] P3B3_h7_C DNSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGDDYDYWGQGTLVTVSS (SEQ ID NO: 75)

[0285] QVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSSGSSGYSDPVKDRFTISR

[0286] P3B3_h7_D DNSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGDDYDYWGQGTQVTVSS (SEQ ID NO: 76)

[0287]

[0288] Attorney Docket No.: 45817-0163WO1

[0289] Table IB provides the sequences of exemplary humanized and affinity matured VHHs that specifically bind to PD-1 (human PD-1, or human and cynomolgus PD-1). Each of these humanized VHHs are encompassed by this disclosure.

[0290] Table 1B. Exemplary Humanized and Affinity Matured Antibodies

[0291] VHH ID Amino Acid Sequence of Humanized and Affinity Matured Anti-PD-1 VHH QVQLVESGGGLVQPGGSLRLSCAASGITFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSFPVKDRFTISRD

[0292] P3B3_v.1 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGDDYDYWGQGTQVTVSS (SEQ ID NO:77)

[0293] QVQLVESGGGLVQPGGSLRLSCAASGITFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSHPVKDRFTISRD

[0294] P3B3_v.2 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGDDYDYWGQGTQVTVSS (SEQ ID NO:78)

[0295] QVQLVESGGGLVQPGGSLRLSCAASGITFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSRPVKDRFTISRD

[0296] P3B3_v.3 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGDDYDYWGQGTQVTVSS (SEQ ID NO:79)

[0297] QVQLVESGGGLVQPGGSLRLSCAASGITFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSYPVKDRFTISRD

[0298] P3B3_v.4 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGDDYDYWGQGTQVTVSS (SEQ ID NO:80)

[0299] QVQLVESGGGLVQPGGSLRLSCAASGITFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSRPVKDRFTISRD

[0300] P3B3_v.5 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGEDYDYWGQGTQVTVSS (SEQ ID NO:81)

[0301] QVQLVESGGGLVQPGGSLRLSCAASGRHFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSFPVKDRFTISRD

[0302] P3B3_v.6 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGEDYDYWGQGTQVTVSS (SEQ ID NO:82)

[0303] QVQLVESGGGLVQPGGSLRLSCAASLRTFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSRPVKDRFTISRD

[0304] P3B3_v.7 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGEDYDYWGQGTQVTVSS (SEQ ID NO:83)

[0305] QVQLVESGGGLVQPGGSLRLSCAASLRTFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSHPAKDRFTISRD

[0306] P3B3_v.8 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGDDYDYWGQGTQVTVSS (SEQ ID NO:84)

[0307] QVQLVESGGGLVQPGGSLRLSCAASLRTFSSYAMGWFRQAPGKEREFVAAITWSSGSQGYSRPVKDRFTISRD

[0308] P3B3_v.9 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVIWVFGDDYDYWGQGTQVTVSS (SEQ ID NO:85)

[0309] QVQLVESGGGLVQPGGSLRLSCAASGITFSSYAMGWFRQAPGKEREFVAAITWSSRSSGYSHPVKDRFTISRD

[0310] P3B3_v.10 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFEWVISVFGDDYDYWGQGTQVTVSS (SEQ ID NO:86)

[0311] QVQLVESGGGLVQPGGSLRLSCAASGITFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSRPAKDRFTISRD

[0312] P3B3_v.12 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGEDYDYWGQGTQVTVSS (SEQ ID NO:87)

[0313] QVQLVESGGGLVQPGGSLRLSCAASGITFSSYAMGWFRQAPGKEREFVAAITWSSRSSGYSDPAKDRFTISRD

[0314] P3B3_v.13 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFEGVISVFGEDYDYWGQGTQVTVSS (SEQ ID NO:88)

[0315] QVQLVESGGGLVQPGGSLRLSCAASLRTFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSRPAKDRFTISRD

[0316]

[0317] P3B3_v.11 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVIWVFGEDYDYWGQGTQVTVSS (SEQ ID NO:89)Attorney Docket No.: 45817-0163WO1

[0318] QVQLVESGGGLVQPGGSLRLSCAASGITFSSYAMGWFRQAPGKEREFVAAITWSSRSSGYSFPAKDRFTISRD

[0319] P3B3_v.14 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGEDYDYWGQGTQVTVSS (SEQ ID NO:90)

[0320] QVQLVESGGGLVQPGGSLRLSCAASGRHFSSYAMGWFRQAPGKEREFVAAITWSSRSSGYSFPAKDRFTISRD

[0321] P3B3_v.15 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFEGVISVFGEDYDYWGQGTQVTVSS (SEQ ID NO:91)

[0322] QVQLVESGGGLVQPGGSLRLSCAASGRHFSSYAMGWFRQAPGKEREFVAAITWSSGSQGYSRPAKDRFTISRD

[0323] P3B3_v.16 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFEWVISVFGEDYDYWGQGTQVTVSS (SEQ ID NO:92)

[0324] QVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWSSGSQGYSRPVKDRFTISRD

[0325] P3B3_v.17 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGDDYDYWGQGTQVTVSS (SEQ ID NO:93)

[0326] QVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWSSGSRGYSYPVKDRFTISRD

[0327] P3B3_v.18 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGDDYDYWGQGTQVTVSS (SEQ ID NO:94)

[0328] QVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSEPVKDRFTISRD

[0329] P3B3_v.19 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVISVFGEDYDYWGQGTQVTVSS (SEQ ID NO:95)

[0330] QVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSEPVKDRFTISRD

[0331] P3B3_v.20 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGEDYDYWGQGTQVTVSS (SEQ ID NO:96)

[0332] QVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSYPVKDRFTISRD

[0333] P3B3_v.21 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGEDYDYWGQGTQVTVSS (SEQ ID NO:97)

[0334] QVQLVESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSFPVKDRFTISRD

[0335] P3B3_v.22 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGEDYDYWGQGTQVTVSS (SEQ ID NO:98)

[0336] QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSEPVKDRFTISRD

[0337] P3B3_v.23 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGEDYDYWGQGTQVTVSS (SEQ ID NO:99)

[0338] QVQLVESGGGLVQPGGSLRLSCAASGLTFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSEPVKDRFTISRD

[0339] P3B3_v.24 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGEDYDYWGQGTQVTVSS (SEQ ID NO:100)

[0340] QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSEPAKDRFTISRD

[0341] P3B3_v.25 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGEDYDYWGQGTQVTVSS (SEQ ID NO:101)

[0342] QVQLVESGGGLVQPGGSLRLSCAASGLTFSSYAMGWFRQAPGKEREFVAAITWSSGSSGYSEPAKDRFTISRD

[0343] P3B3_v.26 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGEDYDYWGQGTQVTVSS (SEQ ID NO:102)

[0344] QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMGWFRQAPGKEREFVAAITWSSGSQGYSEPAKDRFTISRD

[0345] P3B3_v.27 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGEDYDYWGQGTQVTVSS (SEQ ID NO:103)

[0346] QVQLVESGGGLVQPGGSLRLSCAASGLTFSSYAMGWFRQAPGKEREFVAAITWSSGSQGYSEPAKDRFTISRD

[0347]

[0348] P3B3_v.28 NSKNTLYLQMNSLRAEDTAVYYCAARRNRVGFESVITVFGEDYDYWGQGTQVTVSS (SEQ ID NO:104)Attorney Docket No.: 45817-0163WO1 This disclosure features a polypeptide comprising a VHH that specifically binds to PD-1 (human, or human and cyno) and comprises the amino acid sequence of any one of SEQ ID NOs: 65 to 104 (e.g., SEQ ID NO: 103, 90, 71, or 74). In some cases, the VHH that specifically binds to PD-1 (human, or human and cyno) comprises an amino acid sequence that is 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 the sequences set forth in SEQ ID NOs: 65 to 104 (e.g., SEQ ID NO: 103, 90, 71, or 74). In certain cases, the VHH that specifically binds to PD-1 (human, or human and cyno) comprises an amino acid sequence that is 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 the sequences set forth in SEQ ID NOs: 65 to 104 (e.g., SEQ ID NO: 103, 90, 71, or 74) and has the three VHH CDRs of the VHH P3. B3 set forth in any one of Tables 3 to 7 with zero to three substitutions in one, two, or all three VHH CDRs. In some cases, the VHH that specifically binds to PD-1 (human, or human and cyno) comprises the amino acid sequence of any one of SEQ ID NOs: 65 to 104 (e.g., SEQ ID NO: 103, 90, 71, or 74) except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions within the VHH, wherein the VHH has the three CDRs of VHH P3. B3 set forth in any one of Tables 3 to 7 with zero to three substitutions in one, two, or all three VHH CDRs. In some cases, the substitutions are in the framework region(s). In other cases, the substitutions are in one or more (e.g., 1, 2, 3) of the three VHH CDRs. In yet other instances, the substitutions are in the framework region(s) and in one or more (e.g., 1, 2, 3) of the three VHH CDRs. In some cases, the substitutions are conservative amino acid substitutions. In other instances, the substitutions are not conservative amino acid substitutions. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as PD-1 agonists. In certain instances, the VHHs have an EC50 of about 5 nM to about 60 nM. In certain instances, the VHHs have an EC50 of about 5 nM to about 35 nM. In other instances, the VHHs have an EC50 of about 5 nM to about 15 nM.Attorney Docket No.: 45817-0163WO1 Also encompassed by this disclosure are binding molecules or polypeptides that comprise a VHH that specifically binds to PD-1 (human, or human and cyno), wherein the VHH comprises the VHH CDR1, VHH CDR2, and VHH CDR3 of the VHH of any one of the clones listed in Table 1A (e.g., SEQ ID NO: 71 or 74) with zero to three 5 substitutions in one, two, or all three VHH CDRs. The VHH CDRs of the VHH can be based on any CDR definition. Tables 2A through 2E provide CDR sequences of two clones from Table IB based on multiple CDR definitions.

[0349] Table 2A: Humanized & Affinity Matured CDRs Based on IMGT Definition

[0350] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0351] P3B3_v.14 GITFSSYA ITWSSRSS AARRNRVGFESVITVFGEDYDY (SEQ ID (SEQ ID NO:107) (SEQ ID NO:108) NO:106)

[0352] P3B3_v.27 GFTFSSYA ITWSSGSQ AARRNRVGFESVITVFGEDYDY (SEQ ID (SEQ ID NO: 110) (SEQ ID NO: 108)

[0353]

[0354] NO: 109)

[0355] w Table 2B: Humanized & Affinity Matured CDRs Based on Kabat Definition

[0356] VHH ID VHH- VHH-CDR2 VHH-CDR3

[0357] CDR1

[0358] P3B3_v.14 SYAMG AITWSSRSSGYSFPAKD RRNRVGFESVITVFGEDYDY (SEQ ID (SEQ ID NO:111) (SEQ ID NO:112)

[0359] NO:23)

[0360] P3B3_v.27 SYAMG AITWSSGSQGYSEPAKD RRNRVGFESVITVFGEDYDY (SEQ ID (SEQ ID NO: 113) (SEQ ID NO: 112) NO:23)

[0361]

[0362] Table 2C: Humanized & Affinity Matured CDRs Based on Enhanced Chothia Definition VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0363] P3B3_v.14 GITFSSYAMG AITWSSRSSG RRNRVGFESVITVFGEDYDY

[0364]

[0365] (SEQ ID NO: 115) (SEQ ID NO: 112)Attorney Docket No.: 45817-0163WO1 (SEQ ID

[0366] NO: 114)

[0367] P3B3_v.27 GFTFSSYAMG AITWSSGSQG RRNRVGFESVITVFGEDYDY (SEQ ID (SEQ ID NO: 117) (SEQ ID NO: 112)

[0368] NO: 116)

[0369]

[0370] Table 2D: Humanized & Affinity Matured CDRs Based on Contact Definition VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0371] P3B3_v.14 SSYAMG FVAAITWSSRSSG AARRNRVGFESVITVFGEDYD (SEQ ID (SEQ ID NO:118) (SEQ ID NO:515)

[0372] NO:54)

[0373] P3B3_v.27 SSYAMG FVAAITWSSGSQG AARRNRVGFESVITVFGEDYD (SEQ ID (SEQ ID NO: 119) (SEQ ID NO:515)

[0374] NO:54)

[0375]

[0376] Table 2E: Humanized & Affinity Matured CDRs Based on Chothia Definition VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0377] P3B3_v.14 GITFSSY TWSSRS RRNRVGFESVITVFGEDYDY (SEQ ID (SEQ ID NO:121) (SEQ ID NO:112)

[0378] NO: 120)

[0379] P3B3_v.27 GFTFSSY TWSSGS RRNRVGFESVITVFGEDYDY (SEQ ID (SEQ ID NO:36) (SEQ ID NO: 112)

[0380] NO: 122)

[0381]

[0382] In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to PD-1 (human, or human and cyno), wherein the VHH comprises the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of any one clone listed in Table 2A, 2B, 2C, 2D, or 2E. Thus, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds PD-1 (human, or human and cyno), wherein the VHH comprises:Attorney Docket No.: 45817-0163WO1 (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 106, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 107, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 108;

[0383] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 111, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112;

[0384] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 114, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 115, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112;

[0385] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:54, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 118, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 515; or

[0386] (e) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 120, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 121, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112.

[0387] The disclosure also features a binding molecule or polypeptide comprising a VHH that specifically binds PD-1 (human, or human and cyno), wherein the VHH comprises: (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 109, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 110, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 108;

[0388] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 113, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112;

[0389] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 116, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 117, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112;Attorney Docket No.: 45817-0163WO1 (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:54, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 119, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:515; or

[0390] (e) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:36, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112.

[0391] In some instances, the VHH that binds PD-1 (human or human and cynomolgus) comprises a VHH CDR1 sequence set forth in SEQ ID NO: 9 with zero to 3 amino acid substitutions; a VHH CDR2 sequence set forth in SEQ ID NO: 10 with zero to 3 amino acid substitutions; and a VHH CDR3 sequence set forth in SEQ ID NO: 11 with zero to 3 amino acid substitutions. In certain instances, the VHH that binds PD-1 (human or human and cynomolgus) comprises a VHH CDR1 sequence set forth in SEQ ID NO: 9 with zero to 1 amino acid substitutions; a VHH CDR2 sequence set forth in SEQ ID NO: 10 with zero to 1 amino acid substitutions; and a VHH CDR3 sequence set forth in SEQ ID NO: 11 with zero to 1 amino acid substitutions. In some cases, substitutions may be conservative amino acid substitutions. In certain cases, the substitutions can be made at one or more of the underlined residues in: GRTFSSYA (SEQ ID NO:9); ITWSSGSS (SEQ ID NO: 10); and AARRNRVGFESVISVFGDDYDY (SEQ ID NO: 11). In certain the VHH that binds PD-1 (human or human and cynomolgus) comprises the following CDRs: (G / L)(R / I / L / F)(T / H)FSSYA (SEQ ID NO: 123); I(T / S)WSS(G / R)S(S / Q / R) (SEQ ID NO: 124); and AARRNRVGFE(S / W / G)VI(S / I / W)VFG(D / E)DYDY (SEQ ID NO: 125).

[0392] In some instances, the above VHHs are in a monovalent format. In some instances, the above VHHs are in a bivalent format (e.g., by linking the VHH to a hinge-1g Fc domain). In some instances, the above VHHs are linked to a hinge-Fc of a human Ig (e.g., IgA, IgGl, IgG2, IgG3, or IgG4). In some instances, the above VHHs are linked to a hinge from human IgA. In some instances, the above VHHs are linked to a hinge from human IgGl or IgG4. In some instances, the above VHHs comprise a Fc regionAttorney Docket No.: 45817-0163WO1 from human IgGl or human IgG4. In some instances, any of the above VHHs can be linked to a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of the hinge and Fc domain of human IgGl (SEQ ID NO:468). In some instances, any of the above VHHs can be linked to a sequence that is identical to the amino acid sequence of the hinge and Fc domain of human IgGl (SEQ ID NO:468) In some instances, the human Ig Fc region is modified to e.g., alter (increase or decrease) the effector function and / or to promote heterodimerization.

[0393] Bispecific Constructs

[0394] Other binding molecules are also encompassed by this disclosure. For example, the PD-1 binding VHHs or polypeptides of this disclosure may be part of a bispecific construct (i.e., a construct that binds to two different epitopes). A VHH of this disclosure can be linked to itself or to one or more VHHs of this disclosure. In some instances, a VHH of this disclosure may be linked to a VHH that binds to a different epitope of PD-1 and / or to one or more other antigens. See, e.g., Verhaar et al., Semin. Immunol., 2021; 52: 101425 (incorporated by reference herein). For example, the other antigen may be a T cell antigen, a NK cell antigen, a tumor cell membrane protein, a tumor microenvironment target, a ligand, cytokine or chemokine, or immune cell marker. In some cases, the other antigen may be VEGF, PD-L1, TIM-3, or CTLA-4.

[0395] In some instances, a VHH of this disclosure may be linked to a VHH that binds to albumin (e.g., human serum albumin). The linker used to join the VHHs preserves the functionality of the different VHH domains. Non-limiting examples of linkers suitable for linking the VHH domains in the polypeptides of this disclosure include, but are not limited to, (GS)n (SEQ ID NO: 516), (GGS)n (SEQ ID NO: 517), (GGGS)n (SEQ ID NO:470), (GGSG)n (SEQ ID NO:471), (GGSGG)n (SEQ ID NO:472), or (GGGGS)n (SEQ ID NO: 473), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some instances the length of the linker can be about 15 amino acids. In one instance the linker is (G4S)3(SEQ ID NO:474)Attorney Docket No.: 45817-0163WO1 In some instances, the bispecific construct comprises a first VHH of this disclosure (that specifically binds to PD-1) linked to a first human Ig hinge-Fc region (e.g., from IgGl, IgG2, IgG3, IgG4, or IgA) and a second VHH (that binds to a different epitope of PD-1 than the first VHH, or that specifically binds to a different antigen) linked to a second human Ig hinge-Fc region (e.g., from IgGl, IgG2, IgG3, IgG4, or IgA). In certain instances, the human Ig hinge-Fc region is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:468. In certain instances, the human Ig hinge-Fc region used in the bispecific constructs have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions relative to SEQ ID NO:468. In certain instances, the Fc region is engineered to promote heterodimerization. This can be done by any method known in the art such as the use of knob-into-hole, electrostatic steering, or any other design strategies for heterodimerization of Fc. See, Ha et al., Front. Immunol. 2016; 7: 394, Moore et al., Methods, 154:38-50 (2019); WO 2017 / 106462, and Yu et al., Nat Methods, 16, 1095-1100 (2019) (all incorporated by reference herein). In certain instances, the Fc region is also engineered to improve its therapeutic activity. See e.g., Mimoto et al., Curr Pharm Biotechnol., 2016; 17(15): 1298-1314; Chen et al., Theranostics, 2021; 11(4): 1901—1917; Delidakis et al., Ann Rev. Biomed Engg., 24:249-274 (2022) (all incorporated by reference herein).

[0396] Multispecific Constructs

[0397] Another binding molecule encompassed by this disclosure is a multispecific construct. These multispecific constructs bind to three or more different epitopes. In one instance, three or more different VHHs are linked together. In some instances, one VHH is linked by its C-terminal to the N-terminal of the heavy chain of a whole antibody. In another instance, two VHHs are linked by their C-terminals to the N-terminals of the two heavy chains of a whole antibody. In some cases, the two VHHs are identical. In other cases, the two VHHs are different. In yet another instance, one VHH is linked by its C-terminal to the C-terminal of the heavy chain of a whole antibody. In another instance, two VHHs are linked by their C-terminals to the C-terminals of the two heavy chains of a whole antibody. In some cases, the two VHHs are identical. In other cases, the two VHHsAttorney Docket No.: 45817-0163WO1 are different. In some instances, a multispecific construct is based on a bispecific construct described above, wherein the bispecific construct is conjugated at the C-terminal of one or both of its heavy chains to one or two VHHs. The C-terminally conjugated VHH or VHHs can bind to a different epitope than the VHHs of the bispecific construct. In all of the above, at least one antigen binding domain specifically binds to PD-1 (human, or human and cyno).

