LAG3 binding molecules, nucleic acids encoding same, and methods of use

Abstract

Described herein are LAG3 specific single domain antibodies and binding molecules comprising such single domain antibodies, as well as nucleic acids encoding the same. Also provided are nucleic acids (e.g., mRNA) encoding such single domain antibodies and binding molecules and delivery vehicles (e.g., lipid nanoparticles) formulated with said nucleic acids. The disclosure also features methods of using such antibodies, binding molecules, nucleic acids, and delivery vehicles formulated with said nucleic acids to treat various diseases such as inflammatory diseases, T cell driven autoimmune diseases, and cancer (e.g., solid tumors or hematological malignancies).

Description

[0001] Attorney Docket No.: 45817-0164WO1 LAG3 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 / 742,586 filed January 7, 2025, the content of which is incorporated by reference in its 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 45817-0164WOl_SL.xml and is 398,591 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] Lymphocyte activation gene 3 (LAG3) is an immune checkpoint receptor protein belonging to the immunoglobulin superfamily, which is expressed on activated immune cells, mainly activated T cells. LAG3 negatively regulates the function of T cells thereby maintaining the homeostasis of the immune system under normal physiological conditions and promoting tumor cells immune escape in the tumor microenvironment. In view of its important, biological roles, LAG3 has been regarded as a promising target for immunotherapy.

[0008] Accordingly, as LAG3 is a promising immunotherapeutic target there is a need for new compositions that can bind to and agonize or antagonize this receptor.

[0009] SUMMARYAttorney Docket No.: 45817-0164WO1 The present disclosure provides, among other things, binding molecules that specifically bind to LAG3 (human or human and cyno), nucleic acids (e.g., mRNA) encoding same, delivery vehicles that are formulated to contain such nucleic acids, and methods of using these agents in treating diseases such as inflammatory diseases.

[0010] Accordingly, featured herein is a binding molecule comprising a polypeptide that specifically binds to LAG3 (human or human and cyno), i.e., the polypeptide binds at essentially background levels to other proteins (i.e., proteins other than LAG3). In some cases, the LAG3 binding polypeptide is an agonist of human LAG3. In other instances, the LAG3 binding polypeptide is an antagonist of human LAG3. The LAG3 binding polypeptide can comprise a 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 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 LAG3 expression or overexpression (e.g., LAG3 agonists can be used to treat inflammatory diseases and T cell driven autoimmune diseases, whereas LAG3 antagonists can be used to treat cancer (e.g., solid tumors, lymphomas)).

[0011] 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 LAG3 (human or human and cyno), the VHH comprising VHH complementarity determining region (CDR) 1, VHH CDR2, and VHH CDR3 of any one of the VHH amino acid sequences set forth in SEQ ID NOs.: 281, 264, 257, 21, 1 to 20, 237-256, 258-263, or 265-280.

[0012] In one aspect, the disclosure relates to a polypeptide comprising a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds to human LAG3, the VHH comprising VHH complementarity determining region (CDR) 1, VHH CDR2, and VHH CDR3, wherein the VHH binds to amino acids 174, 182, 184-Attorney Docket No.: 45817-0164WO1 186, 188 (with its associated glycan), 196, 220-223, and 225-229 of human LAG3 (numbering according to SEQ ID NO: 465).

[0013] In another aspect, the disclosure features a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds to LAG3 (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.: 281, 264, 257, 21, 1 to 20, 237-256, 258-263, or 265-280.

[0014] In yet another aspect, the disclosure provides a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds to human LAG3, the VHH comprising VHH complementarity determining region (CDR) 1, VHH CDR2, and VHH CDR3, wherein the VHH binds to amino acids 174, 182, 184-186, 188 (with its associated glycan), 196, 220-223, and 225-229 of human LAG3 (numbering according to SEQ ID NO: 465).

[0015] In another aspect, the disclosure relates to a. polypeptide comprising a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds to human LAG3, the VHH comprising the three VHH CDRs set forth in SEQ ID NOs: 71, 295, and 303, respectively, except that the VHH-CDR1, VHH-CDR2 and VHH-CDR3 are substituted at one or more (e.g., 1, 2, 3, 4, 5, 6) of the positions that are not shown in bold: AFTLDYYA (SEQ ID NO: 71); FSSAGYT (SEQ ID NO: 295), and ALGIHGPFLDQRIYDY (SEQ ID NO: 303). In some cases, 1, 2, or 3 amino acids in one or more (1, 2, or 3) of VHH-CDR1, VHH-CDR2, and VHH-CDR3 are substituted. In some cases, the substitutions are conservative amino acid substitutions. In some cases, the VHH comprises aVHH-CDRl comprising the sequence:

[0016] AF(T / P)(L / F)D(Y / W)(A / V); a VHH-CDR2 comprising the sequence:

[0017] (L / F / W / Y)SS(A / P)GYT (SEQ ID NO: 284); and a VHH-CDR3 comprising the sequence: A(I / L)(G / S)IHGPFLD(Q / 1 / K)R(I / L)YDY (SEQ ID NO: 285), wherein the residues shown in bold are not substituted. In some instances, the VHH binds to an epitope within the D2 domain of LAG3.Attorney Docket No.: 45817-0164WO1 In some instances of the above aspects, the VHH CDR1, VHH CDR2, and VHH CDR3 are based on CDR definitions according to any one of the Kabat, Chothia, enhanced Chothia, contact, IMGT, or AbM CDR definitions. In certain cases, the VHH of this disclosure comprises the three VHH-CDRs of any one VHH set forth in any one of Tables 3, 3A, 4, 4A, 5, 5A, 6, 6A, 7, or 7A. In some cases, the VHH of this disclosure comprises the three VHH-CDRs of the VHH L3G4_hu_v.7 or L3G4_hu_v.25. In some instances, the VHH-CDR1 comprises the sequence AF(T / P)(L / F)D(Y / W)(A / V); the VHH-CDR2 comprises the sequence set forth in SEQ ID NO: 284; and VHH-CDR3 comprises the sequence set forth in SEQ ID NO: 285. In some instances, the VHH binds to an epitope within the D2 domain of LAG3.

[0018] In some instances, the polypeptide or VHH binds to human LAG3. In other instances, the polypeptide or VHH binds to both human and cynomolgus LAG3. In some cases, the polypeptide or VHH bind to an epitope within the D2 domain of human LAG3 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 LAG3 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 LAG3 of about 0.1 nM to about 25 nM (e.g., about 0.3 nM to about 0.8 nM).

[0019] 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 21 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 21 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 IMGTAttorney Docket No.: 45817-0164WO1 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 50 (numbering based on IMGT numbering) are not humanized. In certain cases, positions 49 and / or 50 (numbering based on IMGT numbering) are humanized. In some cases, the humanized VHHs comprise an amino acid sequence that is 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.: 237-257 (e.g., SEQ ID NO: 257). In some cases, the humanized VHHs comprise an amino acid sequence that has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions relative to the sequence set forth in any one of SEQ ID NOs.: 237-257 (e.g., SEQ ID NO: 257). These humanized VHHs bind to human LAG3 (e.g., within the D2 domain).

[0020] In some instances, the VHH is humanized and affinity matured. In some cases, 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 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.: 237-257 are substituted in the process of affinity maturation. In certain cases, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) of the VHHs set forth in SEQ ID NOs.: 237-257 are substituted in the process of humanization. In some cases, the humanized and affinity matured VHHs comprise an amino acid sequence that is 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.: 258-281 (e.g., SEQ ID NO: 264 or 281). In some cases, the humanized VHHs comprise an amino acid sequence that has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions relative to the sequence set forth in any one of SEQ ID NOs.: 258-281 (e.g., SEQ ID NO: 264 or 281). Generally, when substitutions are made in one or more of the three VHH CDRs, they are not made at the paratope residues identified in FIG. 17. These humanized and affinity matured VHHs bind to human LAG3 (e.g., within the D2 domain).Attorney Docket No.: 45817-0164WO1 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.

[0021] 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 another instance, the human Ig is human IgG4. In certain 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 IgG4 PAA hinge and Fc domain sequence set forth in SEQ ID NO: 469. In certain cases, the polypeptide or VHH is conjugated, fused, or linked to an amino acid sequence that is identical to the human IgG4 PAA hinge and Fc domain sequence set forth in SEQ ID NO:469 except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some cases, the Fc domain includes mutations that reduce or abrogate FcyR binding. In some cases, the Fc domain includes mutations that promote heterodimerization (e.g., knob-into hole mutations, electrostatic steering mutations).Attorney Docket No.: 45817-0164WO1 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 LAG3 (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 21, 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 LAG3 (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 21, 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 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, or to an amino acid sequence that is identical to the human IgG4 PAA hinge and Fc domain sequence set forth in SEQ ID NO:468 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some 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 IgG4 PAA hinge and Fc domain sequence set forth in SEQ ID NO:469, or to an amino acid sequence that is identical to the human IgG4 PAA hinge and Fc domain sequence set forth in SEQ ID NO:469 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In certain instances, the polypeptide specifically binds to human LAG3. In some cases, theAttorney Docket No.: 45817-0164WO1 polypeptide binds to both human and cynomolgus LAG3. In certain cases, the polypeptide binds to an epitope within the D1-D2 domain or the D3-D4 domain of human LAG3.

[0022] In another aspect, provided herein is a bispecific binder comprising a polypeptide or VHH that specifically binds to LAG3 (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 a NK 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 certain instances, the polypeptide specifically binds to human LAG3. In some cases, the polypeptide binds to both human and cynomolgus LAG3. In certain cases, the polypeptide binds to an epitope within the D1-D2 domain or the D3-D4 domain of human LAG3.

[0023] In some instances, the VHH specifically binds to LAG3 (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, or 7. In some instances, the VHH binds to human LAG3. In some instances, the VHH binds to human and cynomolgus LAG3. In certain cases, the polypeptide binds to an epitope within the D1-D2 domain or the D3-D4 domain of human LAG3.

[0024] In certain instances, the VHH specifically binds to LAG3 (human or human and cyno) and comprises:

[0025] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:22, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:23, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:24;Attorney Docket No.: 45817-0164WO1 (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25, 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;

[0026] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25, 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;

[0027] (d) 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;

[0028] (e) 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:35;

[0029] (f) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:36, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:23, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:37;

[0030] (g) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, 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:39;

[0031] (h) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:40, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:41;

[0032] (i) 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:44;Attorney Docket No.: 45817-0164WO1 (j) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:45, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:46, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:47;

[0033] (k) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:48, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:49, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50;

[0034] (l) 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;

[0035] (m) 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;

[0036] (n) 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;

[0037] (o) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 60, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:61, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:62;

[0038] (p) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:45, 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:47;

[0039] (q) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:64, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:65, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:66;Attorney Docket No.: 45817-0164WO1 (r) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:67, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:68, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:69;

[0040] (s) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:64, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:68, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:70;

[0041] (t) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:71, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 72, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:73; or

[0042] (u) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:71, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:72, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:74.

[0043] The VHHs bind human LAG3. In some instances, the VHHs when linked to an Ig hinge-Fc domain (e.g., from IgGl) act as LAG3 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 0.1 nM to about 25 nM. In some cases, the VHHs bind to an epitope within the D2 domain of human LAG3.

[0044] In some instances, the VHH specifically binds to LAG3 (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 1A, Table IB, or Table 1C. In certain cases, the VHH comprises a sequence that is set forth in any one of SEQ ID NOs.: 1 to 21. In certain cases, the VHH is a LAG3 agonist and comprises a sequence that is set forth in any one of SEQ ID NOs.: 1, 4, 6, 10, 11, 14, 15, 16, 17, 20, or 21. In certain cases, the VHH comprises a sequence that is set forth in any one of SEQ ID NOs.: 10, 11, 16, 17, or 20. In one case, the VHH comprises a sequence that is set forth in SEQ ID NO.: 17. In certain cases, the VHH is a LAG3 agonist andAttorney Docket No.: 45817-0164WO1 comprises a sequence that is set forth in any one of SEQ ID NOs.: 258-281. In one case, the VHH comprises a sequence that is set forth in SEQ ID NO.:264 or 281. The VHHs bind to human LAG3. In some instances, the VHHs when linked to an Ig hinge-Fc domain (e.g., from IgGl) act as LAG3 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 0.1 nM to about 25 nM. In some cases, the VHHs binds to an epitope within the D1-D2 domain of human LAG3. In other cases, the VHHs binds to an epitope within the D2 domain of human LAG3.

[0045] In some instances, the VHH specifically binds to LAG3 (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 21 (e g., SEQ ID NOs: 1, 4, 6, 10, 11, 14, 15, 16, 17, 20, or 21), 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 LAG3. In other instances, the VHH specifically binds to both human LAG3 and cynomolgus LAG3.

[0046] In some instances, the VHH binds to LAG3 expressed on a c cell with an EC50 of about 0.1 nM to about 25 nM. In other instances, the VHH binds to LAG3 expressed on a cell with an EC50 of about 0.1 nM to about 20 nM. In certain instances, the VHH binds to LAG3 expressed on a cell with an EC 50 of about 0.1 nM to about 15 nM. In some instances, the VHH binds to LAG3 expressed on a cell with an EC50 of about 0.1 nM to about 10 nM. In other instances, the VHH binds to LAG3 expressed on a cell with an EC 50 of about 0.1 nM to about 5 nM. In yet other instances, the VHH binds to LAG3 expressed on a cell with an EC50 of about 0.1 nM to about 4 nM. In yet other instances, the VHH binds to LAG3 expressed on a cell with an EC50 of about 0.1 nM to about 3 nM. In other instances, the VHH binds to LAG3 expressed on a cell with an EC50 of about 0.1 nM to about 2 nM. In other instances, the VHH binds to LAG3 expressed on a cell with an EC50 of about 0.1 nM to about 1 nM. In some other instances, the VHH binds to LAG3 expressed on a cell with an EC50 of about 0.2 nM to about 1 nM. In some otherAttorney Docket No.: 45817-0164WO1 instances, the VHH binds to LAG3 expressed on a cell with an EC50of about 0.2 nM to about 0.8 nM. Tn 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 or 469, except for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 substitutions). In certain cases, the LAG3 is human LAG3.