[0398] Conjugates

[0399] The polypeptides, binding molecules, and VHHs of this disclosure can be fused, linked, or conjugated to other moieties. These moieties may be for detection, purification, to measure agonism (e.g., by linking to IgGl hinge-Fc where binding to FcR is required) or to add another therapeutic agent, radionuclide, or toxin to the polypeptides, binding molecules, and VHHs. In certain instances, the moiety is linked or conjugated to the C-terminal of the polypeptide, binding molecule, or VHH. In some instances, the linker can be any of the Gly Ser linkers discussed above (e.g., (G4S)3(SEQ ID NO:474)) or the natural hinge of human IgA. In some instances, a radionuclide may be directly conjugated to the polypeptide, binding molecule, or VHH, or conjugated via a small chelator.

[0400] In some instances, a polypeptide, binding molecule, or VHH is linked to a detection or purification tag. In some instances, the tag moiety is His6 (SEQ ID NO:475), c-myc, hemagglutinin (HA), Flag, headlock tag, C-tag, ALFA tag, Avi tag, alkaline phosphatase or horseradish peroxidase. In some instances, a polypeptide, binding molecule, or VHH is linked to a fluorophore (GFP, RFP). In some instances, a polypeptide, binding molecule, or VHH is linked to a drug. In some instances, a polypeptide, binding molecule, or VHH is linked to a nanoparticle (e.g., liposomes, micelles, albumin-based NPs, polymer based polymerosomes). In some instances, a polypeptide, binding molecule, or VHH is linked to a toxic agent. In some instances, a polypeptide, binding molecule, or VHH is linked to a radionuclide (e.g.,89Zr,68 / 67Ga,18F,99mTc). In some instances, a polypeptide, binding molecule, or VHH is linked to a VHH,Attorney Docket No.: 45817-0163WO1 a Fab, a scFv, or a whole antibody. In some instances, a polypeptide, binding molecule, or VHH is linked to a multimerization module (e.g. dimerization, trimerization, tetramerization, or pentamerization module). See e.g., Sadeghnezhad, G. et al., Int J Mol Sci., 20, 4818, 2019; Zhang et al., J. Mol Biol., 335, 49-56, 2004 (both incorporated by reference herein). In some cases, the dimerization module is a human Ig hinge-Fc domain (e.g., from IgGl, IgG4, IgA). In some cases, the dimerization module is the hinge-Fc domain of IgGl (e.g., SEQ ID NO:468). In some cases, the trimerization module, is e.g. a collagen trimerization moiety, particularly a human collagen moiety, and a lung surfactant protein D moiety. In some instances, a polypeptide, binding molecule, or VHH is linked to a moiety that facilitates the polypeptide crossing the blood brain barrier (e.g., anti-transferrin antibody, FC5, FC44, a VHH that binds IGF-1R, VHHs E9, ni3a, and pa2H). In some instances, a polypeptide, binding molecule, or VHH is linked to a half-life extension moiety (e.g., PEG (linear and branched), human serum albumin, a VHH that binds human serum albumin, PASylation (PA600), human Ig Fc).

[0401] Methods of Making and Pharmaceutical Compositions

[0402] The disclosure provides a nucleic acid or nucleic acids that encode the polypeptides, binding molecules, and VHHs of this disclosure. The nucleic acid may be DNA or mRNA. The nucleic acid may be modified. The coding sequence may be modified to take into account the preferred codon usage of the expression host.

[0403] Also provided are vectors that comprise the above nucleic acids. In some instances, the vector is an expression vector. In other instances, the vector is a viral vector.

[0404] Also provided are host cells that comprise the nucleic acid or vectors above. In some instances, the host cells are bacterial cells, yeast cells, insect cells, or mammalian cells. In some instances, the host cell is E. coli. In some instances, the host cell is Pichia pastoris or Saccharomyces cerevisiae. In some instances, the host cell is a CHO cell, a COS cell, a HEK293T cell, or a NIH3T3 cell.Attorney Docket No.: 45817-0163WO1 The disclosure also features methods of making the polypeptides, binding molecules, or VHHs described herein. The method involves culturing the host cell under conditions that promote the expression of the polypeptide, binding molecule, or VHH. In some instances, the polypeptide, binding molecule, or VHH is purified. In certain instances, the polypeptide, binding molecule, or VHH is formulated as a sterile pharmaceutical composition.

[0405] Also provided are pharmaceutical compositions comprising the polypeptides, binding molecules, or VHHs described herein, and a pharmaceutically acceptable carrier. In some instances, the pharmaceutically acceptable carrier is phosphate buffered saline. In some instances, the pharmaceutically acceptable carrier is sterile distilled water.

[0406] mRNA Constructs

[0407] In some aspects, the polynucleotides disclosed herein are or function as a messenger RNA (mRNA). As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide which encodes at least one peptide or polypeptide of interest and which is capable of being translated to produce the encoded peptide polypeptide of interest in vitro, in vivo, in situ or ex vivo.

[0408] The disclosure features an mRNA comprising an ORF encoding any of the polypeptides described above. The mRNA may be a modified mRNA (e.g., some or all of the uracils in the mRNA may be N1 -methylpseudouracils).

[0409] In some aspects, provided is a polynucleotide comprising an mRNA comprising: (i) a 5' UTR; (ii) an open reading frame (ORF) encoding a polypeptide, a VHH, a bispecific antibody, or a multispecific antibody described herein; (iii) a stop codon; and (iv) a 3' UTR. In certain instances, the mRNA can include a 5’-cap. In certain instances, the mRNA can also include a poly A region.

[0410] The disclosure features compositions including an mRNA encoding a polypeptide described herein (e.g., an anti-PD-1 VHH, a polypeptide comprising an anti-PD-1 VHH, a bivalent anti-PD-1 VHH-human IgGl (i.e., the VHH is linked at its C-terminal to aAttorney Docket No.: 45817-0163WO1 hinge-Fc domain of human IgGl), a bivalent anti-PD-1 VHH-IgG4PAA (i.e., the VHH is linked at its C-terminal to a hinge-Fc domain of human IgG4PAA), a bispecific antibody comprising an anti-PD-1 VHH, or a multispecific antibody comprising an anti-PD-1 VHH). In one instance, the mRNAs include (i) a 5 ’-cap structure; (ii) a 5’-UTR; (iii) an open reading frame encoding a polypeptide described herein; (iv) a 3’-untranslated region (3’-UTR); and (v) a poly A region.

[0411] mRNA: 5 '-cap

[0412] The 5 '-cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly A binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns removal during mRNA splicing.

[0413] mRNA molecules may be 5 '-end capped generating a 5 '-ppp-5 '-triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA. This 5 '-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-O-methylated. 5 '-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[0414] Alterations to the nucleic acids of the present disclosure may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5 '-ppp-5' phosphorodiester linkages, alternative nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer’s instructions to create a phosphorothioate linkage in the 5 '-ppp-5' cap. Additional alternative guanosineAttorney Docket No.: 45817-0163WO1 nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.

[0415] Additional alterations include, but are not limited to, 2'-O-methylation of the ribose sugars of 5 '-terminal and / or 5 '-anteterminal nucleotides of the mRNA (as mentioned above) on the 2'-hydroxyl group of the sugar ring. Multiple distinct 5'-cap structures can be used to generate the 5 '-cap of a nucleic acid molecule, such as an mRNA molecule.

[0416] 5 ’-cap structures include those described in International Patent Publication Nos. WO2008 / 127688, W02008 / 016473, and WO2011 / 015347, each of which is incorporated herein by reference in its entirety.

[0417] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (z.e., endogenous, wild-type or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and / linked to a nucleic acid molecule.

[0418] For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanosines linked by a 5 '-5 '-triphosphate group, wherein one guanosine contains an N7 methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5 '-triphosphate-5 guanosine (m7G-3 ’mppp-G; which may equivalently be designated 3' O-Me-m7G(5')ppp(5')G)). The 3'-0 atom of the other, unaltered, guanosine becomes linked to the 5 '-terminal nucleotide of the capped nucleic acid molecule (e.g., an mRNA or mmRNA). The N7- and 3'-O-methlyated guanosine provides the terminal moiety of the capped nucleic acid molecule (e.g., mRNA or mmRNA).

[0419] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O-methyl group on guanosine i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).Attorney Docket No.: 45817-0163WO1 In some instances, the cap is a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog may be modified at different phosphate positions with a boranophosphate group or a phophoroselenoate group such as the dinucleotide cap analogs described in US Patent No. US 8,519, 110, the contents of which are herein incorporated by reference in its entirety.

[0420] In some instances, the cap analog is a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog known in the art and / or described herein. Nonlimiting examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog include a N7-(4-chlorophenoxyethyl)-G(5’)ppp(5’)G and a N7-(4-chlorophenoxyethyl)-m3'oG(5’)ppp(5’)G cap analog (see, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 21:4570-4574 (2013); the contents of which are herein incorporated by reference in its entirety). In some instances, a cap analog of the present disclosure is a 4-chloro / bromophenoxyethyl analog.

[0421] While cap analogs allow for the concomitant capping of a nucleic acid molecule in an tn vitro transcription reaction, up to 20% of transcripts remain uncapped. This, as well as the structural differences of a cap analog from endogenous 5 '-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.

[0422] Nucleic acids of the disclosure (e.g., mRNAs of the disclosure) may also be capped post-transcriptionally, using enzymes. 5’ cap structures produced by enzymatic capping may enhance binding of cap binding proteins, increase half-life, reduce susceptibility to 5' endonucleases and / or reduce 5' decapping, as compared to synthetic 5 '-cap structures known in the art (or to a wild-type, natural or physiological 5 '-cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-O-methyltransferase enzyme can create a canonical 5 '-5 '-triphosphate linkage between the 5 '-terminal nucleotide of an mRNA and a guanosine cap nucleotide wherein the cap guanosine contains an N7 methylation and the 5 '-terminal nucleotide of the mRNAAttorney Docket No.: 45817-0163WO1 contains a 2'-O-methyl. Such a structure is termed the Capl structure. This cap results in a higher translational -competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5 'cap analog structures known in the art. Cap structures include 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), 7mG(5')-ppp(5')NlmpN2mp (cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (cap 4). In one instance, the 5’ terminal cap comprises m7GpppGm.

[0423] 5' terminal caps may include endogenous caps or cap analogs. A 5' terminal cap may include a guanosine analog. Useful guanosine analogs include inosine, N1 -methylguanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0424] In some instances, the nucleic acids described herein may contain a modified 5’-cap. A modification on the 5 ’-cap may increase the stability of mRNA, increase the halflife of the mRNA, and could increase the mRNA translational efficiency. The modified 5 ’-cap may include, but is not limited to, one or more of the following modifications: modification at the 2’ and / or 3’ position of a capped guanosine triphosphate (GTP), a replacement of the sugar ring oxygen (that produced the carbocyclic ring) with a methylene moiety (CH2), a modification at the triphosphate bridge moiety of the cap structure, or a modification at the nucleobase (G) moiety.

[0425] Untranslated Regions (UTRs)

[0426] Untranslated regions (UTRs) are nucleic acid sections of a polynucleotide before a start codon (5′ UTR) and after a stop codon (3′ UTR) that are not translated. In some instances, a polynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of the disclosure comprising an open reading frame (ORF) encoding an antibody further comprises UTR (e.g., a 5 'UTR or functional fragment thereof, a 3 'UTR or functional fragment thereof, or a combination thereof).Attorney Docket No.: 45817-0163WO1 Translation of a polynucleotide comprising an open reading frame encoding a polypeptide can be controlled and regulated by a variety of mechanisms that are provided by various cis-acting nucleic acid structures. For example, naturally-occurring, cis-acting RNA elements that form hairpins or other higher-order (e.g., pseudoknot) intramolecular mRNA secondary structures can provide a translational regulatory activity to a polynucleotide, wherein the RNA element influences or modulates the initiation of polynucleotide translation, particularly when the RNA element is positioned in the 5' UTR close to the 5’-cap structure (Pelletier and Sonenberg (1985) Cell 40(3):515-526; Kozak (1986) Proc Natl Acad Sci 83:2850-2854).

[0427] Cis-acting RNA elements can also affect translation elongation, being involved in numerous frameshifting events (Namy et al., (2004) Mol Cell 13(2): 157-168). Internal ribosome entry sequences (IRES) represent another type of cis-acting RNA element that are typically located in 5' UTRs, but have also been reported to be found within the coding region of naturally-occurring mRNAs (Holcik et al. (2000) Trends Genet 16(10):469-473). In cellular mRNAs, IRES often coexist with the 5'-cap structure and provide mRNAs with the functional capacity to be translated under conditions in which cap-dependent translation is compromised (Gebauer et al., (2012) Cold Spring Harb Perspect Biol 4(7):a012245). Another type of naturally-occurring cis-acting RNA element comprises upstream open reading frames (uORFs). Naturally-occurring uORFs occur singularly or multiply within the 5' UTRs of numerous mRNAs and influence the translation of the downstream major ORF, usually negatively (with the notable exception of GCN4 mRNA in yeast and ATF4 mRNA in mammals, where uORFs serve to promote the translation of the downstream major ORF under conditions of increased eIF2 phosphorylation (Hinnebusch (2005) Annu Rev Microbiol 59:407-450)). Additional exemplary translational regulatory activities provided by components, structures, elements, motifs, and / or specific sequences comprising polynucleotides (e.g., mRNA) include, but are not limited to, mRNA stabilization or destabilization (Baker & Parker (2004) Curr Opin Cell Biol 16(3):293-299), translational activation (Villalba et al., (2011) Curr Opin Genet Dev 21 (4):452-457), and translational repression (Blumer et al.,Attorney Docket No.: 45817-0163WO1 (2002) Mech Dev 110(1-2):97-112). Studies have shown that naturally-occurring, cisacting RNA elements can confer their respective functions when used to modify, by incorporation into, heterologous polynucleotides (Goldberg-Cohen et al., (2002) J Biol Chem 277(16): 13635-13640).

[0428] The present disclosure provides synthetic polynucleotides comprising a modification (e.g., an RNA element), wherein the modification provides a desired translational regulatory activity. In some instances, the disclosure provides a polynucleotide comprising a 5’ untranslated region (UTR), an initiation codon, a full open reading frame encoding a polypeptide, a 3’ UTR, and at least one modification, wherein the at least one modification provides a desired translational regulatory activity, for example, a modification that promotes and / or enhances the translational fidelity of mRNA translation. In some instances, the desired translational regulatory activity is a cis-acting regulatory activity. In some instances, the desired translational regulatory activity is an increase in the residence time of the 43 S pre-initiation complex (PIC) or ribosome at, or proximal to, the initiation codon. In some instances, the desired translational regulatory activity is an increase in the initiation of polypeptide synthesis at or from the initiation codon. In some instances, the desired translational regulatory activity is an increase in the amount of polypeptide translated from the full open reading frame. In some instances, the desired translational regulatory activity is an increase in the fidelity of initiation codon decoding by the PIC or ribosome. In some instances, the desired translational regulatory activity is inhibition or reduction of leaky scanning by the PIC or ribosome. In some instances, the desired translational regulatory activity is a decrease in the rate of decoding the initiation codon by the PIC or ribosome. In some instances, the desired translational regulatory activity is inhibition or reduction in the initiation of polypeptide synthesis at any codon within the mRNA other than the initiation codon. In some instances, the desired translational regulatory activity is inhibition or reduction of the amount of polypeptide translated from any open reading frame within the mRNA other than the full open reading frame. In some instances, the desired translational regulatory activity is inhibition or reduction in the production of aberrantAttorney Docket No.: 45817-0163WO1 translation products. In some instances, the desired translational regulatory activity is a combination of one or more of the foregoing translational regulatory activities.

[0429] Accordingly, the present disclosure provides a polynucleotide, e.g., an mRNA, comprising an RNA element that comprises a sequence and / or an RNA secondary structure(s) that provides a desired translational regulatory activity as described herein. In some aspects, the mRNA comprises an RNA element that comprises a sequence and / or an RNA secondary structure(s) that promotes and / or enhances the translational fidelity of mRNA translation. In some aspects, the mRNA comprises an RNA element that comprises a sequence and / or an RNA secondary structure(s) that provides a desired translational regulatory activity, such as inhibiting and / or reducing leaky scanning. In some aspects, the disclosure provides an mRNA that comprises an RNA element that comprises a sequence and / or an RNA secondary structure(s) that inhibits and / or reduces leaky scanning thereby promoting the translational fidelity of the mRNA.

[0430] In some instances, the RNA element comprises natural and / or modified nucleotides. In some instances, the RNA element comprises of a sequence of linked nucleotides, or derivatives or analogs thereof that provides a desired translational regulatory activity as described herein. In some instances, the RNA element comprises a sequence of linked nucleotides, or derivatives or analogs thereof that forms or folds into a stable RNA secondary structure, wherein the RNA secondary structure provides a desired translational regulatory activity as described herein. RNA elements can be identified and / or characterized based on the primary sequence of the element (e.g., GC-rich element), by RNA secondary structure formed by the element (e.g. stem-loop), by the location of the element within the RNA molecule (e.g., located within the 5’ UTR of an mRNA), by the biological function and / or activity of the element (e.g., “translational enhancer element”), and any combination thereof.

[0431] In some aspects, the disclosure provides an mRNA having one or more structural modifications that inhibits leaky scanning and / or promotes the translational fidelity of mRNA translation, wherein at least one of the structural modifications is a GC-rich RNAAttorney Docket No.: 45817-0163WO1 element. In some aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA. In one embodiment, the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5’ UTR of the mRNA. In another embodiment, the GC-rich RNA element is located 15-30, 15-20, 15-25, 10-15, or 5-10 nucleotides upstream of a Kozak consensus sequence. In another embodiment, the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5’ UTR of the mRNA.

[0432] In any of the foregoing or related aspects, the disclosure provides a GC-rich RNA element which comprises a sequence of 3-30, 5-25, 10-20, 15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about 3 nucleotides, derivatives or analogs thereof, linked in any order, wherein the sequence composition is 70-80% cytosine, 60-70% cytosine, 50%-60% cytosine, 40-50% cytosine, 30-40% cytosine bases. In any of the foregoing or related aspects, the disclosure provides a GC-rich RNA element which comprises a sequence of 3-30, 5-25, 10-20, 15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about 3 nucleotides, derivatives or analogs thereof, linked in any order, wherein the sequence composition is about 80% cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine.

[0433] In any of the foregoing or related aspects, the disclosure provides a GC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is 70-80% cytosine, 60-70% cytosine, 50%-60% cytosine, 40-50% cytosine, or 30-40% cytosine. In any of the foregoing or related aspects, the disclosure provides a GC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is about 80% cytosine,Attorney Docket No.: 45817-0163WO1 about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine.

[0434] In some instances, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA, wherein the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5’ UTR of the mRNA, and wherein the GC-rich RNA element comprises a sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is >50% cytosine. In some instances, the sequence composition is >55% cytosine, >60% cytosine, >65% cytosine, >70% cytosine, >75% cytosine, >80% cytosine, >85% cytosine, or >90% cytosine.

[0435] In other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA, wherein the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5’ UTR of the mRNA, and wherein the GC-rich RNA element comprises a sequence of about 3-30, 5-25, 10-20, 15-20 or about 20, about 15, about 12, about 10, about 6 or about 3 nucleotides, or derivatives or analogues thereof, wherein the sequence comprises a repeating GC-motif, wherein the repeating GC -motif is [CCG]n (SEQ ID NO: 518), wherein n = 1 to 10, n= 2 to 8, n= 3 to 6, or n= 4 to 5. In some instances, the sequence comprises a repeating GC-motif [CCG]n (SEQ ID NO: 519), wherein n = 1, 2, 3, 4 or 5. In some instances, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 1, 2, or 3. In some instances, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 1. In some instances, the sequence comprises a repeating GC-motif [CCG]n,Attorney Docket No.: 45817-0163WO1 wherein n = 2. In some instances, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 3. In some instances, the sequence comprises a repeating GC-motif [CCG]n (SEQ ID NO: 520), wherein n = 4. In some instances, the sequence comprises a repeating GC-motif [CCG]n (SEQ ID NO: 521), wherein n = 5.