[0047] In another aspect, the disclosure features a bispecific antibody comprising the polypeptide or VHH described above that specifically binds to LAG3; and a binding molecule that binds to a different epitope of LAG3 than the polypeptide or VHH described above, or binds to a different antigen (e.g., as T cell antigen, a NK cell antigen, a tumor associated antigen).

[0048] In a different aspect, the disclosure features a multispecific binding molecule comprising the polypeptide or VHH described above that specifically binds to LAG3; and a binding molecule that binds to a different epitope(s) of LAG3 than the polypeptide or VHH described above, or binds to a different antigen(s) (e.g., as T cell antigen, aNK cell antigen, a tumor associated antigen).

[0049] 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 LAG3 (human or human and cyno LAG3). The binding polypeptide also comprises a polypeptide that binds to a different epitope or antigen (e.g., a T cell antigen, aNK 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 LAG3 (human or human and cyno LAG3) is linked to the polypeptide via a peptide linker (e.g., glycine linker, serine linker, glycineserine 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).

[0050] In some instances, the binding molecule is a VHH. In other instances, the binding molecule is a scFv.Attorney Docket No.: 45817-0164WO1 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 LAG3 (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 other cases, the human Ig hinge domain and 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:469. In some cases, the human Ig Fc domain include one or more mutations that alter (e g., increase or decrease) 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).

[0051] 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.

[0052] In another aspect, provided are a vector or vectors comprising the nucleic acid or nucleic acids described above.

[0053] 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 bispecific antibody. In certain cases, the method further comprises formulating the polypeptide or the bispecific antibody as a sterile pharmaceutical composition.Attorney Docket No.: 45817-0164WO1 In another aspect, provided herein is a pharmaceutical composition comprising the polypeptide or the bispecific antibody described herein, and a pharmaceutically acceptable carrier.

[0054] In yet another aspect, the disclosure relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a means for binding LAG3 (human or human and cyno LAG3).

[0055] In yet another aspect, the disclosure relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a means for agonizing human LAG3.

[0056] 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, 4, 6, 10, 11, 14, 15, 16, 17, 20, 21, or 237-281), 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 some instances, administration is performed intramuscularly. In other instances, administration is performed subcutaneously.

[0057] In a different aspect, the disclosure features a method of treating a cancer (e.g., a solid tumor, a hematological cancer such as a lymphoma) 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 LAG3 antagonist VHH), 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 melanoma, ovarian cancer, breast cancer, or colorectal cancer. In some instances, administration is performedAttorney Docket No.: 45817-0164WO1 intravenously. In some instances, administration is performed intramuscularly. In other instances, administration is performed subcutaneously.

[0058] 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.

[0059] 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, a polypeptide, a bispecific antibody, or multispecific binding molecule described herein; (iii) a stop codon; and (iv) a 3' UTR.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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-Attorney Docket No.: 45817-0164WO1 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.

[0064] 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 90 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.

[0065] 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 (\| / ), N1 -methylpseudouracil (ml\| / ), 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

[0066] 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.

[0067] 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.

[0068] In some instances, the mRNA comprises a 5’terminal cap comprising m7GpppGm and a poly A region 100 nucleotides in length.

[0069] In some instances, all uracils of the polynucleotide are N1 -methylpseudouracils.Attorney Docket No.: 45817-0164WO1 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 some cases, the polynucleotide comprises a signal sequence. The signal sequence is positioned after the 5’UTR.

[0070] 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., LNP).

[0071] In another aspect, the disclosure features a pharmaceutical composition comprising a polynucleotide described herein, wherein the polynucleotide is formulated in a delivery agent.

[0072] In some cases, the delivery agent comprises 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.

[0073] In some cases, the lipid nanoparticle comprises an ionizable amino lipid, a structural lipid, a phospholipid, and a polyethyleneglycol (PEG)-modified lipid.

[0074] In certain cases, the phospholipid is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0075] In some cases, the ionizable amino lipid is a compound of Formula (I). In one case, the ionizable amino lipid is Compound 1-1.

[0076] In certain cases, the PEG-modified lipid is PEG 2000 dimyristoyl glycerol (PEG-DMG) or

[0077] 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,Attorney Docket No.: 45817-0164WO1 93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectyl stearate.

[0078] 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-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.

[0079] In some instances, the lipid nanoparticle 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%.

[0080] 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 aminoAttorney Docket No.: 45817-0164WO1 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%.

[0081] 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%.

[0082] 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%.

[0083] In one instance, the lipid nanoparticle comprises an ionizable amino lipid that is Compound 1-1, a phospholipid that is l,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%.

[0084] 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 inAttorney Docket No.: 45817-0164WO1 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, 4, 6, 10, 11, 14, 15, 16, 17, 20, 21, or 237-281), a polypeptide, a bispecific antibody, or a multispecific binding molecule described herein, wherein the mRNA is formulated in a delivery vehicle (e.g., LNP). In some instances, administration is performed intravenously. In some instances, administration is performed intramuscularly. In other instances, administration is performed subcutaneously.

[0085] In another aspect, the disclosure features a method of treating a cancer (e.g., a solid tumor, a hematological cancer such as a lymphoma) in a human subject in need thereof. The method comprises administering to the human subject a therapeutically effective amount of a nucleic acid (e.g., mRNA) that encodes a polypeptide (e.g., a polypeptide comprising a VHH comprising the three VHH CDRs of any LAG3 antagonist VHH), a bispecific antibody, a multispecific antibody, or a pharmaceutical composition described herein, wherein the nucleic acid (e.g., mRNA) is formulated in a delivery vehicle (e.g., LNP). In one instance, the human subject in need thereof has or is at risk of developing melanoma, ovarian cancer, breast cancer, or colorectal cancer. In some instances, administration is performed intravenously. In some instances, administration is performed intramuscularly. In other instances, administration is performed subcutaneously.

[0086] 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,Attorney Docket No.: 45817-0164WO1 the human subject in need thereof has an inflammatory disease or disorder pr an autoimmune disease. In other cases, the human subject in need thereof has a cancer.

[0087] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 provides graphical depictions of the binding of LAG3 VHHs that were conjugated to the hinge and Fc domains of human IgGl to LAG3+ Jurkat cells.

[0088] FIG. 2 provides data showing that anti-LAG3 VHH can suppress CD4 (left panel) and CD8 (central and right panels) proliferation in a Staphylococcal enterotoxin B (SEB) stimulation assay.

[0089] FIG. 3 are graphs that show that LAG3 agonists are reliant on FcyR binding to exert suppressive effects.

[0090] FIG. 4A is a graph showing that treatment of transgenic mice (human PD-1 and LAG3 knock in) with L3F4 VHH linked to murine IgGl hinge + Fc domain confers full protection from disease in the collagen induced arthritis model.

[0091] FIG. 4B are graphs that treatment of transgenic mice (human PD-1 and LAG3 knock in) with L3G4v2 demonstrate marked reduction in synovial inflammation and periosteal bone formation.

[0092] FIG. 5 is a table summarizing the properties of some of the anti -LAG3 -VHHs of this disclosure. Figure discloses SEQ ID NOS 50, 47, 47, 73-74, 59, 66, 27, 37, 24, 62, 32, 29, 41, 53, 35 and 69-70, respectively, in order of appearance.

[0093] FIG. 6 shows cell binding of indicated humanization variants of the L3F4 antibody on a Jurkat cell line engineered to express human LAG3.

[0094] FIG. 7 shows cell binding of indicated humanization variants of the L3G4v2 antibody on a Jurkat cell line engineered to express human LAG3.

[0095] FIG. 8 shows inhibition of CD8 T cell proliferation by humanization variants of the L3F4 antibody.Attorney Docket No.: 45817-0164WO1 FIG. 9 shows inhibition of CD8 T cell proliferation by humanization variants of the L3G4v2 antibody and indicated controls.

[0096] FIG.10A shows binding affinity and binding sensogram as determined for the monovalent VHH of L3G4v2_A using surface plasmon resonance.

[0097] FIG. 10B shows the melting temperature of L3G4v2 and its humanization variants determined using nano-DSF.

[0098] FIG. 11 is a graph showing monovalent binding affinity measurements of L3G4v2_h7_A and progeny from affinity maturation process against the human and cyno ectodomains of LAG3.

[0099] FIG. 12A is a graph showing melting temperature of select affinity maturation progeny of L3G4v2_A.

[0100] FIG. 12B is a graph showing polyreactivity of select affinity maturation progeny ofL3G4v2_A.

[0101] FIG. 13 is a graph showing inhibition of CD8+T cell proliferation in the SEB stimulation assay with human PBMCs and the respective IC50 values for each of the indicated antibodies.

[0102] FIG. 14 is a table that summarizes data on the most improved affinity matured antibodies.

[0103] FIG. 15A shows the overall complex, with the human LAG3 D1-D2 ectodomain displayed in surface representation and the anti-LAG3 VHH as grey ribbons, binding within Domain 2 of human LAG3.

[0104] FIG. 15B shows an open-book view of the epitope-paratope interface. Epitope residues are labeled on LAG3, and paratope residues are labeled on the VHH21 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 LAG3 that form the epitope are residues for the anti-LAG3 VHH21 areAttorney Docket No.: 45817-0164WO1 amino acids 174, 182, 184-186, 188 (and associated glycan), 196, 220-223, and 225-229 of human LAG3 as set forth in SEQ ID NO:465.

[0105] FIG. 16 shows the amino acid sequence of the LAG3 ectodomain used for structural studies of the antibody: target complex, with epitope residues shown in enlarged font with grey background shading. Figure discloses SEQ ID NO: 282. The sequence includes a LAG3 D1 / D2 construct containing the M171I mutation.

[0106] FIG. 17 shows the amino acid sequence of VHH21 with IMGT-defined CDRs indicated in enlarged font and grey background shading. Contact residues defining the paratope are underlined and bolded. Figure discloses SEQ ID NO: 21.

[0107] FIG. 18A shows the effect of L3G4v2 VHH-Fc twice-weekly treatment with human IgGl Fc on human CD8+ T cell expansion in NSG mice over time.

[0108] FIG. 18B shows the effect of L3G4v2 antibody treatment on production of intracellular Granzyme B by CD8+ T cells on day 28 post human T cell transfer.

[0109] FIG. 18C shows a representative flow plot from day 28 post human T cell transfer gated on CD8 and LAG3+ CD8 T cells.

[0110] DETAILED DESCRIPTION

[0111] The compositions and methods of the disclosure feature LAG3 specific single domain antibodies (anti-LAG3 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 LAG3. The VHHs bind human LAG3 or human and cynomolgus LAG3. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as LAG3 agonists. In certain instances, the VHHs have an EC50 of about 0.1 nM to about 25 nM. In some cases, the VHHs bind to an epitope within the D1-D2 domain of LAG3. In other cases, the VHHs bind an epitope within the D3-D4 domain of human LAG3. In one case, the VHHs bind to an epitope within the D2 domain of LAG3. The disclosure also provides nucleic acids (e.g., mRNA) encoding the disclosed proteins, and such nucleic acids formulated in delivery vehicles (e.g., lipidAttorney Docket No.: 45817-0164WO1 nanoparticles). Also featured are methods of using such VHHs, binding proteins, nucleic acids, and such nucleic acids formulated in delivery vehicles in the treatment of inflammatory diseases, autoimmune diseases (e.g., T cell driven autoimmune diseases) and cancer (e.g., solid tumors such as ovarian cancer and hematological cancers such as lymphomas).

[0112] Definitions

[0113] “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 or an antigen-binding fragment. VHHs are also referred to as single domain antibodies and nanobodies.

[0114] “Monovalent” means a binding molecule with a single antigen binding domain (e g., a single VHH).

[0115] “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).

[0116] “Multivalent” means a binding molecule with more than two antigen binding domains.

[0117] “% 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 inAttorney Docket No.: 45817-0164WO1 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.

[0118] “about” means + or - 10% of the recited value, so “about 250 nM” means 225 nM to 275 nM.

[0119] LAG3

[0120] LAG3 is a type I transmembrane glycoprotein comprising four extracellular immunoglobulin (Ig)-like domains (referred to as DI, D2, D3, and D4), a connecting peptide, and an intracellular region that transmits inhibitory signals to the T cell upon binding to MHC class II and other ligands. LAG3 has a similar domain architecture as the T cell co-receptor CD4 (~25% identical at the amino acid level) and binds to MHC class II. In addition to MHC class II, other proposed LAG3 -binding partners include galnectin-3 (Gal-3), liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin), a-synuclein, fibrinogen-like protein 1 (FGL1), and the TCR-CD3 complex.