[0436] In another aspect, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA, wherein the GC-rich RNA element comprises any one of the sequences set forth in Table 8. In one embodiment, the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5’ UTR of the mRNA. In another embodiment, the GC-rich RNA element is located about 15-30, 15-20, 15-25, 10-15, or 5-10 nucleotides upstream of a Kozak consensus sequence. In another embodiment, the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5’ UTR of the mRNA.

[0437] In other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence VI [CCCCGGCGCC (SEQ ID NO:476)] as set forth in Table 8, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA. In some instances, the GC-rich element comprises the sequence VI as set forth in Table 8 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In some instances, the GC-rich element comprises the sequence VI as set forth in Table 8 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In other instances, the GC-rich element comprises the sequence VI as set forth in Table 8 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA.Attorney Docket No.: 45817-0163WO1 In other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence V2 [CCCCGGC] as set forth in Table 8, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA. In some instances, the GC-rich element comprises the sequence V2 as set forth in Table 8 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In some instances, the GC-rich element comprises the sequence V2 as set forth in Table 8 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In other instances, the GC-rich element comprises the sequence V2 as set forth in Table 8 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA.

[0438] In other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence EK [GCCGCC] as set forth in Table 8, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA. In some instances, the GC-rich element comprises the sequence EK as set forth in Table 8 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In some instances, the GC-rich element comprises the sequence EK as set forth in Table 8 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In other instances, the GC-rich element comprises the sequence EK as set forth in Table 8 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA.

[0439] In yet other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence VI [CCCCGGCGCC (SEQ ID NO: 476)] as set forth in Table 8, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA, wherein the 5’ UTR comprises the following sequence shown in Table 8: GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA (SEQ ID NO: 479). The skilled artisan will of course recognize that all Us in the RNA sequencesAttorney Docket No.: 45817-0163WO1 described herein will be Ts in a corresponding template DNA sequence, for example, in DNA templates or constructs from which mRNAs of the disclosure are transcribed, e.g., via in vitro transcription (IVT).

[0440] In some instances, the GC-rich element comprises the sequence VI as set forth in Table 8 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5’ UTR sequence shown in Table 8. In some instances, the GC-rich element comprises the sequence VI as set forth in Table 8 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA, wherein the 5’ UTR comprises the following sequence shown in Table 8:

[0441] GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA (SEQ ID NO:

[0442] 479).

[0443] In other instances, the GC-rich element comprises the sequence VI as set forth in Table 8 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA, wherein the 5’ UTR comprises the following sequence shown in Table 8:

[0444] GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA (SEQ ID NO:

[0445] 479).

[0446] In some instances, the 5’ UTR comprises the following sequence set forth in

[0447] Table 8:

[0448] GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGC CGCCACC (SEQ ID NO: 236)

[0449] Table 8

[0450] 5' UTRs 5' UTR Sequence GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAG

[0451] Standard

[0452] AAAUAUAAGAGCCACC (SEQ ID NO:481)

[0453]

[0454] Attorney Docket No.: 45817-0163WO1

[0455] GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAG AAAUAUAAGACCCCGGCGCCGCCACC (SEQ ID Vl-UTR

[0456] NO: 480) GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAG AAAUAUAAGACCCCGGCGCCACC (SEQ ID NO:

[0457] V2-UTR

[0458] 482)

[0459] GC-Rich RNA Elements Sequence

[0460] K0 (Traditional Kozak consensus) [GCCA / GCC]

[0461] EK [GCCGCC]

[0462] VI [CCCCGGCGCC] (SEQ ID NO:476)

[0463] V2 [CCCCGGC]

[0464] (CCG)n, where n=1-10 [CCG]n(SEQ ID NO: 518)

[0465] (GCC)n, where n=1-10 [GCC]n(SEQ ID NO: 522)

[0466]

[0467] In another aspect, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element

[0468] comprising a stable RNA secondary structure comprising a sequence of nucleotides, or

[0469] 5 derivatives or analogs thereof, linked in an order which forms a hairpin or a stem-loop.

[0470] In one embodiment, the stable RNA secondary structure is upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is

[0471] located about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary w structure is located about 20, about 15, about 10 or about 5 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located about 5, about 4, about 3, about 2, about 1 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure isAttorney Docket No.: 45817-0163WO1 located about 15-30, about 15-20, about 15-25, about 10-15, or about 5-10 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located 12-15 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure has a deltaG of about -30 kcal / mol, about -20 to -30 kcal / mol, about -20 kcal / mol, about -10 to -20 kcal / mol, about -10 kcal / mol, about -5 to -10 kcal / mol.

[0472] In another embodiment, the modification is operably linked to an open reading frame encoding a polypeptide and wherein the modification and the open reading frame are heterologous.

[0473] In another embodiment, the sequence of the GC-rich RNA element is comprised exclusively of guanine (G) and cytosine (C) nucleobases.

[0474] RNA elements that provide a desired translational regulatory activity as described herein can be identified and characterized using known techniques, such as ribosome profiling. Ribosome profiling is a technique that allows the determination of the positions of PICs and / or ribosomes bound to mRNAs (see e.g., Ingolia et al., (2009) Science 324(5924):218-23, incorporated herein by reference). The technique is based on protecting a region or segment of mRNA, by the PIC and / or ribosome, from nuclease digestion. Protection results in the generation of a 30-bp fragment of RNA termed a ‘footprint’. The sequence and frequency of RNA footprints can be analyzed by methods known in the art (e.g., RNA-seq). The footprint is roughly centered on the A-site of the ribosome. If the PIC or ribosome dwells at a particular position or location along an mRNA, footprints generated at these position would be relatively common. Studies have shown that more footprints are generated at positions where the PIC and / or ribosome exhibits decreased processivity and fewer footprints where the PIC and / or ribosome exhibits increased processivity (Gardin et al., (2014) eLife 3:e03735). In some instances, residence time or the time of occupancy of the PIC or ribosome at a discrete position or location along an polynucleotide comprising any one or more of the RNA elements described herein is determined by ribosome profiling.Attorney Docket No.: 45817-0163WO1 A UTR can be homologous or heterologous to the coding region in a polynucleotide. In some instances, the UTR is homologous to the ORF encoding the antibody. In some instances, the UTR is heterologous to the ORF encoding the antibody. In some instances, the polynucleotide comprises two or more 5'UTRs or functional fragments thereof, each of which have the same or different nucleotide sequences. In some instances, the polynucleotide comprises two or more 3'UTRs or functional fragments thereof, each of which have the same or different nucleotide sequences.

[0475] In some instances, the 5'UTR or functional fragment thereof, 3' UTR or functional fragment thereof, or any combination thereof is sequence optimized.

[0476] In some instances, the 5'UTR or functional fragment thereof, 3' UTR or functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, e.g., N1 methylpseudouracil or 5-methoxyuracil. In some instances, all the uracils of the 5’UTR and 3 ’UTR are N1 methylpseudouracils.

[0477] UTRs can have features that provide a regulatory role, e.g., increased or decreased stability, localization and / or translation efficiency. A polynucleotide comprising a UTR can be administered to a cell, tissue, or organism, and one or more regulatory features can be measured using routine methods. In some instances, a functional fragment of a 5'UTR or 3'UTR comprises one or more regulatory features of a full length 5' or 3' UTR, respectively.

[0478] Natural 5'UTRs bear features that play roles in translation initiation. They harbor signatures like Kozak sequences that are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'. 5'UTRs also have been known to form secondary structures that are involved in elongation factor binding.

[0479] By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of a polynucleotide. For example, introduction of 5'UTR of liver-expressed mRNA, such asAttorney Docket No.: 45817-0163WO1 albumin, serum amyloid A, Apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, or antibody, can enhance expression of polynucleotides in hepatic cell lines or liver. Likewise, use of 5'UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (e.g., Tie-1, CD36), for myeloid cells (e.g., C / EBP, AML1, G-CSF, GM-CSF, CDllb, MSR, Fr-1, i-NOS), for leukocytes (e.g., CD45, CD18), for adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (e.g., SP-AZB / C / D).

[0480] In some instances, UTRs are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, an encoded polypeptide can belong to a family of proteins (i.e., that share at least one function, structure, feature, localization, origin, or expression pattern), which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of the genes or mRNA can be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.

[0481] In some instances, the 5’UTR and the 3 ’UTR can be heterologous. In some instances, the 5'UTR can be derived from a different species than the 3'UTR. In some instances, the 3'UTR can be derived from a different species than the 5'UTR.

[0482] Co-owned International Patent Application No. PCT / US2014 / 021522 (Publ. No. WO / 2014 / 164253, incorporated herein by reference in its entirety) provides a listing of exemplary UTRs that can be utilized in the polynucleotide of the present disclosure as flanking regions to an ORF.

[0483] Exemplary UTRs of the application include, but are not limited to, one or more 5'UTR and / or 3'UTR derived from the nucleic acid sequence of: a globin, such as an a-or P-globin (e.g., aXenopus, mouse, rabbit, or human globin); a strong Kozak translational initiation signal; a CYBA (e.g., human cytochrome b-245 a polypeptide); an albumin (e.g., human albumin7); aHSD17B4 (hydroxysteroid ( 17-0) dehydrogenase); a virus (e.g., a tobacco etch virus (TEV), a Venezuelan equine encephalitis virus (VEEV), aAttorney Docket No.: 45817-0163WO1 Dengue virus (DENV), a cytomegalovirus (CMV) (e.g., CMV immediate early 1 (IE1)), a hepatitis virus (e.g., hepatitis B virus), a sindbis virus, or a PAV barley yellow dwarf virus); a heat shock protein (e.g., Hsp70); a translation initiation factor (e.g., elF4G); a glucose transporter (e.g., hGLUTl (human glucose transporter 1)); an actin (e.g., human a or P actin); a GAPDH; a tubulin; a histone; a citric acid cycle enzyme; a topoisomerase (e.g., a 5'UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract)); a ribosomal protein Large 32 (L32); a ribosomal protein (e.g., human or mouse ribosomal protein, such as, for example, rps9); an ATP synthase (e.g., ATP5A1 or the subunit of mitochondrial H+-ATP synthase); a growth hormone (e.g., bovine (bGH) or human (hGH)); an elongation factor (e.g., elongation factor 1 al (EEF1 Al)); a manganese superoxide dismutase (MnSOD); a myocyte enhancer factor 2A (MEF2A); a P-Fl-ATPase, a creatine kinase, a myoglobin, a granulocyte-colony stimulating factor (G-CSF); a collagen (e.g., collagen type I, alpha 2 (Col1A2), collagen type I, alpha 1 (Col1A1), collagen type VI, alpha 2 (Col6A2), collagen type VI, alpha 1 (Col6A1)); a ribophorin (e.g., ribophorin I (RPN1)); a low density lipoprotein receptor-related protein (e.g., LRP1); a cardiotrophin-like cytokine factor (e.g., Nntl); calreticulin (Calr); a procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plodl); and a nucleobindin (e.g., Nucbl).

[0484] In some instances, the 5'UTR is selected from the group consisting of a P-globin 5’UTR; a 5'UTR containing a strong Kozak translational initiation signal; a cytochrome b-245 a polypeptide (CYBA) 5'UTR; a hydroxysteroid (17-P) dehydrogenase (HSD17B4) 5'UTR; a Tobacco etch virus (TEV) 5'UTR; a Venezuelan equine encephalitis virus (VEEV) 5'UTR; a 5' proximal open reading frame of rubella virus (RV) RNA encoding nonstructural proteins; a Dengue virus (DEN) 5'UTR; a heat shock protein 70 (Hsp70) 5'UTR; a eIF4G 5'UTR; a GLUT1 5'UTR; functional fragments thereof and any combination thereof.

[0485] In some instances, the 3'UTR is selected from the group consisting of a P-globin 3’UTR; a CYBA 3'UTR; an albumin 3'UTR; a growth hormone (GH) 3'UTR; a VEEV 3'UTR; a hepatitis B virus (HBV) 3'UTR; a-globin 3 TR; a DEN 3'UTR; a PAV barleyAttorney Docket No.: 45817-0163WO1 yellow dwarf virus (BYDV-PAV) 3'UTR; an elongation factor 1 al (EEF1A1) 3'UTR; a manganese superoxide dismutase (MnSOD) 3'UTR; a 0 subunit of mitochondrial H(+)-ATP synthase (0-mRNA) 3'UTR; a GLUT1 3'UTR; a MEF2A 3'UTR; a 0-Fl-ATPase 3'UTR; functional fragments thereof and combinations thereof.

[0486] Wild-type UTRs derived from any gene or mRNA can be incorporated into the polynucleotides of the present disclosure. In some instances, a UTR can be altered relative to a wild type or native UTR to produce a variant UTR, e.g., by changing the orientation or location of the UTR relative to the ORF; or by inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. In some instances, variants of 5' or 3' UTRs can be utilized, for example, mutants of wild type UTRs, or variants wherein one or more nucleotides are added to or removed from a terminus of the UTR.

[0487] Additionally, one or more synthetic UTRs can be used in combination with one or more non-synthetic UTRs. See, e.g., Mandal and Rossi, Nat. Protoc. 2013 8(3):568-82, the contents of which are incorporated herein by reference in their entirety.

[0488] UTRs or portions thereof can be placed in the same orientation as in the transcript from which they were selected or can be altered in orientation or location. Hence, a 5' and / or 3' UTR can be inverted, shortened, lengthened, or combined with one or more other 5' UTRs or 3' UTRs.

[0489] In some instances, the polynucleotide comprises multiple UTRs, e.g., a double, a triple or a quadruple 5’UTR or 3’UTR. For example, a double UTR comprises two copies of the same UTR either in series or substantially in series. For example, a double betaglobin 3'UTR can be used (see US2010 / 0129877, the contents of which are incorporated herein by reference in its entirety).

[0490] In certain instances, the polynucleotides of the disclosure comprise a 5' UTR and / or a 3' UTR selected from any of the UTRs disclosed herein. In some instances, the 5' UTR comprises:Attorney Docket No.: 45817-0163WO1 5' UTR-001 (Upstream UTR) (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:486);

[0491] 5’ UTR-002 (Upstream UTR) (GGGAGAUCAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:487);

[0492] 5' UTR-004 (Upstream UTR) (GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAGCCACC) (SEQ ID NO:488);

[0493] 5' UTR-005 (Upstream UTR) (GGGAGAUCAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:489);

[0494] 5’ UTR-007 (Upstream UTR) (GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAGCCACC) (SEQ ID NO:490);

[0495] 5' UTR-008 (Upstream UTR) (GGGAAUUAACAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:491);

[0496] 5' UTR-009 (Upstream UTR) (GGGAAAUUAGACAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:492);

[0497] 5' UTR-010, Upstream (GGGAAAUAAGAGAGUAAAGAACAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:493);

[0498] 5' UTR-011 (Upstream UTR) (GGGAAAAAAGAGAGAAAAGAAGACUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:494);Attorney Docket No.: 45817-0163WO1 5' UTR-012 (Upstream UTR) (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAUAUAUAAGAGCCACC) (SEQ ID NO:495);

[0499] 5’ UTR-013 (Upstream UTR) (GGGAAAUAAGAGACAAAACAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:496);

[0500] 5' UTR-014 (Upstream UTR) (GGGAAAUUAGAGAGUAAAGAACAGUAAGUAGAAUUAAAAGAGCCACC) (SEQ ID NO:497);

[0501] 5' UTR-015 (Upstream UTR) (GGGAAAUAAGAGAGAAUAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:498);

[0502] 5’ UTR-016 (Upstream UTR) (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAAUUAAGAGCCACC) (SEQ ID NO:499);

[0503] 5' UTR-017 (Upstream UTR); or (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUUUAAGAGCCACC) (SEQ ID NO:500);

[0504] 5' UTR-018 (Upstream UTR) 5' UTR (UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGG AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:501)

[0505] In some instances, the 3' UTR comprises:

[0506] 3 'UTR:

[0507] UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGG GCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUG GUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:502)Attorney Docket No.: 45817-0163WO1

[0508] 142-3p 3' UTR (UTR including miR142-3p binding site) UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGC CAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGC ACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:503);

[0509] 142-3p 3' UTR (UTR including miR142-3p binding site) UGAUAAUAGGCUGGAGCCUCGGUGGCUCCAUAAAGUAGGAAACACUACA CAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGC ACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:504); or

[0510] 142-3p 3' UTR (UTR including miR142-3p binding site) UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUCCAUAA AGUAGGAAACACUACAUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGC ACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:505);

[0511] 142-3p 3' UTR (UTR including miR142-3p binding site) UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCU CCCCCCAGUCCAUAAAGUAGGAAACACUACACCCCUCCUCCCCUUCCUGC ACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:506);

[0512] 142-3p 3' UTR (UTR including miR142-3p binding site) UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCUCCAUAAAGUAGGAAACACUACACUGC ACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:507);Attorney Docket No.: 45817-0163WO1 142-3p 3' UTR (UTR including miR142-3p binding site) UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUA GGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:508)

[0513] 142-3p 3' UTR (UTR including miR142-3p binding site) UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGA AUAAAGUUCCAUAAAGUAGGAAACACUACACUGAGUGGGCGGC (SEQ ID NO:509);

[0514] 3’UTR-018 GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAG GGUGUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG (SEQ ID NO:510)

[0515] 3' UTR (miR142 and miR126 binding sites variant 1)

[0516] UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGC CAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGC ACCCGUACCCCCCGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUAA AGUCUGAGUGGGCGGC (SEQ ID NO:511)

[0517] 3' UTR (miR142 and miR126 binding sites variant 2)

[0518] UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGC CUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGC ACCCGUACCCCCCGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUAA AGUCUGAGUGGGCGGC) (SEQ ID NO:512); or

[0519] 3’UTR (miR142-3p binding site variant 3)Attorney Docket No.: 45817-0163WO1 UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUA GGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:513)

[0520] 3’UTR (miR142-3p binding site variant 3, DNA sequence)

[0521] TGATAATAGGCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCC CCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCTCCATAAAGTAGGA AACACTACAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO:514)

[0522] In certain instances, the 5'UTR and / or 3'UTR sequence of the present disclosure comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of 5'UTR sequences comprising any of SEQ ID NOs: 486-501 and / or 3'UTR sequences comprises any of SEQ ID NOs: 502-514, and any combination thereof.

[0523] The polynucleotides of the present disclosure can comprise combinations of features. For example, the ORF can be flanked by a 5'UTR that comprises a strong Kozak translational initiation signal and / or a 3'UTR comprising an oligo(dT) sequence for templated addition of a poly A tail. A 5'UTR can comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different UTRs (see, e.g., US2010 / 0293625, herein incorporated by reference in its entirety).

[0524] Other non-UTR sequences can be used as regions or subregions within the polynucleotides of the present disclosure. For example, introns or portions of intron sequences can be incorporated into the polynucleotides of the present disclosure.

[0525] Incorporation of intronic sequences can increase protein production as well as polynucleotide expression levels. In some instances, the polynucleotide of the presentAttorney Docket No.: 45817-0163WO1 disclosure comprises an internal ribosome entry site (IRES) instead of or in addition to a UTR (see, e g., Yakubov et al., Biochem. Biophys. Res. Commun. 2010 394(1):189-193, the contents of which are incorporated herein by reference in their entirety). In some instances, the polynucleotide comprises an IRES instead of a 5 ’UTR sequence. In some instances, the polynucleotide comprises an ORF and a viral capsid sequence. In some instances, the polynucleotide comprises a synthetic 5'UTR in combination with a nonsynthetic 3 'UTR.