[0121] The DI domain of LAG3 is a V-type domain whereas the D2, D3, and D4 domains are C2-type domains. DI and D2 fold as discrete domains, while D3 and D4 form a contiguous unit linked by an extended P-strand. LAG3 forms a V-shaped dimer via D2-D2 interactions. Dimerization of hLAG3 is mediated by a cluster of hydrophobic residues: Trpl84, Ilel 86, Phe225, andPhe227. LAG3 dimerization is critical forLAG3 function.Attorney Docket No.: 45817-0164WO1 LAG3, is an immune checkpoint receptor that downregulates T cell activation, proliferation, and cytokine production, rendering T cells dysfunctional. Thus, agonizing LAG3 has utility in treating autoimmune and inflammatory disorders. Conversely, antagonizing LAG3 has benefits in treating cancers.

[0122] The amino acid sequence of human LAG3 (Uniprot: Pl 8627) is provided below:

[0123]

[0124] LLRRAGVTWQHQPD SGPP AAAPGHPL APGPHP AAP S S WGPRPRRYTVL S VGPGG LRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCR LRLRLGQ ASMTASPPGSLRASDWVILNC SF SRPDRP AS VHWFRNRGQGRVP VRE SPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTV YAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVS QAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERF VWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPG AQRSGRAPGALPAGHLLLFLILGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQ GIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL (SEQ ID NO:465)

[0125] It is noted that an M1711 (numbering according to SEQ ID NO:465) mutation can be introduced in the above sequence to improve expression and crystallizability.

[0126] The amino acid sequence of cynomolgus LAG3 (NCBI Reference Sequence: XP_045220576.2) is provided below:

[0127] MDSPSTPKASLGREGTETQALPAPCFSLGKHPGSFSTPASRLTPPPLGLSFAHLVK PPQP GAEISVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAPAPGHPP APGHRPAAPYSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSL WLRPARRADAGEYRATVHLRDRALSCRLRLRVGQASSMWGGTTGEGLGVTAS PPGSLRTSDWVILNCSFSRPDRPASVHWFRSRGQGRVPVQGSPHHHLAESFLFLP HVGPMDSGLWGCILTYRDGFNVSIMYNLTVLGLEPATPLTVYAGAGSRVELPCR LPPAVGTQSFLTAKWAPPGGGPDLLVAGDNGDFTLRLEDVSQAQAGTYICHIRLAttorney Docket No.: 45817-0164WO1 QGQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPASGQEHFVWSPLNTPSQRSF SGPWLEAQEAQLLSQPWQCQLHQGETLLGAAVYFTELSSPGAQRSGRAPGALR AGHLPLFLILGVLFLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEEL EQEPELEPEPELERELGPEPEPEQL (SEQ ID NO:466)

[0128] The amino acid sequence of murine LAG3 (Uniprot: Q61790) is provided below: MREDLLLGFLLLGLLWEAPVVS SGPGKELPVVWAQEGAPVHLPC SLKSPNLDPN FLRRGGVIWQHQPDSGQPTPIPALDLHQGMPSPRQPAPGRYTVLSVAPGGLRSGR QPLHPHVQLEERGLQRGDFSLWLRPALRTDAGEYHATVRLPNRALSCSLRLRVG QASMIASPSGVLKLSDWVLLNCSFSRPDRPVSVHWFQGQNRVPVYNSPRHFLAE TFLLLPQVSPLDSGTWGCVLTYRDGFNVSITYNLKVLGLEPVAPLTVYAAEGSRV ELPCHLPPGVGTPSLLIAKWTPPGGGPELPVAGKSGNFTLHLEAVGLAQAGTYTC SIHLQGQQLNATVTLAVITVTPKSFGLPGSRGKLLCEVTPASGKERFVWRPLNNL SRSCPGPVLE1QEARLLAERWQCQLYEGQRLLGATVYAAESSSGAHSARR1SGDL KGGHLVLVLILGALSLFLLVAGAFGFHWWRKQLLLRRFSALEHGIQPFPAQRKIE ELERELETEMGQEPEPEPEPQLEPEPRQL (SEQ ID NO:467)

[0129] VHHs that Specifically Bind LAG3

[0130] Provided herein are novel VHHs that specifically bind to LAG3 (human or human and cyno); i.e., the VHHs bind to a protein or proteins other than LAG3 at essentially background levels. In some instances, these VHHs bind to human LAG3. In some instances, some of these VHH bind to human and cynomolgus LAG3. Tables 1 A and IB provide the amino acid sequences of several examples of novel VHHs that bind to LAG3.

[0131] This disclosure features a binding molecule or polypeptide that specifically binds to LAG3 (human or human and cyno) and comprises the VHH amino acid sequence of any of the clones listed in Table 1A or IB. In some instances, the binding molecule or polypeptide comprises a VHH that specifically binds to LAG3 (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 least 90%, at least 91%, at least 92%, at least 93%, atAttorney Docket No.: 45817-0164WO1 least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 21. Tn certain instances, the binding molecule or polypeptide comprises a VHH that specifically binds to LAG3 (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 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% or at least 99% identical to an amino acid sequence set forth in any one of SEQ ID NOs.: 1, 10, 11, 16, 17, or 20. In some instances, the binding molecule or polypeptide comprises a VHH that specifically binds to LAG3 (human, or human and cyno) and comprises an amino acid sequence set forth in any one of SEQ ID NO:1 to 21 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 some instances, the binding molecule or polypeptide comprises a VHH that specifically binds to LAG3 (human, or human and cyno) and comprises an amino acid sequence set forth in any one of SEQ ID NOs: 10, 11, 16, 17, or 20 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 LAG3 (human, or human and cyno) and comprises an amino acid sequence set forth in any one of SEQ ID NO:1 to 21 except for having 1, 2, 34, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions. In other instances, the binding molecule or polypeptide comprises a VHH that specifically binds to LAG3 (human, or human and cyno) and comprises an amino acid sequence set forth in any one of SEQ ID NOs: 10, 11, 16, 17, or 20 except for having 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, or 12 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 21 may be due to humanization of the VHH. These humanized VHHs still specifically bind to LAG3. In some instances, the binding molecule or polypeptide specifically binds to LAG3 (human, or human and cyno) and comprises a sequence that is identical to anAttorney Docket No.: 45817-0164WO1 amino acid sequence set forth in any one of SEQ ID NOs.: 10, 11, 16, 17, or 20. In some instances, the above-described binding molecule or polypeptide specifically binds to human LAG3. In some instances, the above-described binding molecule or polypeptide binds to both human LAG3 and cynomolgus LAG3. In some instances, the VHHs act as LAG3 agonists. In certain instances, the VHHs have an EC50 of about 0.1 nM to about 25 nM. In certain instances, the VHHs have an EC50 of about 0.3 nM to about 5 nM.

[0132] In some instances, the binding molecule or polypeptide that binds LAG3 (human, or human and cyno) is in a monovalent format. In some instances, the binding molecule or polypeptide that binds LAG3 (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 IgA. In another 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):

[0133] EPKSADK. THTCPPCPAPEEEGGPSVFLFPPKPKDTLM1SRTPEFTCPWDFSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLWLHQDWLNGKJLYKCKVSNKAL

[0134] / <4 / 7 / AV7S’ VGOPREPOVYTLPPSREEMTI< NOVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO:468).