[0526] In some instances, the UTR can also include at least one translation enhancer polynucleotide, translation enhancer element, or translational enhancer elements (collectively, " TEE," which refers to nucleic acid sequences that increase the amount of polypeptide or protein produced from a polynucleotide. As a non-limiting example, the TEE can be located between the transcription promoter and the start codon. In some instances, the 5'UTR comprises a TEE.

[0527] In one aspect, a TEE is a conserved element in a UTR that can promote translational activity of a nucleic acid such as, but not limited to, cap-dependent or capindependent translation.

[0528] In some instances, a 5'UTR and / or 3'UTR comprising at least one TEE described herein can be incorporated in a monocistronic sequence such as, but not limited to, a vector system or a nucleic acid vector.

[0529] In some instances, a 5'UTR and / or 3'UTR of a polynucleotide of the present disclosure comprises a TEE or portion thereof described herein. In some instances, the TEEs in the 3'UTR can be the same and / or different from the TEE located in the 5'UTR.

[0530] In some instances, a 5'UTR and / or 3'UTR of a polynucleotide of the present disclosure can include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences. In one embodiment, the 5'UTR of aAttorney Docket No.: 45817-0163WO1 polynucleotide of the present disclosure can include 1-60, 1-55, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 TEE sequences. The TEE sequences in the 5'UTR of the polynucleotide of the present disclosure can be the same or different TEE sequences. A combination of different TEE sequences in the 5'UTR of the polynucleotide of the present disclosure can include combinations in which more than one copy of any of the different TEE sequences are incorporated.

[0531] In some instances, the 5'UTR and / or 3UTR comprises a spacer to separate two TEE sequences. As a non-limiting example, the spacer can be a 15 nucleotide spacer and / or other spacers known in the art. As another non-limiting example, the 5'UTR and / or 3UTR comprises a TEE sequence-spacer module repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, or more than 10 times in the 5'UTR and / or 3'UTR, respectively. In some instances, the 5'UTR and / or 3UTR comprises a TEE sequence-spacer module repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0532] In some instances, the spacer separating two TEE sequences can include other sequences known in the art that can regulate the translation of the polynucleotide of the present disclosure, e.g., miR binding site sequences described herein (e.g., miR binding sites and miR seeds). As a non-limiting example, each spacer used to separate two TEE sequences can include a different miR binding site sequence or component of a miR sequence (e.g., miR seed sequence).

[0533] In some instances, a polynucleotide of the present disclosure comprises a miR and / or TEE sequence. In some instances, the incorporation of a miR sequence and / or a TEE sequence into a polynucleotide of the present disclosure can change the shape of the stem loop region, which can increase and / or decrease translation. See e.g., Kedde et al., Nature Cell Biology 2010 12(10): 1014-20, herein incorporated by reference in its entirety).

[0534] mRNA: C oding regionAttorney Docket No.: 45817-0163WO1 Provided herein are mRNA that encode the coding regions of the VHHs, polypeptides, bispecific antibodies, and multispecific antibodies, of the disclosure. In some instances, the disclosure provides mRNA that encode coding regions of a polypeptide which is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any of the VHH sequences described herein. In certain instances, the disclosure provides coding regions that encode a polypeptide which includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substitutions in any of the sequences provided or referenced herein. In some instances, the mRNA comprises a codon optimized nucleic acid sequence, wherein the open reading frame (ORF) of the codon optimized nucleic acid sequence is derived from a polypeptide comprising an anti-PD-1 VHH. For example, the polynucleotides can comprise a codon optimized ORF encoding a polypeptide comprising an anti-PD-1 VHH.

[0535] The nucleic acid molecules of the disclosure may include one or more alterations. Herein, in a nucleotide, nucleoside, or polynucleotide (such as the mRNA of the disclosure), the terms “alteration” or, as appropriate, “alternative” refer to alteration with respect to A, G, U or C ribonucleotides. The alterations may be various distinct alterations. In some instances, where the nucleic acid is an mRNA, the coding region, the flanking regions, and / or the terminal regions may contain one, two, or more (optionally different) nucleoside or nucleotide alterations. In some instances, an alternative polynucleotide introduced to a cell may exhibit reduced degradation in the cell, as compared to an unaltered polynucleotide. The polynucleotides can include any useful alteration, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage, or to the phosphodiester backbone). In certain instances, alterations (e.g., one or more alterations) are present in each of the sugar and the internucleoside linkage. Alterations according to the present disclosure may be alterations of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs) (e.g., the substitution of the 2’OH of the ribofuranosyl ring to 2’H), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof. Additional alterations are described herein.Attorney Docket No.: 45817-0163WO1 Modified Nucleic Acids

[0536] According to Aduri et al., (Aduri, R. et al., Journal of Chemical Theory and Computation. 3(4): 1464-75(2006)), there are 107 naturally occurring nucleosides, including 1 -methyladenosine, 2-methylthio-N6-hydroxynorvalyl carbamoyl adenosine, 2-methyladenosine, 2-O-ribosylphosphate adenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-acetyladenosine, N6-glycinylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, N6, N6-dimethyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6-hydroxynorvalylcarbamoyladenosine, 1,2-O-dimethyladenosine, N6,2-O-dimethyladenosine, 2-O-methyladenosine, N6, N6, O-2-trimethyladenosine, 2-methylthio-N6-(cis-hydroxy isopentenyl) adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6-isopentenyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, 2-thiocytidine, 3 -methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5 -methyl cytidine, 5-hydroxymethylcytidine, lysidine, N4-acetyl-2-O-methylcytidine, 5-formyl-2-O-methylcytidine, 5,2-O-dimethylcytidine, 2-O-methylcytidine, N4,2-O-dimethylcytidine, N4, N4,2-O-trimethylcytidine, 1 -methylguanosine, N2,7-dimethylguanosine, N2-methylguanosine, 2-O-ribosylphosphate guanosine, 7-methylguanosine, under modified hydroxywybutosine, 7-aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, N2, N2-dimethylguanosine, 4-demethylwyosine, epoxy queuosine, hydroxywybutosine, isowyosine, N2, 7,2-0-trimethylguanosine, N2,2-O-dimethylguanosine, 1,2-O-dimethylguanosine, 2-0-methylguanosine, N2, N2,2-O-trimethylguanosine, N2, N2,7-trimethylguanosine, peroxywybutosine, galactosyl-queuosine, mannosyl-queuosine, queuosine, archaeosine, wybutosine, methylwyosine, wyosine, 2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 3 -methyluridine, 4-thiouridine, 5-methyl-2-thiouridine, 5-methylaminomethyluridine, 5-carboxymethyluridine, 5-carboxymethylaminomethyluridine, 5-hydroxyuridine, 5-methyluridine, 5-taurinomethyluridine, 5-carbamoylmethyluridine, 5-(carboxyhydroxymethyl)uridine methyl ester, dihydrouridine, 5-methyldihydrouridine, 5-methylaminomethyl-2-Attorney Docket No.: 45817-0163WO1 thiouridine, 5-(carboxyhydroxymethyl)uridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine, 3,2-O-dimethyluridine, 5-carboxymethylaminomethyl-2-O-methyluridine, 5-carbamoylmethyl-2-O-methyluridine, 5-methoxycarbonylmethyl-2-O-methyluridine, 5-(isopentenylaminomethyl)-2-O-methyluridine, 5,2-O-dimethyluridine, 2-O-methyluridine, 2-thio-2-O-methyluridine, uridine 5-oxyacetic acid, 5-methoxycarbonylmethyluridine, uridine 5-oxyacetic acid methyl ester, 5-methoxyuridine, 5-aminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2 -thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-taurinomethyl-2 -thiouridine, pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 1 -methylpseudouridine, 3-m ethylpseudouridine, 2-O-m ethylpseudouridine, inosine, 1 -methylinosine, 1,2-0-dimethylinosine, and 2-0-methylinosine. Each of these may be components of nucleic acids (e.g., mRNA) of the present disclosure.

[0537] Nucleosides Containing Modified Sugars

[0538] The alternative nucleosides and nucleotides (e.g., building block molecules), which may be incorporated into a polynucleotide (e.g., RNA or mRNA, as described herein), can be altered on the sugar of the ribonucleic acid. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different substituents.

[0539] Exemplary substitutions at the 2'-position include, but are not limited to, H, halo, optionally substituted C1-6alkyl; optionally substituted C1-6alkoxy; optionally substituted C6-10aryloxy; optionally substituted C3-8cycloalkyl; optionally substituted C3-8cycloalkoxy; optionally substituted C6-10aryloxy; optionally substituted C6-10aryl-C1-6alkoxy, optionally substituted C1-12(heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), -O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20); “locked” nucleic acids (LN A) in which the 2'-hydroxyl is connected by a C1-6 alkylene or C1-6 heteroalkylene bridge to theAttorney Docket No.: 45817-0163WO1 4’-carbon of the same ribose sugar, where exemplary bridges included methylene, propylene, ether, or amino bridges; aminoalkyl, as defined herein; aminoalkoxy, as defined herein; amino as defined herein; and amino acid, as defined herein

[0540] Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary, non-limiting alternative nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replace with a-L-threofuranosyl-(3'— >2')), and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone). The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a polynucleotide molecule can include nucleotides containing, e.g., arabinose, as the sugar.

[0541] Alterations on the Nucleobase

[0542] The present disclosure provides for alternative nucleosides and nucleotides. As described herein “nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). As described herein, “nucleotide” is defined as a nucleoside including a phosphate group.

[0543] Exemplary non-limiting alterations include an amino group, a thiol group, an alkyl group, a halo group, or any described herein. The alternative nucleotides may byAttorney Docket No.: 45817-0163WO1 synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more alternative or alternative nucleosides).

[0544] In some instances, a nucleic acid of the disclosure (e.g., an mRNA) includes one or more 2’-0Me nucleotides, 2’ -methoxy ethyl nucleotides (2’-M0E nucleotides), 2’-F nucleotide, 2’-NH2 nucleotide, 2’ fluoroarabino nucleotides (FANA nucleotides), locked nucleic acid nucleotides (LNA nucleotides), or 4’-S nucleotides.

[0545] The alternative nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, and guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or alternative nucleotides including non-standard or alternative 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. One example of such non-standard base pairing is the base pairing between the alternative nucleotide inosine and adenine, cytosine, or uracil.

[0546] The alternative nucleosides and nucleotides can include an alternative nucleobase. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. Examples of nucleobase found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine. These nucleobases can be altered or wholly replaced to provide polynucleotide molecules having enhanced properties (e.g., resistance to nucleases and stability), and these properties may manifest through disruption of the binding of a major groove binding partner.

[0547] In some instances, the alternative nucleobase is an alternative uracil. Exemplary nucleobases and nucleosides having an alternative uracil include pseudouridine ( ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy -uridine (ho’U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyaceticAttorney Docket No.: 45817-0163WO1 acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm’U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm’U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2 -thio-uridine (nm3s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2 -thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm’U), 5-carboxymethylaminomethyl-2 -thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (rm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm3s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m’s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (

[0548]

[0549] m3ψ), 2-thio-l-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy -pseudouridine, 4-methoxy-2-thio-pseudouridine, N1 -methylpseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3y), 5-(isopentenylaminomethyl)uridine (inm’U), 5-(isopentenylaminomethyl)-2 -thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m’Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm’Um), 3,2'-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2’-F-ara-uridine, 2’-F-uridine, 2’-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l-E-propenylamino)uridine.

[0550] In one instance, the nucleic acid is modified to contain 1-methylpseudouridine (m1ψ) in lieu of uridine at each instance.Attorney Docket No.: 45817-0163WO1 In some instances, the alternative nucleobase is an alternative cytosine.

[0551] Exemplary nucleobases and nucleosides having an alternative cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1 -thio-cytidine, 2’-F-ara-cytidine, 2’-F-cytidine, and 2’-OH-ara-cytidine.

[0552] In some instances, the alternative nucleobase is an alternative adenine. Exemplary nucleobases and nucleosides having an alternative adenine include 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine e.g., 2-amino-6-chloro-purine), 6-halo-purine e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m1A), 2-m ethyl -adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6, N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-Attorney Docket No.: 45817-0163WO1 adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6, N6,2'-O-trimethyl-adenosine (m62Am), l,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido-adenosine, 2’-F-ara-adenosine, 2’-F-adenosine, 2’-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0553] In some instances, the alternative nucleobase is an alternative guanine. Exemplary nucleobases and nucleosides having an alternative guanine include inosine (I), 1 -methylinosine (m1!), wyosine (imG), methylwyosine (mimG), 4-dem ethyl -wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxy queuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m’G), N2-m ethylguanosine (m2G), N2, N2-dimethyl-guanosine (m2? G), N2,7-dimethyl-guanosine (m2,7G), N2, N2,7-dimethyl-guanosine (m2’2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2, N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2, N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1 -thio-guanosine, O6-methyl-guanosine, 2’-F-ara-guanosine, and 2’-F-guanosine.

[0554] The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine, or pyrimidine analog. For example, the nucleobase can each be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In someAttorney Docket No.: 45817-0163WO1 instances, the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g, 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3 -deazaguanine, deazaadenine, 7-deazaadenine, 3 -deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[l,5-a] 1,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine, pyridazine; and 1,3,5 triazine. When the nucleotides are depicted using the shorthand A, G, C, T or U, each letter refers to the representative base and / or derivatives thereof (e.g., A includes adenine or adenine analogs (e.g, 7-deaza adenine)).

[0555] In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-methyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-trifluoromethyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-hydroxymethyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-bromo-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-iodo-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5 -methoxy-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-ethyl-cytosine, and cytosine as the only uracils and cytosines. InAttorney Docket No.: 45817-0163WO1 some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-phenyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-ethnyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, N4-methyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-fluoro-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, N4-acetyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, pseudoisocytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-formyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-aminoallyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uracil, uracil, 5-carboxy-cytosine, and cytosine as the only uracils and cytosines.

[0556] In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-methyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-trifluoromethyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-hydroxymethyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-bromo-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-iodo-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-methoxy-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosureAttorney Docket No.: 45817-0163WO1 contain 1-methyl-pseudouracil, uracil, 5-ethyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-phenyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-ethnyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, N4-methyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-fluoro-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, N4-acetyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, pseudoisocytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-formyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-aminoallyl-cytosine, and cytosine as the only uracils and cytosines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouracil, uracil, 5-carboxy-cytosine, and cytosine as the only uracils and cytosines.

[0557] In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-methyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-trifluoromethyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-hydroxymethyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-bromo-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-iodo-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides ofAttorney Docket No.: 45817-0163WO1 the disclosure contain 5-methoxy-uridine, uridine, 5-methoxy-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-ethyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-phenyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-ethnyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, N4-methyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-fluoro-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, N4-acetyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, pseudoisocytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-formyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-aminoallyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 5-methoxy-uridine, uridine, 5-carboxy-cytidine, and cytidine as the only uridines and cytidines.

[0558] In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-methyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-trifluoromethyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-hydroxymethyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-bromo-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine,Attorney Docket No.: 45817-0163WO1 5-iodo-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-methoxy-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-ethyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-phenyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-ethnyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, N4-methyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-fluoro-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, N4-acetyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, pseudoisocytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-formyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-aminoallyl-cytidine, and cytidine as the only uridines and cytidines. In some instances, the polynucleotides of the disclosure contain 1-methyl-pseudouridine, uridine, 5-carboxy-cytidine, and cytidine as the only uridines and cytidines.

[0559] In some instances, the polynucleotides of the disclosure contain the uracil of one of the nucleosides of Table 9 and uracil as the only uracils. In other instances, the polynucleotides of the disclosure contain a uridine of Table 9 and uridine as the only uridines.Attorney Docket No.: 45817-0163WO1 Table 9 - Exemplary Uracil-containing Nucleosides

[0560] Nucleoside Name

[0561] 5-methoxy -uridine

[0562] 1 -Methyl -pseudo-uridine

[0563] Pseudouridine

[0564] 5-methyl-uridine

[0565] 5-bromo-uridine

[0566] 2-thio-uridine

[0567] 4-thiouridine

[0568] 2'-O-methyluridine

[0569] 5-methyl-2 -thiouridine

[0570] 5,2'-O-dimethyluridine

[0571] 5-aminomethyl-2-thiouridine

[0572] 5-(1-Propynyl)ara-uridine

[0573] 2'-O-Methyl-5-(l-propynyl)uridine

[0574] 5-Vinylarauridine

[0575] (Z)-5-(2-Bromo-vinyl)ara-uridine

[0576] (E)-5-(2-Bromo-vinyl)ara-uridine

[0577] (Z)-5-(2-Bromo-vinyl)uridine

[0578] (E)-5-(2-Bromo-vinyl)uridine

[0579] 5-Cyanouridine

[0580] 5-Formyluridine

[0581] 5-Dimethylaminouridine

[0582] 5-Trideuteromethyl-6-deuterouridine

[0583] 5-(2-Furanyl)uridine

[0584] 5-Phenylethynyluridine

[0585] 4'-Carbocyclic uridine

[0586] 4'-Ethynyluridine

[0587] 4'-Azidouridine

[0588] 2'-Deoxy-2',2'-difluorouridine

[0589] 2'-Deoxy-2'-b-fluorouridine

[0590] 2'-Deoxy-2'-b-chlorouridine

[0591] 2'-Deoxy-2'-b-bromouridine

[0592] 2'-Deoxy-2'-b-iodouridine

[0593] 5'-Homo-uridine

[0594] 2'-Deoxy-2'-a-mercaptouridine

[0595] 2'-Deoxy-2'-a-thiomethoxyuridine

[0596] 2'-Deoxy-2'-a-azidouridine

[0597]

[0598] 2'-Deoxy-2'-a-aminouridineAttorney Docket No.: 45817-0163WO1

[0599] Nucleoside Name

[0600] 2'-Deoxy-2'-b-mercaptouridine

[0601] 2'-Deoxy-2'-b-thiomethoxyuridine

[0602] 2'-Deoxy-2'-b-azidouridine

[0603] 2'-Deoxy-2'-b-aminouridine

[0604] 2'-b-Trifluoromethyluridine

[0605] 2'-a-Trifluoromethyluridine

[0606] 2'-b-Ethynyluridine

[0607] 2'-a-Ethynyluridine

[0608] 1 -ethyl -p seudo-uri dine

[0609] 1 -propyl-pseudo-uridine

[0610] 1-iso-propyl-pseudo-uridine

[0611] l-(2,2,2-trifluoroethyl)-pseudo-uridine

[0612] 1 -cyclopropyl-pseudo-uridine

[0613] 1-cyclopropylmethyl-pseudo-uridine

[0614] 1 -phenyl-pseudo-uridine

[0615] 1 -benzyl-pseudo-uridine

[0616] 1 -aminomethyl-pseudo-uridine

[0617] pseudo-uridine-l-2-ethanoic acid

[0618] 1 -(3 -amino-3 -carboxypropyl )pseudo-uri dine

[0619] l-methyl-3-(3-amino-3-carboxypropyl)pseudo-uridine

[0620] 6-methyl-pseudo-uridine

[0621] 6-trifluoromethyl-pseudo-uridine

[0622] 6-methoxy-pseudo-uridine

[0623] 6-phenyl-pseudo-uridine

[0624] 6-iodo-pseudo-uridine

[0625] 6-bromo-pseudo-uridine

[0626] 6-chloro-pseudo-uridine

[0627] 6-fluoro-pseudo-uridine

[0628] 4-Thio-pseudo-uridine

[0629] 2-Thio-pseudo-uridine

[0630] Alpha-thio-pseudo-uridine

[0631] 1 -Me-alpha-thio-pseudo-uridine

[0632] 1 -butyl-pseudo-uridine

[0633] 1 -tert-butyl -pseudo-uridine

[0634] 1 -pentyl-pseudo-uridine

[0635] 1 -hexyl-pseudo-uridine

[0636] 1 -trifluoromethyl-pseudo-uridine

[0637] 1 -cyclobutyl-pseudo-uridine

[0638]