[0135] In another case, the hinge and Fc domain is from human IgG4. In some cases, the hinge and Fc domain comprises an amino acid sequence that is at least 80%, at least 85%, atAttorney Docket No.: 45817-0164WO1 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 IgG4PAA (SEQ ID NO:469). IgG4PAA is human IgG4 with mutations S228P / F234A / L235A and has reduced binding to FcyR. The amino acid sequence of the hinge and Fc domain of human IgG4PAA 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):

[0136] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC WDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSS

[0137] / AA / AS' AGOPREPOVYTLPPSOEEMTKNOVSLTCLVKGFYPSDIAVEWESNGOP ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWOEGNVFSCSVMHEALHNHYTQKSL SLSLGK (SEQ ID NO:469)

[0138] In some cases, the hinge and Fc domain comprises an amino acid sequence set forth in SEQ ID NO:468 or 469 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, Sidhu SS and Wu D (2017) Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds. Front. Immunol. 8:38 doi: 10.3389 / fimmu.2017.00038; Wilkinson 1, 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. or / 10.1 71 / journal pone.0260954, all of which are incorporated by reference herein.

[0139] 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 LAG3 with an EC50 of about 0.1 nM to about 25 nM. In some other cases, the binding molecule or polypeptide comprising a VHH described herein inAttorney Docket No.: 45817-0164WO1 bivalent format binds a cell expressing human LAG3 with an EC50 of about 0.1 nM to about 20 nM. In certain cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human LAG3 with an EC50 of about 0.1 nM to about 15 nM. In other cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human LAG3 with an EC50 of about 0.1 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 LAG3 with an EC50 of about 0.1 nM to about 5 nM. In other cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human LAG3 with an EC50 of about 0.1 nM to about 4 nM. In some cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human LAG3 with an EC50 of about 0.1 nM to about 3 nM. In other cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human LAG3 with an EC50 of about 0.1 nM to about 2 nM. In some other cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human LAG3 with an EC50 of about 0.3 nM to about 5 nM. In some other cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human LAG3 with an EC50 of about 0.3 nM to about 4 nM. In some other cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human LAG3 with an EC50 of about 0.3 nM to about 3 nM. In yet other cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human LAG3 with an EC50 of about 0.3 nM to about 2 nM. In further cases, the binding molecule or polypeptide comprising a VHH described herein in bivalent format binds a cell expressing human LAG3 with an EC50 of about 0.3 nM to about 1 nM.

[0140] Table 1 A provides the sequences of exemplary VHHs that specifically bind to LAG3 (human LAG3, or human and cynomolgus LAG3). The LAG3 agonists are set forth in SEQ ID NOs.: 1, 4, 6, 10, 11, 14, 15,16, 17, 20, or 21, respectively.Attorney Docket No.: 45817-0164WO1

[0141] Table 1A: Amino Acid Sequences of VHHs that Bind LAG3

[0142] VHH VHH Amino Acid Sequence

[0143] ID VHH1 / QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRD

[0144] L3G11 RGKNMVFLQMNNVKVEDTAVYYCATPDGSRCTGARWEPDAWGQGTLVTVSS

[0145] (SEQ ID NO:1)

[0146] VHH2 QVQLVESGGGLVQTGGSLRLSCAAYGRDFSSYAVGWFRQAPGKEREFVATISMSGVYTRYADSVTGRFTISRD

[0147] L3G3v2 / NAKNTVYLQMNSLKPGDTAVYYCAADLSSALYGSSWDEEYDFWGQGTQVTVSS

[0148] (SEQ ID NO 2)

[0149] VHH3 / QVQLVESGGGLVQTGGSLRLSCTAYGRDFSSYAVGWFRQAPGKEREFVATITISGTNTRYRDSVKGRFTISRD

[0150] L3F10 NAKNTVYLQMNSLKPEDTAVYYCGADLSSALFGSSWDEEYDYWGQGTQVTVSS

[0151] (SEQ ID NO:3)

[0152] VHH4 / QVQLVESGGGLVQPGGSLRLSCVASGFTLDDYGIGWFRQAPGKERLAVSCTTGSDGKTYYADSVKGRFTISRD

[0153] L3D4 NAKNTVYLQMNSLKPEDTAVYYCATDQYRDCRGPYKNWGQGTQVTVSS

[0154] (SEQ ID NO:4)

[0155] VHH5 / QVQLVESGGGLVQAGGSLRLSCEASGDTDTVAWFRQAPGSERECVTCITWNGAEKYYAPSVKGRFTISRD

[0156] L3H4 NAKKTVYLQLSSLKPEDTAVYYCAAVGAWSGRGLATRESEYQYWGQGTQVTVSS

[0157] (SEQ ID NO:5)

[0158] VHH6 / QVQLVESGGGLVQAGGSLRLSCAASGFADDDYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISSD

[0159] L3E5 KAKNTVYLQMNSLKPEDTAVYYCAAALLWCVSDLLGDEYDYWGQGTQVTVSS

[0160] (SEQ ID NO:6)

[0161] VHH7 / QVQLVESGGGLVQAGGSLRLSCAASGFADDDYAIGWFRQAPWKELVSDAGFTLICGTTCYTDSAKGRFTISRD

[0162] L3D7 NGTNTVFLQMNSLKPVDTAIYYCAARRRLRIEVGGGEYDYWGQGTQVTVSS

[0163] (SEQ ID NO:7 )

[0164] VHH8 / QVQLVESGGGLVQPGGSLRLSCTAYGRDFSSYAVGWFRQAPGKEREFVATITISGGSGERYRDSVKGRFTISRD

[0165] L3F11 NAKNTVYLQMNSLKPEDTAVYYCGADLSSALFGSSWDAEYDYWGQGIQVTVSS

[0166] (SEQ ID NO:8)

[0167] VHH9 / QVQLVESGGGLVQAGGSLRLSCKAYGRNFSSNYAMGWFRQAPGKERESVATITIRNGSKTHYTDSVKGRFTISRD

[0168] L3F12 NAKNTVYLQMNSLKPEDTAIYYCAAHRAPPMEVRSGDYEYDDQGTQVTVSS

[0169] (SEQ ID NO:9)

[0170] VHH10 / QVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREAVSCIRSSDGNTYYADSVKGRFTISSD

[0171]

[0172] L3F1 SARNVVHLQMNSLKPEDTAVYYCAKDRYSDCKGPYNYWGQGTQVTVSSAttorney Docket No.: 45817-0164WO1

[0173] (SEQ ID NO: 10)

[0174] VHH11 / QVQLVESGGGLVQAGGSLRLSCAASGFTFDNYAIAWFRQAPGKEREAVSCVRSSDGNTYYADSVKGRFTISSD

[0175] L3B6v2 SARNVVHLQMNSLKPEDTAVYYCAKDSYSDCKGPYNYWGQGTQVTVSS

[0176] (SEQ ID NO:11)