[0639] 1-cyclopentyl-pseudo-uridineAttorney Docket No.: 45817-0163WO1

[0640] Nucleoside Name

[0641] 1-cyclohexyl-pseudo-uridine

[0642] 1-cycloheptyl-pseudo-uridine

[0643] 1 -cyclooctyl-pseudo-uridine

[0644] 1-cyclobutylmethyl-pseudo-uridine

[0645] 1-cyclopentylmethyl-pseudo-uridine

[0646] 1-cyclohexylmethyl-pseudo-uridine

[0647] 1-cycloheptylmethyl-pseudo-uridine

[0648] 1-cyclooctylmethyl-pseudo-uridine

[0649] 1-p-tolyl-pseudo-uridine

[0650] l-(2,4,6-trimethyl-phenyl)pseudo-uridine

[0651] l-(4-methoxy-phenyl)pseudo-uridine

[0652] l-(4-amino-phenyl)pseudo-uridine

[0653] 1 (4-nitro-phenyl)pseudo-uridine

[0654] pseudo-uridine-Nl-p-benzoic acid

[0655] l-(4-methyl-benzyl)pseudo-uridine

[0656] l-(2,4,6-trimethyl-benzyl)pseudo-uridine

[0657] l-(4-methoxy-benzyl)pseudo-uridine

[0658] 1 -(4-amino-benzyl)pseudo-uridine

[0659] l-(4-nitro-benzyl)pseudo-uridine

[0660] pseudo-uridine-N 1 -methyl-p-benzoic acid

[0661] 1 -(2-amino-ethyl)pseudo-uridine

[0662] l-(3-amino-propyl)pseudo-uridine

[0663] l-(4-amino-butyl)pseudo-uridine

[0664] l-(5-amino-pentyl)pseudo-uridine

[0665] 1 -(6-amino-hexyl)pseudo-uridine

[0666] pseudo-uridine-N 1 -3-propionic acid

[0667] pseudo-uridine-Nl-4-butanoic acid

[0668] pseudo-uridine-Nl-5-pentanoic acid

[0669] pseudo-uridine-Nl-6-hexanoic acid

[0670] pseudo-uridine-Nl-7-heptanoic acid

[0671] l-(2-amino-2-carboxyethyl)pseudo-uridine

[0672] l-(4-amino-4-carboxybutyl)pseudo-uridine

[0673] 3-alkyl-pseudo-uridine

[0674] 6-ethyl-pseudo-uridine

[0675] 6-propyl-pseudo-uridine

[0676] 6-iso-propyl-pseudo-uridine

[0677] 6-butyl-pseudo-uridine

[0678] 6-tert-butyl-pseudo-uridine

[0679]

[0680] 6-(2,2,2-trifluoroethyl)-pseudo-uridineAttorney Docket No.: 45817-0163WO1

[0681] Nucleoside Name

[0682] 6-ethoxy-pseudo-uridine

[0683] 6-trifluoromethoxy -pseudo-uridine

[0684] 6-phenyl-pseudo-uridine

[0685] 6-(substituted-phenyl)-pseudo-uridine

[0686] 6-cyano-pseudo-uridine

[0687] 6-azido-pseudo-uridine

[0688] 6-amino-pseudo-uridine

[0689] 6-ethylcarboxylate-pseudo-uridine

[0690] 6-hydroxy -pseudo-uridine

[0691] 6-methylamino-pseudo-uridine

[0692] 6-dimethylamino-pseudo-uridine

[0693] 6-hydroxyamino-pseudo-uridine

[0694] 6-formyl-pseudo-uridine

[0695] 6-(4-morpholino)-pseudo-uridine

[0696] 6-(4-thiomorpholino)-pseudo-uridine

[0697] 1 -me-4-thio-pseudo-uridine

[0698] 1 -me-2-thio-pseudo-uridine

[0699] 1, 6-dimethyl -pseudo-uri dine

[0700] l-methyl-6-trifluoromethyl-pseudo-uridine

[0701] 1 -methyl-6-ethyl-pseudo-uridine

[0702] 1 -methyl-6-propyl-pseudo-uridine

[0703] 1-methyl-6-iso-propyl-pseudo-uridine

[0704] 1 -methyl-6-butyl-pseudo-uridine

[0705] l-methyl-6-tert-butyl-pseudo-uridine

[0706] l-methyl-6-(2,2,2-trifluoroethyl)pseudo-uridine

[0707] 1-methyl-6-iodo-pseudo-uridine

[0708] 1 -methyl-6-bromo-pseudo-uridine

[0709] l-methyl-6-chloro-pseudo-uridine

[0710] 1 -methyl-6-fluoro-pseudo-uridine

[0711] l-methyl-6-methoxy-pseudo-uridine

[0712] 1-methyl-6-ethoxy-pseudo-uridine

[0713] l-methyl-6-trifluoromethoxy-pseudo-uridine

[0714] 1 -methyl-6-phenyl-pseudo-uridine

[0715] l-methyl-6-(substituted phenyl)pseudo-uridine

[0716] l-methyl-6-cyano-pseudo-uridine

[0717] 1 -methyl-6-azido-pseudo-uridine

[0718] 1 -methyl-6-amino-pseudo-uridine

[0719] l-methyl-6-ethylcarboxylate-pseudo-uridine

[0720]

[0721] l-methyl-6-hydroxy-pseudo-uridineAttorney Docket No.: 45817-0163WO1

[0722] Nucleoside Name

[0723] l-methyl-6-methylamino-pseudo-uridine

[0724] l-methyl-6-dimethylamino-pseudo-uridine

[0725] l-methyl-6-hydroxyamino-pseudo-uridine

[0726] 1 -methyl-6-formyl-pseudo-uridine

[0727] l-methyl-6-(4-morpholino)-pseudo-uridine

[0728] l-methyl-6-(4-thiomorpholino)-pseudo-uridine

[0729] 1 -alkyl-6-vinyl-pseudo-uridine

[0730] l-alkyl-6-allyl-pseudo-uridine

[0731] l-alkyl-6-homoallyl-pseudo-uridine

[0732] l-alkyl-6-ethynyl-pseudo-uridine

[0733] 1 -alkyl -6-(2-propynyl )-p seudo-uri dine

[0734] 1 -alkyl-6-( 1 -propynyl)-pseudo-uridine

[0735] 1 -Hydroxymethylpseudouridine

[0736] 1 -(2-Hydroxyethyl)pseudouridine

[0737] 1 -Methoxymethylpseudouridine

[0738] 1 -(2 -Methoxy ethyl)pseudouri dine

[0739] l-(2,2-Diethoxyethyl)pseudouridine

[0740] (±)l-(2-Hydroxypropyl)pseudouridine

[0741] (2R)-l-(2-Hydroxypropyl)pseudouridine

[0742] (2S)-l-(2-Hydroxypropyl)pseudouridine

[0743] 1-Cyanomethylpseudouridine

[0744] 1 -Morpholinomethylpseudouridine

[0745] 1-Thiomorpholinomethylpseudouridine

[0746] 1 -Benzyloxymethylpseudouridine

[0747] l-(2,2,3,3,3-Pentafluoropropyl)pseudouridine

[0748] 1-Thiomethoxymethylpseudouridine

[0749] 1-Methanesulfonylmethylpseudouridine

[0750] 1 -Vinylpseudouridine

[0751] 1 -Allylpseudouridine

[0752] 1 -Homoallylpseudouridine

[0753] 1 -Propargylpseudouridine

[0754] 1 -(4-Fluorobenzyl)pseudouridine

[0755] 1 -(4-Chlorobenzyl)pseudouridine

[0756] 1 -(4-Bromobenzyl)pseudouridine

[0757] 1 -(4-Iodobenzyl)pseudouridine

[0758] l-(4-Methylbenzyl)pseudouridine

[0759] l-(4-Trifluoromethylbenzyl)pseudouridine

[0760] l-(4-Methoxybenzyl)pseudouridine

[0761]

[0762] l-(4-Trifluoromethoxybenzyl)pseudouridineAttorney Docket No.: 45817-0163WO1

[0763] Nucleoside Name

[0764] l-(4-Thiomethoxybenzyl)pseudouridine

[0765] l-(4-Methanesulfonylbenzyl)pseudouridine

[0766] Pseudouridine l-(4-methylbenzoic acid)

[0767] Pseudouridine l-(4-m ethylbenzenesulfonic acid)

[0768] l-(2,4,6-Trimethylbenzyl)pseudouridine

[0769] 1 -(4-Nitrobenzyl)pseudouridine

[0770] 1 -(4-Azidobenzyl)pseudouridine

[0771] l-(3,4-Dimethoxybenzyl)pseudouridine

[0772] l-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine

[0773] 1 -Acetylpseudouridine

[0774] 1 -Tri fluoroacetylpseudouridine

[0775] 1 -Benzoylpseudouridine

[0776] 1 -Pivaloylpseudouridine

[0777] l-(3-Cyclopropyl-prop-2-ynyl)pseudouridine

[0778] Pseudouridine 1-methylphosphonic acid diethyl ester Pseudouridine 1-methylphosphonic acid

[0779] Pseudouridine l-[3-(2-ethoxy)]propionic acid

[0780] Pseudouridine l-[3-{2-(2-ethoxy)-ethoxy}] propionic acid Pseudouridine TP l-[3-{2-(2-[2-ethoxy ]-ethoxy)-ethoxy}]propionic acid Pseudouridine l-[3-{2-(2-[2-(2-ethoxy )-ethoxy]-ethoxy )-ethoxy}]propionic acid Pseudouridine l-[3-{2-(2-[2-{2(2-ethoxy )-ethoxy} -ethoxy] -ethoxy )- ethoxy}]propionic acid

[0781] l-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl } pseudouridine l-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine 1 -Biotinylpseudouridine

[0782] 1 -Biotinyl-PEG2 -pseudouridine

[0783] 5-Oxyacetic acid-methyl ester-uridine

[0784] 3-Methyl-pseudo-uridine

[0785] 5-trifluoromethyl-uridine

[0786] 5-methyl-amino-methyl-uridine

[0787] 5-carboxy-methyl-amino-methyl-uridine

[0788] 5 -carb oxy m ethyl aminomethyl -2 ’ -OMe-uri dine

[0789] 5 -carb oxy m ethyl ami nomethyl -2-thi o-uri di ne

[0790] 5-methylaminomethyl-2-thio-uridine

[0791] 5-methoxy-carbonyl-methyl-uridine

[0792] 5-methoxy-carbonyl-methyl-2’-OMe-uridine

[0793] 5-oxyacetic acid-uridine

[0794] 3 -(3 -amino-3 -carboxypropyl )-uridine

[0795]

[0796] 5-(carboxyhydroxymethyl)uridine methyl esterAttorney Docket No.: 45817-0163WO1

[0797] Nucleoside Name

[0798] 5-(carboxyhydroxymethyl)uridine

[0799] 2’-OMe-pseudo-uridine

[0800] 2 ’ - Azi do-2 ’ -deoxy -uri di ne

[0801] 2 ’ - Amino-2 ’ -deoxy -uri dine

[0802] 2 ’-F- 5 -Methyl -2’ -deoxy -uridine

[0803] 5-i odo-2’ -fluoro-deoxyuri dine

[0804] 2’ -bromo-deoxyuridine

[0805] 2,2’-anhydro-uridine

[0806] 2’-Azido-deoxyuridine

[0807] 5-Methoxycarbonylmethyl-2 -thiouridine

[0808] 5 -Methyl ami nomethyl -2-thi ouri dine

[0809] 5-Carbamoylmethyluridine

[0810] 5-Carbamoylmethyl-2'-O-methyluridine

[0811] 1 -Methyl -3 -(3 -amino-3 -carboxypropyl) pseudouridine

[0812] 5-Methylaminomethyl-2-selenouridine

[0813] 5-Carboxymethyluridine

[0814] 5 -Methyl dihy drouri di ne

[0815] 5-Taurinomethyluridine

[0816] 5-Taurinomethyl-2-thiouridine

[0817] 5-(iso-Pentenylaminomethyl)uridine

[0818] 5-(iso-Pentenylaminomethyl)- 2-thiouridine

[0819] 5-(iso-Pentenylaminomethyl)-2'-O-methyluridine

[0820] 2'-O-Methylpseudouridine

[0821] 2-Thio-2'-O-methyluridine

[0822] 3,2'-O-Dimethyluridine

[0823] 5-Methoxy-carbonylmethyl-uridine

[0824] 5-hydroxy-uridine

[0825]

[0826] 5-Isopentenyl-aminomethyl-uridine

[0827] In some instances, the polynucleotides of the disclosure contain the cytosine of one of the nucleosides of Table 10 and cytosine as the only cytosines. In other instances, the polynucleotides of the disclosure contain a cytidine of Table 10 and cytidine as the only cytidines.Attorney Docket No.: 45817-0163WO1 Table 10 - Exemplary Cytosine Containing Nucleosides

[0828] Nucleoside Name

[0829] α-thio-cytidine

[0830] Pseudoisocytidine

[0831] pyrrolo-cytidine

[0832] 5-methyl-cytidine

[0833] N4-acetyl -cytidine

[0834] 5-Bromo-cytidine

[0835] 5-Trifluoromethyl-cytidine

[0836] 5-Hydroxymethyl-cytidine

[0837] 5-Iodo-cytidine

[0838] 5-Ethyl-cytidine

[0839] 5-Methoxy-cytidine

[0840] 5-Ethynyl-cytidine

[0841] 5-Fluoro-cytidine

[0842] 5-Phenyl-cytidine

[0843] N4-Bz-cytidine

[0844] N4-Methyl-cytidine

[0845] 5-Pseudo-iso-cytidine

[0846] 5-Formyl-cytidine

[0847] 5-Aminoallyl-cytidine

[0848] 2'-O-methylcytidine

[0849] 2'-O-Methyl-5-(1-propynyl)cytidine

[0850] 5-( 1 -Propynyl)ara-cytidine

[0851] 5-Ethynylara-cytidine

[0852] 5-Ethynylcytidine

[0853] 5-Cyanocytidine

[0854] 5-(2-Chloro-phenyl)-2 -thiocytidine

[0855] 5-(4-Amino-phenyl)-2 -thiocytidine

[0856] N4,2'-O-Dimethylcytidine

[0857] 3'-Ethynylcytidine

[0858] 4'-Carbocyclic cytidine

[0859] 4'-Ethynylcytidine

[0860] 4'-Azidocytidine

[0861] 2'-Deoxy-2',2'-difluorocytidine

[0862] 2'-Deoxy-2'-b-fluorocytidine

[0863] 2'-Deoxy-2'-b-chlorocytidine

[0864] 2'-Deoxy-2'-b-bromocytidine

[0865]

[0866] 2'-Deoxy-2'-b-iodocytidineAttorney Docket No.: 45817-0163WO1

[0867] Nucleoside Name

[0868] 5'-Homo-cytidine

[0869] 2'-Deoxy-2'-a-mercaptocytidine

[0870] 2'-Deoxy-2'-a-thiomethoxy cytidine

[0871] 2'-Deoxy-2'-a-azidocytidine

[0872] 2'-Deoxy-2'-a-aminocytidine

[0873] 2'-Deoxy-2'-b-mercaptocytidine

[0874] 2'-Deoxy-2'-b-thiomethoxy cytidine

[0875] 2'-Deoxy-2'-b-azidocytidine

[0876] 2'-Deoxy-2'-b-aminocytidine

[0877] 2'-b-Tri fluoromethylcytidine

[0878] 2'-a-Tri fluoromethylcytidine

[0879] 2'-b-Ethynylcytidine

[0880] 2'-a-Ethynylcytidine

[0881] (E)-5-(2-Bromo-vinyl)cytidine

[0882] 2 ’ - Azi do-2 ’ -deoxy-cy ti di ne

[0883] 2 ’ -Amino-2 ’ -deoxy-cy ti dine

[0884] 5-aminoallyl-cytidine

[0885] 2,2’ -anhydro-cytidine

[0886] N4-amino-cytidine

[0887] 2’-O-Methyl-N4-acetyl-cytidine

[0888] 2’-fluoro-N4-acetyl-cytidine

[0889] 2’-fluor-N4-Bz-cytidine

[0890] 2’-O-methyl-N4-Bz-cytidine

[0891] N4,2'-O-Dimethylcytidine

[0892]

[0893] 5-Formyl-2'-O-methylcytidine

[0894] Alterations on the Internucleoside Linkage

[0895] The alternative nucleotides, which may be incorporated into a polynucleotide molecule, can be altered on the internucleoside linkage (e.g., phosphate backbone).

[0896] Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be altered by replacing one or more of the oxygen atoms with a different substituent.

[0897] The alternative nucleosides and nucleotides can include the wholesale replacement of an unaltered phosphate moiety with another intemucleoside linkage asAttorney Docket No.: 45817-0163WO1 described herein. Examples of alternative phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be altered by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0898] The alternative nucleosides and nucleotides can include the replacement of one or more of the non-bridging oxygens with a borane moiety (BH3), sulfur (thio), methyl, ethyl and / or methoxy. As a non-limiting example, two non-bridging oxygens at the same position (e.g., the alpha (α), beta (β) or gamma (γ) position) can be replaced with a sulfur (thio) and a methoxy.

[0899] The replacement of one or more of the oxygen atoms at the a position of the phosphate moiety (e.g., a-thio phosphate) is provided to confer stability (such as against exonucleases and endonucleases) to RNA and DNA through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. While not wishing to be bound by theory, phosphorothioate linked polynucleotide molecules are expected to also reduce the innate immune response through weaker binding / activation of cellular innate immune molecules.

[0900] In specific instances, an alternative nucleoside includes an alpha-thio-nucleoside (e.g., 5'-O-(l-thiophosphate)-adenosine, 5'-O-(l-thiophosphate)-cytidine (α-thio-cytidine), 5'-O-(l-thiophosphate)-guanosine, 5'-O-(l-thiophosphate)-uridine, or 5'-O-(l-thi ophosphate)-p seudouri di ne).

[0901] Other internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein below.Attorney Docket No.: 45817-0163WO1 Combinations of Alternative Sugars, Nucleobases, and Internucleoside Linkages The polynucleotides of the disclosure can include a combination of alterations to the sugar, the nucleobase, and / or the internucleoside linkage. These combinations can include any one or more alterations described herein.

[0902] mRNA: Poly A tail

[0903] During RNA processing, a long chain of adenosine nucleotides (poly A tail) is normally added to mRNA molecules to increase the stability of the mRNA. Immediately after transcription, the 3' end of the transcript is cleaved to free a 3' hydroxyl. Then poly A polymerase adds a chain of adenosine nucleotides to the RNA. The process, called polyadenylation, adds a poly A tail that is between 100 and 250 residues long (SEQ ID NO: 523).

[0904] Methods for the stabilization of RNA by incorporation of chain-terminating nucleosides at the 3 ’-terminus include those described in International Patent Publication No. WO2013 / 103659, incorporated herein in its entirety.

[0905] Poly A tail deadenylation by 3' exonucleases is a key step in cellular mRNA degradation in eukaryotes. By blocking 3' exonucleases, the functional half-life of mRNA can be increased, resulting in increased protein expression. Chemical and enzymatic ligation strategies to modify the 3' end of mRNA with reverse chirality adenosine (LA10) and / or inverted deoxythymidine (IdT) are known to those of skill in the art and have been demonstrated to extend mRNA half-life in cellular and in vivo studies. In some instances, the poly A tail of the mRNA includes a 3’ LA10 or IdT modification. For example, as described in International Patent Publication No. WO2017 / 049275, the tail modifications of which are incorporated by reference in their entirety.

[0906] Additional strategies have been explored to further stabilize mRNA, including: chemical modification of the 3’ nucleotide (e.g., conjugation of a morpholino to the 3’ end of the poly A tail); incorporation of stabilizing sequences after the poly A tail (e.g., aAttorney Docket No.: 45817-0163WO1 co-polymer, a stem-loop, or a triple helix); and / or annealing of structured oligos to the 3' end of an mRNA, as described, for example, in International Patent Publication No. WO2017 / 049286, the stabilized linkages of which are incorporated by reference in their entirety.