[0177] VHH12 / QVQLVESGGGLVQAGGSLRLSCEASGDTDTVGWFRQAPGSEREFVTCINWSGDSKYVATSVRGRFTISRD

[0178] L3B3 NAKHTVYLQLSSLKPEDTAVYYCAAVRAWSGGGLAAREDEYEYWGQGTQVTVSS

[0179] (SEQ ID NO: 12)

[0180] VHH13 / EVQLVESGGGLVQAGGSLRLSCEASGRTFSSTSAGYWLHAARGKDLFSVATITCDGGTTYYTYSMKGLFTISRN

[0181] L3A2 NAKNTVYLLMNSLKHDDTVIYYCAERPALLIEIRCGEYDYWGQGTQVTVSS

[0182] (SEQ ID NO:13)

[0183] VHH14 / QVQLVESGGGLVQPGGSLRLSCAASGASLDYSVIGWFRQAPGKEREAVSCIDNSDGSPYFADSVKGRFAISRD

[0184] L3F10vl NAKNTVYLQMNSLKPEDTAIYYCATPHSGRRCVGAQWRSWGQGTQVTVSS

[0185] (SEQ ID NO:14)

[0186] VHH15 / QVQLVESGGELVQAGGSLRLSCAASGRTFNIAAMGWFRQTPGKEREFVAAITWISSNTYYADSVKGRFTISRD

[0187] L3G8 GAKSTVFLQMNSLKPEDTAVYYCAANEAYSSNYYYTLTGTYPYWGQGTQVTVSS

[0188] (SEQ ID NO:15)

[0189] VHH16 / EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREAVSCIRSSDGHTYYADSVKGRFTISSD

[0190] L3E7 SARNVVHLQMNSLKPEDTAIYYCAKDRYSDCKGPYNYWGQGTQVTVSS

[0191] (SEQ ID NO:16)

[0192] VHH17 / QVQLVESGGGLVQPGGSLRLSCAASGRTLDYYAVGWFRQAPGKEREGVSCISAIDDSTYYADSVKGRFTISRD

[0193] L3F4 NAKNTVYLQMNSLKPEDTAVYYCVTDRYPQVFSGRLCPSDYWGQGTQVTVSS

[0194] (SEQ ID NO: 17)

[0195] VHH18 / QVQLVESGGGLVQAGGSLRLSCAASGRTFSMYAMGWFRQAPGKEREFVAAITWSGGSTYYADSVKGRFTISRD

[0196] L3F12vl NANNTVYLQMNNLKPDDTAVYYCASKLTTFPILPTLYDYWGRGTGVTVSS

[0197] (SEQ ID NO:18)

[0198] VHH19 / QVQLVESGGGLVQPGGSLRLSCAASGRTLDYYAVGWFRQAPGKEREFVAAITWSGGSTYYADSVKGRFTISRD

[0199] L3F12v2 NAKNMEYLQMNSLKPEDTAVYYCAAKLTTVSLLPTHYDYWGQGTQVTVSS

[0200] (SEQ ID NO:19)

[0201] VHH20 / QVQLVESGGGLVQPGGSLRLSCVASAFTLDYYAIAWFRQAPGKEREGVSCLSSAGYTYYADSVKGRFTISRD

[0202] L3G4vl NAKNTVYLQMNSLKPEDTAVYYCAIGIHGPFLDHRLYDYWGQGTQVTVSS

[0203] (SEQ ID NO:20)

[0204] VHH21 / QVQLVESGGGLVQPGGSLRLSCVASAFTLDYYAIAWFRQAPGKEREGVSCLSSAGYTYYADSVKGRFTISRD

[0205]

[0206] L3G4v2 NAKNTVYLQMNSLKPEDTAVYYCAIGIHGPFLDQRLYDYWGQGTQVTVSSAttorney Docket No.: 45817-0164WO1

[0207] (SEQ ID NO:21)

[0208]

[0209] Attorney Docket No.: 45817-0164WO1 This disclosure features a polypeptide comprising a VHH that specifically binds to LAG3 (human, or human and cyno) and comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 21. In certain cases, the VHH that specifically binds to LAG3 (human, or human and cyno) comprises the amino acid sequence of any one of SEQ ID NOs: 10, 11, 16, 17, or 20. In some cases, the VHH that specifically binds to LAG3 (human, or human and cyno) comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 21 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 certain cases, the VHH that specifically binds to LAG3 (human or human and cyno) comprises the amino acid sequence of any one of SEQ ID NOs: 10, 11, 16, 17, or 20 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 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 LAG3 agonists. In certain instances, the VHHs have an EC50 of about 0.1 nM to about 25 nM. In certain instances, the VHHs have an EC50 of about 0.3 nM to about 5 nM.

[0210] Also encompassed by this disclosure are binding molecules or polypeptides that comprise a VHH that specifically binds to LAG3 (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 1 A. The VHH CDRs of the VHH can be based on any 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)).

[0211] Table 2: Exemplary CDR Definitions

[0212] Kabat + IMGT Kabat AbM Chothia Contact Chothia

[0213]

[0214] Attorney Docket No.: 45817-0164WO1 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

[0215] CDR3

[0216]

[0217] Provided in Table 3 are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the clones of Table 1 A according to the IMGT definition. The

[0218] disclosure encompasses polypeptides, single domain antibodies, and binding molecules that specifically bind to LAG3 (human, or human and cyno) and which comprise the

[0219] three VHH CDRs of any one of the VHHs listed in Table 3.

[0220] Table 3: Anti-LAG3 VHH CDRs Based on IMGT CDR Definition

[0221] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0222] GFTLDYYA ISSSDGST ATPDGSRCTGARWEPDA VHH1 / L3G11 (SEQ ID NO:22) (SEQ ID NO:23) (SEQ ID NO:24)

[0223] GRDFSSYA ISMSGVYT AADLSSALYGSSWDEEYDF VHH2 / L3G3v2 (SEQ ID NO:25) (SEQ ID NO:26) (SEQ ID NO:27)

[0224] GRDFSSYA ITISGTNT GADLS S ALFGS S WDEE YD Y VHH3 / L3F10 (SEQ ID NO:25) (SEQ ID NO:28) (SEQ ID NO:29)

[0225] GFTLDDYG TTGSDGKT ATDQYRDCRGPYKN VHH4 / L3D4 (SEQ ID NO: 30) (SEQ ID NO:31) (SEQ ID NO:32)

[0226] GDTDTVA ITWNGAEK AAVGAWSGRGLATRESEYQY VHH5 / L3H4 (SEQ ID NO:33) (SEQ ID NO:34) (SEQ ID NO:35)

[0227] GFADDDYA ISSSDGST AAALLWCVSDLLGDEYDY VHH6 / L3E5 (SEQ ID NO:36) (SEQ ID NO:23) (SEQ ID NO:37)