[0907] Annealing an oligonucleotide (e.g., an oligonucleotide conjugate) with a complex secondary structure (e.g., a triple-helix structure or a stem-loop structure) at the 3 ’end may provide nuclease resistance and increase half-life of mRNA.

[0908] Unique poly A tail lengths may provide certain advantages to the RNAs of the present disclosure. Generally, the length of a poly A tail of the present disclosure is greater than 30 nucleotides in length. In some instances, the poly A tail is greater than 35 nucleotides in length. In some instances, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In some instances, the length is at least 50 nucleotides. In some instances, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 65 nucleotides. In another embodiment, the length is at least 70 nucleotides. In some instances, the length is at least 80 nucleotides. In some instances, the length is at least 90 nucleotides. In some instances, the length is at least 100 nucleotides. In some instances, the length is at least 120 nucleotides. In some instances, the length is at least 140 nucleotides. In some instances, the length is at least 160 nucleotides. In some instances, the length is at least 180 nucleotides. In some instances, the length is at least 200 nucleotides. In some instances, the length is at least 250 nucleotides. In some instances, the length is at least 300 nucleotides. In some instances, the length is at least 350 nucleotides. In some instances, the length is at least 400 nucleotides. In some instances, the length is at least 450 nucleotides. In some instances, the length is at least 500 nucleotides. In some instances, the length is at least 600 nucleotides. In some instances, the length is at least 700 nucleotides. In some instances, the length is at least 800 nucleotides. In some instances, the length is at least 900 nucleotides. In some instances, the length is at least 1000 nucleotides. In some instances, the length is at least 1100Attorney Docket No.: 45817-0163WO1 nucleotides. In some instances, the length is at least 1200 nucleotides. In some instances, the length is at least 1300 nucleotides. In some instances, the length is at least 1400 nucleotides. In some instances, the length is at least 1500 nucleotides. In some instances, the length is at least 1600 nucleotides. In some instances, the length is at least 1700 nucleotides. In some instances, the length is at least 1800 nucleotides. In some instances, the length is at least 1900 nucleotides. In some instances, the length is at least 2000 nucleotides. In some instances, the length is at least 2500 nucleotides. In some instances, the length is at least 3000 nucleotides.

[0909] In some instances, the poly A tail may be 80 nucleotides, 120 nucleotides, or 160 nucleotides in length. In some instances, the poly A tail may be 20, 40, 80, 100, 120, 140 or 160 nucleotides in length.

[0910] In some instances, the poly A tail is designed relative to the length of the mRNA. This design may be based on the length of the coding region of the mRNA, the length of a particular feature or region of the mRNA, or based on the length of the ultimate product expressed from the RNA. When relative to any additional feature of the RNA (e.g., other than the mRNA portion which includes the poly A tail), poly A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% greater in length than the additional feature. The poly A tail may also be designed as a fraction of the mRNA to which it belongs. In this context, the poly A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly A tail.

[0911] In some instances, engineered binding sites and / or the conjugation of nucleic acids or mRNA for poly A binding protein may be used to enhance expression. The engineered binding sites may be sensor sequences which can operate as binding sites for ligands of the local microenvironment of the nucleic acids and / or mRNA. As a nonlimiting example, the nucleic acids and / or mRNA may include at least one engineered binding site to alter the binding affinity of poly A binding protein (PABP) and analogs thereof. The incorporation of at least one engineered binding site may increase the binding affinity of the PABP and analogs thereof.Attorney Docket No.: 45817-0163WO1 Additionally, multiple distinct nucleic acids or mRNA may be linked together to the PABP (poly A binding protein) through the 3 '-end using nucleotides at the 3'-terminus of the poly A tail. Transfection experiments can be conducted in relevant cell lines and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72hr, and day 7 post-transfection. As a non-limiting example, the transfection experiments may be used to evaluate the effect on PABP or analogs thereof binding affinity as a result of the addition of at least one engineered binding site.

[0912] In some instances, a poly A tail may be used to modulate translation initiation. While not wishing to be bound by theory, the poly A tail recruits PABP which in turn can interact with translation initiation complex and thus may be essential for protein synthesis.

[0913] In some instances, a poly A tail may also be used in the present disclosure to protect against 3 ’-5’ exonuclease digestion.

[0914] In some instances, the nucleic acids or mRNA of the present disclosure are designed to include a poly A-G Quartet. The G-quartet is a cyclic hydrogen bonded array of four guanosine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly A tail. The resultant nucleic acid or mRNA may be assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the poly A-G quartet results in protein production equivalent to at least 75% of that seen using a poly A tail of 120 nucleotides alone (SEQ ID NO: 524).

[0915] In some instances, the nucleic acids or mRNA of the present disclosure may include a poly A tail and may be stabilized by the addition of a chain terminating nucleoside. The nucleic acids and / or mRNA with a poly A tail may further include a 5 ’cap structure.Attorney Docket No.: 45817-0163WO1 In some instances, the nucleic acids or mRNA of the present disclosure may include a poly A-G Quartet. The nucleic acids and / or mRNA with a poly A-G Quartet may further include a 5 ’cap structure.

[0916] In some instances, the chain terminating nucleoside which may be used to stabilize the nucleic acid or mRNA including a poly A tail or poly A-G Quartet may be, but is not limited to, those described in International Patent Publication No.

[0917] WO2013103659, incorporated herein by reference in its entirety. In some instances, the chain terminating nucleosides which may be used with the present disclosure includes, but is not limited to, 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3 '-deoxy cytosine, 3 '-deoxy guanosine, 3 '-deoxythymine, 2', 3 '-dideoxynucleosides, such as 2', 3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2', 3'- dideoxy guanosine, 2', 3 '-dideoxythymine, a 2'-deoxynucleoside, or a -O- methylnucleoside.

[0918] In some instances, the mRNA which includes a poly A tail or a poly A-G Quartet may be stabilized by an alteration to the 3 ’region of the nucleic acid that can prevent and / or inhibit the addition of oligo(U) (see, e.g., International Patent Publication No. WO2013 / 103659, incorporated herein by reference in its entirety).

[0919] In yet another embodiment, the mRNA, which includes a poly A tail or a poly A-G Quartet, may be stabilized by the addition of an oligonucleotide that terminates in a 3’-deoxynucleoside, 2’, 3’ -di deoxy nucleoside 3'-O-methylnucleosides, 3'-O-ethylnucleosides, 3 '-arabinosides, and other alternative nucleosides known in the art and / or described herein.

[0920] Synthesis of Polynucleotides

[0921] The polynucleotide molecules (e.g., mRNA) for use in accordance with the disclosure may be prepared according to any useful technique, as described herein. The alternative nucleosides and nucleotides used in the synthesis of polynucleotide molecules disclosed herein can be prepared from readily available starting materials using the following general methods and procedures. Where typical or preferred process conditionsAttorney Docket No.: 45817-0163WO1 (e.g., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are provided, a skilled artisan would be able to optimize and develop additional process conditions. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

[0922] The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g.,1H or13C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography (e.g., high performance liquid chromatography (HPLC) or thin layer chromatography).

[0923] Preparation of polynucleotide molecules of the present disclosure can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, (1991), which is incorporated herein by reference in its entirety.

[0924] The reactions of the processes described herein can be carried out in suitable solvents, which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out (i.e., temperatures which can range from the solvent’s freezing temperature to the solvent’s boiling temperature). A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected.

[0925] Resolution of racemic mixtures of alternative polynucleotides or nucleic acids (e.g., polynucleotides or mRNA molecules) can be carried out by any of numerousAttorney Docket No.: 45817-0163WO1 methods known in the art. An example method includes fractional recrystallization using a “chiral resolving acid” which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids.

[0926] Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art.

[0927] Alternative nucleosides and nucleotides (e.g., building block molecules) can be prepared according to the synthetic methods described in Ogata et al., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res. 22(1): 72-78 (1994); Fukuhara et al., Biochemistry, 1(4): 563-568 (1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each of which are incorporated by reference in their entirety.

[0928] If the polynucleotide includes one or more alternative nucleosides or nucleotides, the polynucleotides of the disclosure may or may not be uniformly altered along the entire length of the molecule. For example, one or more or all types of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may or may not be uniformly altered in a polynucleotide of the disclosure, or in a given predetermined sequence region thereof. In some embodiments, all nucleotides X in a polynucleotide of the disclosure (or in a given sequence region thereof) are altered, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

[0929] Different sugar alterations, nucleotide alterations, and / or internucleoside linkages (e.g., backbone structures) may exist at various positions in the polynucleotide. One of ordinary skill in the art will appreciate that the nucleotide analogs or other alteration(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased. An alteration may also be a 5' or 3' terminal alteration. The polynucleotide may contain from 1% to 100% alternative nucleosides,Attorney Docket No.: 45817-0163WO1 nucleotides, or internucleoside linkages (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. In some embodiments, the remaining percentage is accounted for by the presence of A, G, U, or C.

[0930] When referring to percentage incorporation by an alternative nucleoside, nucleotide, or internucleoside linkage, in some embodiments the remaining percentage necessary to total 100% is accounted for by the corresponding natural nucleoside, nucleotide, or intemucleoside linkage. In other embodiments, the remaining percentage necessary to total 100% is accounted for by a second alternative nucleoside, nucleotide, or intemucleoside linkage.

[0931] Nanoparticles

[0932] The present disclosure also provides LNP compositions. The lipid nanoparticle compositions described herein may be used for the delivery of therapeutic and / or prophylactic agents, e.g., mRNAs, to mammalian cells or organs. For example, the lipid nanoparticles described herein have little or no immunogenicity. For example, the lipid compounds disclosed herein have a lower immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or DLinDMA). For example, a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent, e.g., mRNA, has an increased therapeutic index as compared to a corresponding formulation which comprises aAttorney Docket No.: 45817-0163WO1 reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.

[0933] In some instances, the present application provides pharmaceutical compositions comprising: (a) a delivery agent (e.g., a nanoparticle such as a lipid nanoparticle); and (b) a polynucleotide (e.g., mRNA) encoding a VHH, polypeptide, bispecific antibody, or multispecific antibody of the disclosure. The polynucleotide is formulated with the delivery agent. The mRNA may be a modified mRNA (e.g., some or all uracils in the mRNA may be N1 -methylpseudouracils).

[0934] Lipid Nanoparticles

[0935] In some instances, polynucleotides of the present disclosure e.g., mRNA) are included in a lipid nanoparticle (LNP). Lipid nanoparticles according to the present disclosure may comprise: (i) an ionizable lipid e.g., an ionizable amino lipid); (ii) a sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; and (iv) a PEG-modified lipid. In some instances, lipid nanoparticles according to the present disclosure further comprise one or more polynucleotides of the present disclosure (e.g., mRNA).

[0936] In some cases, the lipid nanoparticle comprises an ionizable amino lipid, a structural lipid, a phospholipid, and a polyethyleneglycol (PEG)-modified lipid. In certain cases, the phospholipid is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1, 2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE). In some cases, the ionizable amino lipid is a compound of Formula (I). In one case, the ionizable amino lipid is Compound 1-1. In certain cases, the PEG-modified lipid is PEG 2000 dimyristoyl glycerol (PEG-DMG) or 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90, 93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate. In some cases, the structural lipid is cholesterol, adosterol, agosterol A, atheronals, avenasterol, azacosterol, blazein, cerevisterol, colestolone, cycloartenol, daucosterol, 7-dehydrocholesterol, 5-Attorney Docket No.: 45817-0163WO1 dehydroepisterol, 7-dehydrositosterol, 20a,22R-di hydroxy cholesterol, dinosterol, epibrassicasterol, episterol, ergosterol, ergosterol, fecosterol, fucosterol, fungisterol, ganoderenic acid, ganoderic acid, ganoderiol, ganodermadiol, 7a-hydroxy cholesterol, 22R-hydroxycholesterol, 27-hydroxycholesterol, inotodiol, lanosterol, lathosterol, lichesterol, lucidadiol, lumisterol, oxycholesterol, oxysterol, parkeol, saringosterol, spinasterol, sterol ester, trametenolic acid, zhankuic acid, or zymosterol. In some cases, the structural lipid is cholesterol. In one instance, lipid nanoparticle comprises an ionizable amino lipid that is Compound 1-1, a phospholipid that is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), a PEG-modified lipid that is 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90, 93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate, and a structural lipid that is cholesterol.

[0937] The lipid nanoparticles according to 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;

[0938] PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016000129;

[0939] PCT / US2016 / 014280; PCT / US2016 / 014280; PCT / US2017 / 038426;

[0940] PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 52117;

[0941] PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and

[0942] PCT / US2016 / 069491 all of which are incorporated by reference herein in their entirety.

[0943] In some instances, the lipid nanoparticle comprises: (i) 20 to 60 mole ratio % ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 25 to 55 mole ratio % sterol or other structural lipid, (iii) 5 to 25 mole ratio % non-cationic lipid (e.g, phospholipid), and (iv) 0.5 to 15 mole ratio % PEG-modified lipid.

[0944] In some instances, the lipid nanoparticle comprises: (i) 40 to 50 mole ratio % ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 30 to 45 mole ratio % sterol or other structural lipid, (iii) 5 to 15 mole ratio % non-cationic lipid (e.g, phospholipid), and (iv) 1 to 5 mole ratio % PEG-modified lipid.Attorney Docket No.: 45817-0163WO1 In some instances, the lipid nanoparticle comprises: (i) 45 to 50 mole ratio % ionizable cationic lipid (e.g., ionizable amino lipid), (ii) 35 to 45 mole ratio % sterol or other structural lipid, (iii) 8 to 12 mole ratio % non-cationic lipid (e.g., phospholipid), and (iv) 1.5 to 3.5 mole ratio % PEG-modified lipid.

[0945] In the following sections, “Compounds” numbered with an “I-” prefix (e.g., “Compound 1-1,” “Compound 1-2,” “Compound 1-3,” “Compound I- VI,” etc., indicate specific ionizable lipid compounds. Likewise, compounds numbered with a “P-” prefix (e.g., “Compound P-I,” etc.) indicate a specific PEG-modified lipid compound.

[0946] Ionizable Amino Lipids

[0947] In some instances, the lipid nanoparticle of the present disclosure comprises an ionizable cationic lipid e.g., an ionizable amino lipid) that is a compound of Formula (I):

[0948]

[0949] (I) or its N-oxide, or a salt or isomer thereof,

[0950] wherein R’ais R’branched; wherein

[0951] RaaRa7

[0952] R

[0953]

[0954] ’ branched js; RapRas. denotes a point of attachment; wherein Ra“, Ra|3, Ray, and RaSare each independently selected from the group consisting of H, C2-12alkyl, and C2-12alkenyl;

[0955] R2and R3are each independently selected from the group consisting of C1-14alkyl and

[0956] C2-14alkenyl;

[0957] R4is selected from the group consisting of -(CH2)n0H, wherein n is selected from O. zP

[0958] Rio N \ / X

[0959] the group consisting of 1, 2, 3, 4, and 5, and

[0960]

[0961] H n2Attorney Docket No.: 45817-0163WO1

[0962] wherein

[0963]

[0964] denotes a point of attachment; wherein

[0965] R10is N(R)2; each R is independently selected from the group consisting of C1-6alkyl, C2-3alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0966] each R5is independently selected from the group consisting of C1-3alkyl, C2-3alkenyl, and H;

[0967] each R6is independently selected from the group consisting of C1-3alkyl, C2-3alkenyl, and H;

[0968] M and M’ are each independently selected from the group consisting of -C(O)O-and

[0969] -OC(O)-;

[0970] R’ is a C1-12 alkyl or C2-12 alkenyl;

[0971] 1 is selected from the group consisting of 1, 2, 3, 4, and 5; and

[0972] m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0973] Ra«RaY

[0974] In some instances, in Formula (I), R’ais R’branched; R

[0975]

[0976] ,branched -spapRa«. denotes a point of attachment; Ra“, Rap, Ray, and RaSare each H; R2and R3are each Ci-14 alkyl; R4is -(CH2)nOH; n is 2; each R5is H; each R6is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; 1 is 5; and m is 7.

[0977] In some instances, in Formula (I), R’ais R’branched; R’branched js

[0978] Ra" Ray

[0979]

[0980] RapRae>. =s denotes apoint of attachment; Raa, Ra|3, Ray, and RaSare each H; R2and R3are each C1-14 alkyl; R4is -(CH2)nOH; n is 2; each R5is H; each R6is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; 1 is 3; and m is 7.

[0981] In some instances of the compounds of Formula (I), R’ais R’brancbed;

[0982] Ra“ Ray

[0983] R’ branched js

[0984]

[0985] RapRao. denotes apoint of attachment; Ra“ is C2-12Attorney Docket No.: 45817-0163WO1 alkyl; Raβ, Raγ, and Raδare each H; R2and R3are each C1-14alkyl; R4is

[0986] p 10 N in -

[0987]

[0988] H •RioisNH(CI-6 alkyl); n2 is 2; R is H; each R6is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; 1 is 5; and m is 7.

[0989] In some instances of the compounds of Formula (I), R’ais R,branched;

[0990] Ra" Ray

[0991] R’ branched jspapRas. =s denotes a point of attachment; Raα, Raβ, and Raδare

[0992]

[0993] each H; Rayis C2-12 alkyl; R2and R3are each C1-14 alkyl; R4is -(CH2)nOH; n is 2; each R5is H; each R6is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; 1 is 5; and m is 7.

[0994] In some instances, the compound of Formula (I) is selected from:

[0995] HO

[0996]

[0997] Attorney Docket No.: 45817-0163WO1 HO

[0998]

[0999] In some instances, the compound of Formula (I) is:

[1000] 0

[1001]

[1002] (Compound 1-2).

[1003] In some instances, the compound of Formula (I) is:

[1004]

[1005] (Compound 1-3).

[1006] In one instance, the ionizable amino lipid is or comprises Compound 1-1.

[1007] Phospholipids

[1008] The lipid composition of the lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[1009] A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.

[1010] A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid,Attorney Docket No.: 45817-0163WO1 arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[1011] Particular phospholipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.

[1012] Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid can be functionalized with or cross-linked to one or more alkynes e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety e.g., a dye).

[1013] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidyl glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.

[1014] In some instances, a phospholipid of the present disclosure comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-Attorney Docket No.: 45817-0163WO1 phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3 -phosphocholine, l,2-diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt(DOPG), sphingomyelin, and mixtures thereof.

[1015] In some instances, a phospholipid of the present disclosure comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[1016] In certain instances, a phospholipid useful or potentially useful in the present disclosure is an analog or variant of DSPC.

[1017] Alternative Lipids

[1018] In certain instances, a phospholipid useful or potentially useful in the present disclosure comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in certain instances, a phospholipid useful.

[1019] In certain instances, an alternative lipid is used in place of a phospholipid of the present disclosure.

[1020] In certain instances, an alternative lipid of the present disclosure is oleic acid.

[1021] In certain instances, the alternative lipid is one of the following:Attorney Docket No.: 45817-0163WO1

[1022] 5

[1023]

[1024] Attorney Docket No.: 45817-0163WO1

[1025]

[1026] O Structural Lipids

[1027] The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids. As used herein, the term "structural lipid" refers to sterols and also to lipids containing sterol moieties.

[1028] Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some instances, the structural lipid is a sterol. As defined herein, "sterols" are a subgroup of steroids consisting of steroid alcohols. In certain instances, the structural lipid is a steroid. In certain instances, the structural lipid is cholesterol. In certain instances, the structural lipid is an analog of cholesterol. In certain instances, the structural lipid is alpha-tocopherol.

[1029] In some instances, the structural lipid is cholesterol.

[1030] In some instances, the structural lipids may be one or more of the structural lipids described in U. S. Application No. 62 / 520,530.

[1031] Polyethylene Glycol (PEG)-Lipids

[1032] The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more a polyethylene glycol (PEG) lipid.

[1033] As used herein, the term “PEG-lipid” refers to polyethylene glycol (PEG)-modified lipids. Non-limiting examples ofPEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-Attorney Docket No.: 45817-0163WO1 CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-di acyl oxypropan-3 -amines. Such lipids are also referred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[1034] In some instances, the PEG-lipid includes, but not limited to 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

[1035] In some instances, the PEG-lipid is 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), or 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90, 93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate.

[1036] In some instances, the PEG-lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.

[1037] In some instances, the lipid moiety of the PEG-lipids includes those having lengths of from about C14to about C22, preferably from about C14to about C16. In some instances, a PEG moiety, for example an mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In some instances, the PEG-lipid is PEG2k-DMG.

[1038] In some instances, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE.Attorney Docket No.: 45817-0163WO1 PEG-lipids are known in the art, such as those described in U. S. Patent No.

[1039] 8,158,601 and International Publ. No. WO 2015 / 130584 A2, which are incorporated herein by reference in their entirety.

[1040] 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.

[1041] 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.

[1042] In some instances, PEG lipids useful in the present disclosure 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 instances, 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 instances, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain instances, 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 disclosure.Attorney Docket No.: 45817-0163WO1 In certain instances, a PEG lipid useful in the present disclosure is a PEGylated fatty acid. In certain instances, a PEG lipid useful in the present disclosure is a compound of Formula (VI). Provided herein are compounds of Formula (VI):

[1043] O

[1044]

[1045] r(VI), or a salts thereof, wherein:

[1046] R3is-OR°;

[1047] R° is hydrogen, optionally substituted alkyl or an oxygen protecting group;

[1048] r is an integer between 1 and 100, inclusive;

[1049] R5is optionally substituted C10-40alkyl, optionally substituted C10-40alkenyl, or optionally substituted C10-40alkynyl; and optionally one or more methylene groups of R5are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(RN), O, S, C(O), C(O)N(RN), NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), -NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), -OS(O)N(RN), N(RN)S(O)O, S(O)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), -

[1050]

[1051] OS(O)2N(RN), or N(RN)S(O)2O; and each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.

[1052] In certain instances, the compound of Formula (VI) is of Formula (VI-OH):

[1053] HO^

[1054]

[1055] v / r(VI-OH),

[1056] or a salt thereof. In some instances, r is 45.Attorney Docket No.: 45817-0163WO1 In yet other instances the compound of Formula (VI) is:

[1057]

[1058] or a salt thereof. In one embodiment, r is 40-50.

[1059] In some instances, the compound of Formula (VI) is

[1060] HO

[1061]

[1062] (Compound A).

[1063] In one instance, an LNP of this disclosure comprises Compound A.

[1064] In some aspects, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise aPEG-lipid.

[1065] In some instances, the PEG-lipids may be one or more of the PEG lipids described in U. S. Application No. 62 / 520,530.

[1066] In some instances, a PEG lipid of the present disclosure comprises a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG- modified dialkylglycerol, and mixtures thereof. In some instances, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG and / or PEG-DPG.

[1067] In some instances, a LNP of the present disclosure comprises an ionizable cationic lipid of Formula I, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising PEG-DMG.

[1068] In some instances, a LNP of the present disclosure comprises an ionizable cationic lipid of Formula I, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid.Attorney Docket No.: 45817-0163WO1 In some instances, a LNP of the present disclosure comprises an ionizable cationic lipid of

[1069] O

[1070]

[1071] In some instances, a LNP of the present disclosure comprises an ionizable cationic lipid of

[1072]

[1073] and an alternative lipid comprising oleic acid.

[1074] In some instances, a LNP of the present disclosure comprises an ionizable cationic lipid of

[1075]

[1076] an alternative lipid comprising oleic acid, a structural lipid comprising cholesterol, and a PEG lipid.

[1077] In some instances, a LNP of the present disclosure comprises an ionizable cationic lipid ofAttorney Docket No.: 45817-0163WO1

[1078]

[1079] a phospholipid comprising DOPE, a structural lipid comprising cholesterol, and a PEG lipid.

[1080] In some instances, a LNP of the present disclosure comprises an ionizable cationic lipid of

[1081]

[1082] a phospholipid comprising DOPE, a structural lipid comprising cholesterol, and a PEG lipid.

[1083] In some instances, a LNP of the present disclosure comprises an N: P ratio of from about 2: 1 to about 30: 1. In some instances, a LNP of the present disclosure comprises an N: P ratio of about 6:1. In some instances, a LNP of the present disclosure comprises an N: P ratio of about 3:1. In some instances, a LNP of the present disclosure comprises a wt / wt ratio of the ionizable cationic lipid component to the RNA of from about 10:1 to about 100:1. In some instances, a LNP of the present disclosure comprises a wt / wt ratio of the ionizable cationic lipid component to the RNA of about 20:1. In some instances, a LNP of the present disclosure comprises a wt / wt ratio of the ionizable cationic lipid component to the RNA of about 10: 1. In some instances, a LNP of the present disclosure has a mean diameter from about 50nm to about 150nm. In some instances, a LNP of the present disclosure has a mean diameter from about 70nm to about 120nm.

[1084] Other Lipid Composition ComponentsAttorney Docket No.: 45817-0163WO1 The lipid composition of a pharmaceutical composition disclosed herein can include one or more components in addition to those described above. For example, the lipid composition can include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents (e.g, surfactants), or other components. For example, a permeability enhancer molecule can be a molecule described by U. S. Patent Application Publication No. 2005 / 0222064. Carbohydrates can include simple sugars (e.g, glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).

[1085] A polymer can be included in and / or used to formulate a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition in lipid nanoparticle form). A polymer can be biodegradable and / or biocompatible. A polymer can be selected from, but is not limited to, polyamines, poly ethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.

[1086] The ratio between the lipid composition and the polynucleotide range can be from about 10:1 to about 60:1 (wt / wt).

[1087] In some instances, the ratio between the lipid composition and the polynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt / wt). In some instances, the wt / wt ratio of the lipid composition to the polynucleotide encoding a therapeutic agent is about 20: 1 or about 15:1.

[1088] In some instances, the pharmaceutical composition disclosed herein can contain more than one polypeptides. For example, a pharmaceutical composition disclosed herein can contain two or more polynucleotides (e.g., RNA, e.g., mRNA).Attorney Docket No.: 45817-0163WO1 In some instances, the lipid nanoparticles described herein can comprise polynucleotides (e.g., mRNA) in a lipid:polynucleotide weight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or 70:1, or a range or any of these ratios such as, but not limited to, 5:1 to about 10:1, from about 5:1 to about 15:1, from about 5: 1 to about 20: 1, from about 5: 1 to about 25: 1, from about 5: 1 to about 30:1, from about 5:1 to about 35:1, from about 5: 1 to about 40: 1, from about 5: 1 to about 45: 1, from about 5:1 to about 50: 1, from about 5: 1 to about 55:1, from about 5: 1 to about 60: 1, from about 5:1 to about 70:1, from about 10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 to about 25:1, from about 10:1 to about 30:1, from about 10:1 to about 35:1, from about 10:1 to about 40:1, from about 10:1 to about 45:1, from about 10:1 to about 50:1, from about 10:1 to about 55:1, from about 10:1 to about 60:1, from about 10:1 to about 70:1, from about 15:1 to about 20:1, from about 15:1 to about 25:1, from about 15:1 to about 30:1, from about 15:1 to about 35:1, from about 15:1 to about 40:1, from about 15:1 to about 45:1, from about 15:1 to about 50:1, from about 15:1 to about 55:1, from about 15:1 to about 60:1 or from about 15:1 to about 70:1.

[1089] In some instances, the lipid nanoparticles described herein can comprise the polynucleotide in a concentration from approximately 0.1 mg / ml to 2 mg / ml such as, but not limited to, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, 2.0 mg / ml or greater than 2.0 mg / ml.

[1090] Nanoparticle Compositions

[1091] In some instances, the binding polypeptides and pharmaceutical compositions disclosed herein are formulated as lipid nanoparticles (LNP). Accordingly, the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent such as compound as described herein, and (ii) a polynucleotide encoding a polypeptide. In such nanoparticle composition, the lipidAttorney Docket No.: 45817-0163WO1 composition disclosed herein can be formulated with the polynucleotide encoding a polypeptide.

[1092] Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[1093] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes. In some instances, nanoparticle compositions are vesicles including one or more lipid bilayers. In certain instances, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers can be functionalized and / or crosslinked to one another. Lipid bilayers can include one or more ligands, proteins, or channels.

[1094] In some instances, a lipid nanoparticle comprises an ionizable amino lipid, a structural lipid, a phospholipid, and mRNA. In some instances, the LNP comprises an ionizable amino lipid, a PEG-modified lipid, a sterol and a structural lipid. In some instances, the LNP has a molar ratio of about 40-50% ionizable amino lipid; about 5-15% structural lipid; about 30-45% sterol; and about 1-5% PEG-modified lipid.

[1095] In some instances, the lipid nanoparticle comprises 47-49 mole ratio % ionizable cationic lipid (e.g. ionizable amino lipid, e.g., Compound 1-1, Compound 1-2, or Compound 1-3), 10-12 mole ratio % non-cationic lipid (e.g., phospholipid, e.g., DSPC), 38-40 mole ratio % sterol (e.g., cholesterol) or other structural lipid, and 1-3 mole ratio % PEG-modified lipid (e.g., PEG-DMG or Compound P-I).

[1096] For instance, in some instances, the lipid nanoparticle may comprise the following components at the following molar ratios:

[1097] (i) 45-50 mole ratio % Compound 1-1Attorney Docket No.: 45817-0163WO1 (ii) 35-45 mole ratio % sterol (e.g., cholesterol);

[1098] (iii) 8-12 mole ratio % phospholipid (e.g., DSPC or DOPE); and

[1099] (iv) 1.5-3.5 mole ratio % PEG-lipid (e.g., PEG-DMG or 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75, 78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132- tetratetracontaoxatetratriacontahectyl stearate).

[1100] For instance, in some instances, the lipid nanoparticle may comprise the following components at the following molar ratios:

[1101] (i) 45-50 mole ratio % Compound 1-1

[1102] (ii) 35-45 mole ratio % Cholesterol;

[1103] (iii) 8-12 mole ratio % DSPC; and

[1104] (iv) 1.5-3.5 mole ratio % PEG-lipid (e.g., PEG-DMG or 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75, 78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate).

[1105] For instance, in some instances, the lipid nanoparticle may comprise the following components at the following molar ratios:

[1106] (i) 45-50 mole ratio % Compound 1-1

[1107] (ii) 35-45 mole ratio % Cholesterol;

[1108] (iii) 8-12 mole ratio % DSPC; and

[1109] (iv) 1.5-3.5 mole ratio % PEG-lipid (e.g., PEG-DMG or 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75, 78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132- tetratetracontaoxatetratriacontahectyl stearate).

[1110] In some instances, the lipid nanoparticle may comprise the following:

[1111] (i) 45-50 mole ratio % Compound 1-2;

[1112] (ii) 35-45 mole ratio % sterol (e.g., Cholesterol);

[1113] (iii) 8-12 mole ratio % phospholipid (e.g., DSPC or DOPE); andAttorney Docket No.: 45817-0163WO1 (iv) 1.5-3.5 mole ratio % PEG-lipid (e.g., PEG-DMG or

[1114] 3,6,9, 12, 15, 18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75, 78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132- tetratetracontaoxatetratriacontahectyl stearate).

[1115] In some instances, the lipid nanoparticle may comprise the following:

[1116] (i) 45-50 mole ratio % Compound 1-2;

[1117] (ii) 35-45 mole ratio % Cholesterol;

[1118] (iii) 8-12 mole ratio % DSPC; and

[1119] (iv) 1.5-3.5 mole ratio % PEG-lipid (e.g., PEG-DMG or 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75, 78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132- tetratetracontaoxatetratriacontahectyl stearate).

[1120] For instance, in some instances, the lipid nanoparticle may comprise the following components at the following molar ratios:

[1121] (i) 45-50 mole ratio % Compound 1-2;

[1122] (ii) 35-45 mole ratio % Cholesterol;

[1123] (iii) 8-12 mole ratio % DSPC; and

[1124] (iv) 1.5-3.5 mole ratio % PEG-lipid (e.g., PEG-DMG or

[1125] 3,6,9, 12, 15, 18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75, 78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132- tetratetracontaoxatetratriacontahectyl stearate).

[1126] In some instances, the lipid nanoparticle may comprise the following:

[1127] (i) 45-50 mole ratio % Compound 1-3;

[1128] (ii) 35-45 mole ratio % sterol (e.g., Cholesterol);

[1129] (iii) 8-12 mole ratio % phospholipid e.g., DSPC or DOPE); and

[1130] (iv) 1.5-3.5 mole ratio % PEG-lipid (e.g., PEG-DMG or

[1131] 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,Attorney Docket No.: 45817-0163WO1 78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate).

[1132] In some instances, the lipid nanoparticle may comprise the following:

[1133] (i) 45-50 mole ratio % Compound 1-3;

[1134] (ii) 35-45 mole ratio % Cholesterol;

[1135] (iii) 8-12 mole ratio % DSPC; and

[1136] (iv) 1.5-3.5 mole ratio % PEG-lipid (e.g., PEG-DMG or 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75, 78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132- tetratetracontaoxatetratriacontahectyl stearate).

[1137] In some instances, the lipid nanoparticle may comprise the following:

[1138] (i) 45-50 mole ratio % Compound 1-3;

[1139] (ii) 35-45 mole ratio % Cholesterol;

[1140] (iii) 8-12 mole ratio % DSPC; and

[1141] (iv) 1.5-3.5 mole ratio % PEG-lipid (e.g., PEG-DMG or 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75, 78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132- tetratetracontaoxatetratriacontahectyl stearate).

[1142] In some instances, the lipid nanoparticle may comprise the following components at the following molar ratios:

[1143] (i) 45 to 50 mole ratio % ionizable amino lipid (e.g., Compound 1-1)

[1144] (ii) 35 to 40 mole ratio % structural lipid (e.g., cholesterol);

[1145] (iii) 8 to 12 mole ratio % phospholipid (e.g., DSPC); and

[1146] (iv) 2 to 4 mole ratio % PEG-lipid (e.g., Compound A).

[1147] In other instances, the lipid nanoparticle comprises the following components at the following molar ratios:

[1148] (i) about 47.5 mole ratio % Compound 1-1;

[1149] (ii) about 39 mole ratio % cholesterol;Attorney Docket No.: 45817-0163WO1 (iii) about 10.5 mole ratio % DSPC; and

[1150] (iv) about 3 mole ratio % Compound A.

[1151] In yet other instances, the lipid nanoparticle comprises the following components at the following molar ratios:

[1152] (i) 47.5 mole ratio % Compound 1-1;

[1153] (ii) 39 mole ratio % cholesterol;

[1154] (iii) 10.5 mole ratio % DSPC; and

[1155] (iv) 3 mole ratio % Compound A.

[1156] In some instances, the LNP has a polydispersity value of less than 0.4. In some instances, the LNP has a net neutral charge at a neutral pH. In some instances, the LNP has a mean diameter of 50-150 nm. In some instances, the LNP has a mean diameter of 80-100 nm.

[1157] As generally defined herein, the term “lipid” refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic. Examples of classes of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids. In some instances, the amphiphilic properties of some lipids lead them to form liposomes, vesicles, or membranes in aqueous media.

[1158] In some instances, a lipid nanoparticle (LNP) may comprise an ionizable amino lipid. As used herein, the term “ionizable amino lipid” has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties. In some instances, an ionizable amino lipid may be positively charged or negatively charged. An ionizable amino lipid may be positively charged, in which case it can be referred to as “cationic lipid”. In certain instances, an ionizable amino lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipid. As used herein, a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalentAttorney Docket No.: 45817-0163WO1 (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc. The charged moiety may be anionic (i.e., negatively charged) or cationic (, positively charged). Examples of positively-charged moi eties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively- charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and / or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired.

[1159] It should be understood that the terms “charged” or “charged moiety” does not refer to a “partial negative charge" or “partial positive charge" on a molecule. The terms “partial negative charge" and “partial positive charge" are given their ordinary meaning in the art. A “partial negative charge" may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way.

[1160] The ionizable amino lipid is sometimes referred to in the art as an “ionizable cationic lipid”. In some instances, the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail that are connected via a linker structure.

[1161] In addition to these, an ionizable amino lipid may also be a lipid including a cyclic amine group.

[1162] In some instances, the ionizable amino lipid may be selected from, but not limited to, an ionizable amino lipid described in International Publication Nos. WO2013086354 and WO2013116126; the contents of each of which are herein incorporated by reference in their entirety.Attorney Docket No.: 45817-0163WO1 In yet another embodiment, the ionizable amino lipid may be selected from, but not limited to, Formula CLI-CLXXXXII of US Patent No. 7,404,969; each of which is herein incorporated by reference in their entirety.

[1163] In some instances, the lipid may be a cleavable lipid such as those described in International Publication No. WO2012170889, herein incorporated by reference in its entirety. In some instances, the lipid may be synthesized by methods known in the art and / or as described in International Publication Nos. WO2013086354; the contents of each of which are herein incorporated by reference in their entirety.

[1164] Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) can be used to measure zeta potentials. Dynamic light scattering can also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, poly dispersity index, and zeta potential.

[1165] The size of the nanoparticles can help counter biological reactions such as, but not limited to, inflammation, or can increase the biological effect of the polynucleotide.

[1166] As used herein, “size” or “mean size” in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition.

[1167] In some instances, the polynucleotide encoding a polypeptide(s) described herein are formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm,Attorney Docket No.: 45817-0163WO1 about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and / or about 90 to about 100 nm.

[1168] In some instances, the nanoparticles have a diameter from about 10 to 500 nm. In some instances, the nanoparticle has a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.

[1169] In some instances, the largest dimension of a nanoparticle composition is 1 μm or shorter (e.g., 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter).

[1170] A nanoparticle composition can be relatively homogenous. A poly dispersity index can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition can have a poly dispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some instances, theAttorney Docket No.: 45817-0163WO1 poly dispersity index of a nanoparticle composition disclosed herein can be from about 0.10 to about 0.20.

[1171] The amount of a polynucleotide present in a pharmaceutical composition disclosed herein can depend on multiple factors such as the size of the polynucleotide, desired target and / or application, or other properties of the nanoparticle composition as well as on the properties of the polynucleotide.

[1172] For example, the amount of an mRNA useful in a nanoparticle composition can depend on the size (expressed as length, or molecular mass), sequence, and other characteristics of the mRNA. The relative amounts of a polynucleotide in a nanoparticle composition can also vary.

[1173] The relative amounts of the lipid composition and the polynucleotide present in a lipid nanoparticle composition of the present disclosure can be optimized according to considerations of efficacy and tolerability. For compositions including an mRNA as a polynucleotide, the N: P ratio can serve as a useful metric.

[1174] As the N: P ratio of a nanoparticle composition controls both expression and tolerability, nanoparticle compositions with low N:P ratios and strong expression are desirable. N: P ratios vary according to the ratio of lipids to RNA in a nanoparticle composition.

[1175] In general, a lower N: P ratio is preferred. The one or more RNA, lipids, and amounts thereof can be selected to provide an N: P ratio from about 2: 1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain instances, the N: P ratio can be from about 2:1 to about 8:1. In other instances, the N: P ratio is from about 5: 1 to about 8:1. In certain instances, the N: P ratio is between 5:1 and 6: 1. In one specific aspect, the N: P ratio is about is about 5.67:1.Attorney Docket No.: 45817-0163WO1 In some instances, the polynucleotides described herein can be formulated for controlled release and / or targeted delivery. As used herein, "controlled release" refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome. In some instances, the polynucleotides can be formulated with a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery.

[1176] In some instances, the therapeutic nanoparticle polynucleotide can be formulated for sustained release. As used herein, "sustained release" refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time can include, but is not limited to, hours, days, weeks, months and years. As a non-limiting example, the sustained release nanoparticle of the polynucleotides described herein can be formulated as disclosed in Inti. Pub. No.

[1177] W02010075072 and U. S. Pub. Nos. US20100216804, US20110217377, US20120201859 and US20130150295, each of which is herein incorporated by reference in their entirety.

[1178] In some instances, the therapeutic nanoparticle polynucleotide can be formulated to be target specific, such as those described in Inti. Pub. Nos. WO2008121949, W02010005726, W02010005725, WO2011084521 and WO2011084518; and U. S. Pub. Nos. US20100069426, US20120004293 and US20100104655, each of which is herein incorporated by reference in its entirety.

[1179] The LNPs can be prepared using microfluidic mixers or micromixers. Exemplary microfluidic mixers can include, but are not limited to, a slit interdigital micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and / or a staggered herringbone micromixer (SHM) (see, Zhigaltsev et al., Langmuir. 28:3633-40 (2012); Belliveau et al., Molecular Therapy-Nucleic Acids. I:e37 (2012); Chen et al., J. Am. Chem. Soc. 134(16):6948-51 (2012); each of which is herein incorporated by reference in its entirety). Exemplary micromixers include Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital MicroAttorney Docket No.: 45817-0163WO1 Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (IJMM,) from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany. In some instances, methods of making LNP using SHM further comprise mixing at least two input streams wherein mixing occurs by microstructure-induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other. This method can also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling. Methods of generating LNPs using SHM include those disclosed in U. S. Pub. Nos.

[1180] US20040262223 and US20120276209, each of which is incorporated herein by reference in their entirety.

[1181] In some instances, the polynucleotides described herein can be Formulated in lipid nanoparticles using microfluidic technology (see, Whitesides, George M., Nature 442: 368-373 (2006); and Abraham et al., Science 295: 647-651 (2002); each of which is herein incorporated by reference in its entirety). In some instances, the polynucleotides can be Formulated in lipid nanoparticles using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK). A micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.

[1182] In some embodiment, the nanoparticles described herein are stealth nanoparticles or target-specific stealth nanoparticles such as, but not limited to, those described in U. S. Pub. No. US20130172406, herein incorporated by reference in its entirety. The stealth or target-specific stealth nanoparticles can comprise a polymeric matrix, which can comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polyesters,Attorney Docket No.: 45817-0163WO1 polyanhydrides, polyethers, polyurethanes, polymethacrylates, poly acrylates, polycyanoacrylates, or combinations thereof.

[1183] Methods of Treatment

[1184] The binding polypeptides, nucleic acids, pharmaceutical compositions, or lipid nanoparticles comprising or formulated with the nucleic acids described herein can be administered in a therapeutically effective amount to treat a human subject in need thereof. In some aspects, the human subject in need thereof is a subject with a disease, disorder, and / or condition that may be treated with a therapeutically effective amount of the binding polypeptides, nucleic acids, pharmaceutical compositions, or lipid nanoparticles comprising or formulated with the nucleic acids described herein.

[1185] In some instances, the disease or disorder treated is an autoimmune disease. In autoimmune diseases, abnormal immune responses to self-antigens result in damage to the body’s own tissues. The PD-1 pathway via its inhibitory signals, controls the induction and maintenance of tolerance to self-antigens in the context of autoimmunity. In some instances, the autoimmune disorder is rheumatoid arthritis, ulcerative colitis, and psoriasis, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), Type 1 diabetes (T1D), systemic vasculitis, myositis, autoimmune encephalomyelitis, autoimmune hepatitis, Behcet’s disease, myasthenia gravis, autoimmune uveitis, Sjogren’s syndrome, or ankylosing spondylitis.

[1186] In one aspect, the human subject in need thereof is a subject having or at risk of developing an inflammatory disease (e.g., an immune-mediated inflammatory disease). In another aspect, the human subject in need thereof is a subject having or at risk of developing an autoimmune disease (e.g., a T cell driven autoimmune disease). In some cases, the human subject in need thereof is a subject having or at risk of developing rheumatoid arthritis, ulcerative colitis, Type I diabetes, multiple sclerosis, or psoriasis. In the cases of treating an inflammatory disease, an autoimmune disease, or the disorders mentioned above, a PD-1 agonist is used. In one instance, a PD-1 binding protein of the disclosure, a nucleic acid encoding same, or a LNP formulated with such a nucleic acid isAttorney Docket No.: 45817-0163WO1 used to treat rheumatoid arthritis. In another instance, a PD-1 binding protein of the disclosure, a nucleic acid encoding same, or an LNP formulated with such a nucleic acid is used to treat ulcerative colitis.

[1187] In some aspects, a human subject in need thereof is administered a VHH, a binding protein of the present disclosure, a nucleic acid encoding same, or a nucleic acid encoding a VHH that specifically binds PD-1 formulated in a delivery vehicle (e.g., LNP). In some aspects, a human subject in need thereof is administered a lipid nanoparticle that comprises a nucleic acid (e.g., mRNA(s)) encoding a binding protein disclosed herein). In certain cases, the mRNA encodes a PD-1 agonist. Such agonists are useful in treating autoimmune and inflammatory disorders. In some instances, such agonists are useful in treating rheumatoid arthritis, ulcerative colitis, or psoriasis.

[1188] The methods involve administering to the human subject in need thereof a therapeutically effective amount of a VHH containing polypeptide described herein or an mRNA encoding such a polypeptide (e.g., formulated with a LNP). In some cases, the VHH containing polypeptide is a monovalent polypeptide. In some cases, the VHH containing polypeptide is a bivalent polypeptide. In some cases, the VHH containing polypeptide is a bivalent polypeptide (e.g., a PD-1 binding VHH-linked to the hinge-Fc domain of IgGl). In some cases, the VHH containing polypeptide is a bispecific antibody (e.g., a bispecific antibody that binds both PD-1 and a T cell antigen, an NK cell antigen, or a tumor associated antigen). In some cases, the VHH containing polypeptide is a multispecific antibody construct. In some cases, the VHH containing polypeptide is a conjugate (e.g., an antibody drug conjugate).

[1189] In some instances, the methods involve administering to the human subject in need thereof a therapeutically effective amount of a delivery vehicle comprising or formulated with an mRNA encoding a PD-1 binding polypeptide (e.g., VHH). In some cases, the lipid nanoparticle comprises a neutral lipid, an ionizable amino lipid, a polyethyleneglycol (PEG) lipid, and / or a sterol. In certain cases, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine; the ionizable amino lipid is a compound ofAttorney Docket No.: 45817-0163WO1 Formula (I), optional Compound 1-1; the PEG lipid is PEG 2000 dimyristoyl glycerol or 134-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90, 93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate, and the sterol is cholesterol. In some cases, the ionizable amino lipid is present at about 47.5 mole ratio%, the structural lipid is present at about 39 mole ratio%, the phospholipid is present at about 10.5 mole ratio%, and the PEG-modified lipid is present at about 3 mole ratio%.

[1190] The delivery vehicles comprising or formulated with the nucleic acids (e.g., mRNAs) of this disclosure or the polypeptides of this disclosure may be administered subcutaneously, intramuscularly, or intravenously. In some instances, the antibodies are administered to a subject as a bolus IV injection or bolus. This form of delivery can produce high levels of expressed antibody.

[1191] Kits

[1192] Also included herein are kits that contain a VHH, a polypeptide, a bispecific antibody, a VHH-conjugate,, and / or nucleic acids (e.g., mRNA(s)) encoding the same. In some embodiments, the kits provided herein contain one or more cells engineered to express and secrete a polypeptide(s) comprising an anti-PD-1 VHH of the disclosure, such as a cell containing a nucleic acid molecule(s) of the disclosure.

[1193] A kit described herein may include reagents that can be used to produce a pharmaceutical composition of the invention. Optionally, kits described herein may include reagents that can induce the expression of a polypeptide(s) comprising an anti-PD-1 VHH of the disclosure within cells (e.g., bacterial, yeast, insect, or mammalian cells).

[1194] Other kits described herein may include tools for engineering a prokaryotic or eukaryotic cell (e.g., a CHO cell or a BL21(DE3) E. Coli cell or an immune cell) so as toAttorney Docket No.: 45817-0163WO1 express and secrete a polypeptide(s) comprising an anti-PD-1 VHH of the disclosure. For example, a kit may contain CHO cells stored in an appropriate media and optionally frozen according to methods known in the art. The kit may also contain a nucleic acid encoding the desired VHH antibody as well as reagents for expressing the VHH antibody or binding protein in the cell.

[1195] A kit described herein may also provide a polypeptide(s) comprising an anti-PD-1 VHH of the disclosure, or a nucleic acid encoding the same (e.g., mRNA) in combination with a package insert describing how the VHH antibody, or nucleic acid may be administered to a subject, for example, for the treatment of a disease, disorder and / or condition (e.g., inflammatory disease or autoimmune disease).

[1196] In one instance, a kit comprises a nucleic acid(s) encoding a polypeptide comprising a PD-l-binding VHH, a bispecific antibody comprising a PD-l-binding VHH, or a multispecific polypeptide(s) antibody comprising a PD-l-binding VHH. In certain instances, the nucleic acid is an mRNA. In some cases, the nucleic acid is formulated with a delivery vehicle (e.g., sa nanoparticle such as a LNP). In some cases, the kit comprises a package insert describing how the nucleic acid or delivery vehicle may be administered to a subject, for example, for the treatment of a disease, disorder and / or condition (e.g., inflammatory disease or autoimmune disease).

[1197] EXAMPLES

[1198] Example 1: Materials & Methods

[1199] Immunization of Llamas. Animals were immunized with mRNA encoding human PD-1 extracellular domain, transmembrane domain, and cytosolic domain. Formulated mRNA (200-1000 μg) was administered intramuscularly. Following the initial immunizations, boost immunizations were given approximately every two weeks. Test bleeds were taken on day 52 post immunization, to determine antibody titers against the immunized targets in the serum. A final intravenous immunization was given on day 6...

Claims

Attorney Docket No.: 45817-0163WO1 WHAT IS CLAIMED IS:

1. A polypeptide comprising a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds to PD-1, the VHH comprising VHH complementarity determining region (CDR) 1, VHH CDR2, and VHH CDR3 of any one of the amino acid sequences set forth in any one of SEQ ID NOs.: 103, 90, 71, 74, 2, 1, 3 to 5, 65 to 70, 72, 73, 75-89, 91-102, or 104; optionally wherein:(i) the VHH CDR1, VHH CDR2, and VHH CDR3 are based on CDR definitions according to Kabat, Chothia, enhanced Chothia, contact, IMGT, or AbM; and / or(ii) the PD-1 is human PD-1 or human and cynomolgus PD-1; and / or(iii) the VHH comprises VHH CDR1, VHH CDR2, and VHH CDR3 of the amino acid sequence set forth in SEQ ID NO.: 103, 90, 71, 74, or 2; and / or(iv) the VHH comprises VHH CDR1, VHH CDR2, and VHH CDR3 of the amino acid sequence set forth in SEQ ID NO.: 123, 124, and 125, respectively; and / or(v) the VHH does not inhibit the interaction between PD-1 and PD-L1.

2. A polypeptide comprising a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds to PD-1 (human or human and cyno), wherein the VHHbinds to the following residues of human PD-1: 38-45, 48, 49, 51, 53, 98, 107, 109, and 143 (numbering according to SEQ ID NO:465), optionally wherein the VHH does not inhibit the interaction of PD-1 with PD-L13. The polypeptide of claim 1 or 2, further comprising a human Ig Fc domain, optionally further comprising a human Ig hinge domain, and further optionally comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the human IgGl hinge and Fc domain sequence set forth in SEQ ID NO: 468.Attorney Docket No.: 45817-0163WO1 4. The polypeptide of any one of claims 1 to 3, wherein the VHH in bivalent format is a PD-1 agonist.

5. The polypeptide of any one of claims 1 to 4, wherein the VHH in bivalent format binds to human PD-1 with an EC50 of about 5 nM to about 60 nM, optionally wherein the VHH in bivalent format binds to human PD-1 with an EC50 of about 40 nM to about 55 nM.

6. The polypeptide of any one of claims 1 to 5, wherein the polypeptide comprises at least two VHHs that specifically bind to PD-1.

7. The polypeptide of any one of claims 1 to 6, further comprising a second VHH that specifically binds to a T cell surface antigen, NK cell antigen, or a tumor associated antigen.

8. The polypeptide of any one of claims 1 to 7, wherein the VHH comprises:(a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 109, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 110, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 108;(b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 113, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112;(c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 116, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 117, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112;(d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:54, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 119, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:515;Attorney Docket No.: 45817-0163WO1 (e) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:36, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112;(f) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 106, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 107, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 108;(g) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 111, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112;(h) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 114, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 115, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112;(i) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 54, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 118, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:515;(j) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 120, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 121, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112;(k) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:6, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:8;(l) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:9, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11;Attorney Docket No.: 45817-0163WO1 (m) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14;(n) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:N, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; or(o) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19.

9. The polypeptide of any one of claims 1 to 8, wherein the VHH comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one amino acid sequence set forth in any one of SEQ ID NOs.: 1 to 5 or 65 to 104; optionally wherein the VHH comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 103, 90, 71, 74, or 2.

10. A multispecific antibody comprising (i) the polypeptide of any one of claims 1 to 9; and (ii) a binding molecule that binds to a different epitope(s) of human PD-1 than the polypeptide or binds to a different antigen(s), optionally wherein the different antigen(s) is / are an antigen on a T cell, an NK cell, or a tumor cell, further optionally wherein the different antigen is VEGF, CTLA-4, PD-L1 or TIM-3.

11. A nucleic acid or nucleic acids encoding the polypeptide of any one of claims 1 to 9 or the multispecific antibody of claim 10.

12. A vector or vectors comprising the nucleic acid or nucleic acids of claim 11.

13. A host cell comprising the vector or vectors of claim 12.Attorney Docket No.: 45817-0163WO1 14. A method of making a polypeptide or a multispecific antibody, the method comprising culturing the host cell of claim 13 under conditions that facilitate expression of the polypeptide or the bispecific antibody, and isolating the polypeptide or the multispecific antibody, optionally further comprising formulating the polypeptide or the multispecific antibody as a sterile pharmaceutical composition.

15. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 9, or the multispecific antibody of claim 10, and a pharmaceutically acceptable carrier.

16. A method of treating an inflammatory disorder, a T cell driven autoimmune disease, rheumatoid arthritis, psoriasis, ulcerative colitis, Type 1 diabetes, or multiple sclerosis, in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of the polypeptide of any one of claims 1 to 9, or the multispecific antibody of claim 10, further optionally wherein administration is intravenous, intramuscular, or subcutaneous; yet further optionally wherein the VHH comprises VHH CDR1, VHH CDR2, and VHH CDR3 of the amino acid sequence set forth in SEQ ID NO.: 103, 90, 71, 74, or 2.

17. A messenger RNA (mRNA) comprising an open reading frame (ORF) encoding the polypeptide of any one of claims 1 to 9, or the multispecific antibody of claim 10.

18. A polynucleotide comprising an mRNA comprising:(i) a 5' UTR;(ii) an open reading frame (ORF) comprising a nucleic acid encoding a polypeptide of any one of claims 1 to 8, or the multispecific antibody of claim 9;(iii) a stop codon; and(iv) a 3' UTR, optionally wherein the polynucleotide comprises a nucleic acid sequence encoding a signal sequence positioned between the 3’ end of the 5’ UTR and the 5’ end of the ORF.Attorney Docket No.: 45817-0163WO1 19. The polynucleotide of claim 18, wherein the mRNA comprises a microRNA (miR) binding site.

20. The polynucleotide of any one of claim 18 or 19, wherein the 5' UTR comprises a nucleic acid sequence at least 90%, 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: 479-482 or 486-501.

21. The polynucleotide of any one of claims 18 to 20, wherein the 3' UTR comprises a nucleic acid sequence at least 90%, 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: 502-514.

22. The polynucleotide of any one of claims 18 to 21, wherein the mRNA comprises a 5' terminal cap, optionally wherein the terminal cap is or comprises m7GpppGm.

23. The polynucleotide of any one of claims 17 to 21, wherein the mRNA comprises a poly A region, optionally wherein the poly A region is about 100 nt in length.

24. The polynucleotide of any one of claims 18 to 23, wherein the mRNA comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof, optionally wherein the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil (yr), N1 -methylpseudouracil (mly / ), 1-ethylpseudouracil, 2-thiouracil (s2U), 4’-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof.

25. The polynucleotide of claim 24, wherein at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uracils are N1 -methylpseudouracils, optionally wherein 100% of the uracils areN1-methylpseudouracils.

26. The polynucleotide of any one of claims 18 to 25, wherein the open reading frame consists of nucleosides selected from the group consisting of (i) uridine or a modified uridine, (ii) cytidine or a modified cytidine, (iii) adenosine or a modified adenosine, andAttorney Docket No.: 45817-0163WO1 (iv) guanosine or a modified guanosine, optionally wherein the modified uridine is 1-m ethyl p seudouri dine.

27. The polynucleotide of any one of claims 18 to 26, wherein the mRNA comprises a 5 ’terminal cap comprising m7GpppGm and a poly A region 100 nucleotides in length, optionally wherein all uracils of the polynucleotide are N1 -methylpseudouracils.

28. A pharmaceutical composition comprising the polynucleotide of any one of claims 18 to 27, formulated with a delivery agent, optionally wherein the delivery agent comprises a lipid nanoparticle, further optionally wherein the lipid nanoparticle comprises a neutral lipid, an ionizable amino lipid, a polyethyleneglycol (PEG) lipid, and / or a sterol, and also further optionally wherein the neutral lipid is l,2-distearoyl-sn-glycero-3-phosphocholine; wherein the ionizable amino lipid is a compound of Formula (I), optional Compound 1-1; wherein the PEG lipid is PEG 2000 dimyristoyl glycerol or 134-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90, 93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate, and wherein the sterol is cholesterol, optionally wherein the ionizable amino lipid is present at about 47.5 mole ratio%, the structural lipid is present at about 39 mole ratio%, the phospholipid is present at about 10.5 mole ratio%, and the PEG-modified lipid is present at about 3 mole ratio%.

29. A lipid nanoparticle that is formulated with the polynucleotide of any one of claims 18 to 27, wherein the lipid nanoparticle comprises a neutral lipid, an ionizable amino lipid, a polyethyleneglycol (PEG) lipid, and / or a sterol, and further optionally wherein the neutral lipid is l,2-distearoyl-sn-glycero-3-phosphocholine; wherein the ionizable amino lipid is a compound of Formula (I), optional Compound 1-1; wherein the PEG lipid is PEG 2000 dimyristoyl glycerol or 134-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90, 93,96,99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132-tetratetracontaoxatetratriacontahectyl stearate, and wherein the sterol is cholesterol,Attorney Docket No.: 45817-0163WO1 optionally wherein the ionizable amino lipid is present at about 47.5 mole ratio%, the structural lipid is present at about 39 mole ratio%, the phospholipid is present at about 10.5 mole ratio%, and the PEG-modified lipid is present at about 3 mole ratio%.

30. A method of treating an inflammatory disorder, a T cell driven autoimmune disease, rheumatoid arthritis, psoriasis, ulcerative colitis, Type I diabetes, or multiple sclerosis, in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of the pharmaceutical composition of claim 28, or the LNP of claim 29; optionally wherein administration is intravenous, intramuscular, or subcutaneous; further optionally wherein the VHH comprises VHH CDR1, VHH CDR2, and VHH CDR3 of the amino acid sequences set forth in SEQ ID NO.: 103, 90, 71, 74, or 2.

31. A kit comprising (i) the polypeptide of any one of claims 1 to 9; the multispecific antibody of any one of claim 10; the pharmaceutical composition of claim 15 or 28; the polynucleotide of any one of claims 17 to 27, or the LNP of claim 29 and (ii) a package insert instructing a user of the kit to administer the polypeptide, multispecific antibody, pharmaceutical composition, or polynucleotide to a human subject in need thereof.

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