[0228] GFADDDYA FTLICGTT AARRRLRIEVGGGEYDY VHH7 / L3D7 (SEQ ID NO:36) (SEQ ID NO:38) (SEQ ID NO:39)

[0229] GRDFSSYA ITISGGSGE GADLS S ALFGS S WD AEYD Y VHH8 / L3F11 (SEQ ID NO:25) (SEQ ID NO:40) (SEQ ID NO:41)

[0230] GRNFSSNY ITIRNGSKT AAHRAPPMEVRSGDYEY VHH9 / L3F12 (SEQ ID NO:42) (SEQ ID NO:43) (SEQ ID NO:44)

[0231] GFTFDDYA IRSSDGNT AKDRYSDCKGPYNY VHH10 / L3F1 (SEQ ID NO:45) (SEQ ID NO:46) (SEQ ID NO:47)

[0232] GFTFDNYA VRSSDGNT AKDSYSDCKGPYNY VHH11 / L3B6v2 (SEQ ID NO:48) (SEQ ID NO:49) (SEQ ID NO: 50)

[0233] GDTDTVG INWSGDSK AAVRAWSGGGLAAREDEYEY VHH12 / L3B3 (SEQ ID NO:51) (SEQ ID NO:52) (SEQ ID NO:53)

[0234]

[0235] VHH13 / L3A2 GRTFSSTS ITCDGGTT AERPALLIEIRCGEYDYAttorney Docket No.: 45817-0164WO1 (SEQ ID NO:54) (SEQ ID NO:55) (SEQ ID NO:56) GASLDYSV IDNSDGSP ATPHSGRRCVGAQWRS VHH14 / L3F10vl (SEQ ID NO:57) (SEQ ID NO:58) (SEQ ID NO:59)

[0236] GRTFNIAA ITWISSNT AANEAYS SNYYYTLTGTYP Y VHH15 / L3G8 (SEQ ID NO:60) (SEQ ID NO:61) (SEQ ID NO:62)

[0237] GFTFDDYA IRSSDGHT AKDRYSDCKGPYNY VHH16 / L3E7 (SEQ ID NO:45) (SEQ ID NO:63) (SEQ ID NO: 47)

[0238] GRTLDYYA ISAIDDST VTDRYPQVFSGRLCPSDY VHH17 / L3F4 (SEQ ID NO: 64) (SEQ ID NO: 65) (SEQ ID NO:66)

[0239] GRTFSMYA ITWSGGST ASKLTTFPILPTLYDY VHH18 / L3F12vl (SEQ ID NO: 67) (SEQ ID NO:68) (SEQ ID NO:69)

[0240] GRTLDYYA ITWSGGST AAKLTTVSLLPTHYDY VHH19 / L3F12v2 (SEQ ID NO: 64) (SEQ ID NO:68) (SEQ ID NO:70)

[0241] AFTLDYYA LSSAGYT AIGIHGPFLDHRLYDY VHH20 / L3G4vl (SEQ ID NO:71) (SEQ ID NO: 72) (SEQ ID NO:73)

[0242] AFTLDYYA LSSAGYT AIGIHGPFLDQRLYDY

[0243]

[0244] VHH21 / L3G4v2 (SEQ ID NO:71) (SEQ ID NO: 72) (SEQ ID NO:74)

[0245] In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to LAG3 (human, or human and cyno), wherein the VHH comprises the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid 5 sequences of any one clone listed in Table 3. Thus, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds LAG3 (human, or human and cyno), wherein the VHH comprises:

[0246] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:22, a

[0247] VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:23, and a VHH w CDR3 comprising the amino acid sequence set forth in SEQ ID NO:24;

[0248] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25, a

[0249] 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;

[0250] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25, a

[0251] 15 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;Attorney Docket No.: 45817-0164WO1 (d) 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;

[0252] (e) 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:35;

[0253] (f) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:36, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:23, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:37;

[0254] (g) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:36, 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:39;

[0255] (h) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:40, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:41;

[0256] (i) 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:44;

[0257] (j) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:45, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:46, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:47;

[0258] (k) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:48, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:49, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50;Attorney Docket No.: 45817-0164WO1 (1) 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;

[0259] (m) 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;

[0260] (n) 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;

[0261] (o) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:60, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:61, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:62;

[0262] (p) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:45, 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:47;

[0263] (q) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 64, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:65, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:66;

[0264] (r) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:67, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:68, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:69;

[0265] (s) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:64, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:68, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:70;Attorney Docket No.: 45817-0164WO1 (t) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:71, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:72, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:73; or

[0266] (u) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 71, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:72, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:74.

[0267] The VHHs bind human LAG3. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as LAG3 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for binding human LAG3 of about 0.1 nM to about 25 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for binding human LAG3 of about 0.3 nM to about 5 nM. In some cases, the VHHs bind an epitope within the DI and D2 domains domain of LAG3. In other cases, the VHHs bind an epitope within the D3 and D4 domains of LAG3.

[0268] In some instances, featured herein are single domain antibodies that specifically bind to LAG3 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to LAG3 (human, or human and cyno), which comprise: a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:64, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:65, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:66. 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: 17. In other cases, the single domain antibodies or binding molecules comprise an amino acid sequence set forth in SEQ ID NO: 17 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,Attorney Docket No.: 45817-0164WO1 the substitutions are in the framework regions and one or more of the three VHH CDRs. In some cases, the VHHs bind to human LAG3. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as LAG3 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 5 0.1 nM to about 25 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 0.2 nM to about 0.8 nM. In some cases, the VHHs bind an epitope within the DI domain of LAG3. In other cases, the VEIHs bind an epitope within the D2 domain of LAG3. In yet other cases, the VHHs bin an epitope within the D 1 and D2 domain of LAG3.

[0269] w Provided in Table 4 are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the clones of Table 1A according to the Kabat CDR definition. The disclosure encompasses single domain antibodies that specifically bind to LAG3 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to LAG3 (human, or human and cyno) and which comprise the three 15 VHH CDRs of any one of the VHHs listed in Table 4.Attorney Docket No.: 45817-0164WO1

[0270] Table 4. Anti-LAG3 VHH CDRs Based on Kabat CDR Definition

[0271] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0272] YYAIG CIS S SDGST YYAD S VKG PDGSRCTGARWEPDA VHH1 / L3G11 (SEQ ID NO:75) (SEQ ID NO:76) (SEQ ID NO:77)

[0273] SYAVG TISMSGVYTRYADSVTG DLSSALYGSSWDEEYDF VHH2 / L3G3v2 (SEQ ID NO:78) (SEQ ID NO: 79) (SEQ ID NO: 80)

[0274] SYAVG TITIS GTNTRYRD S VKG DLS S ALFGS S WDEEYD Y VHH3 / L3F10 (SEQ ID NO:78) (SEQ ID NO: 81) (SEQ ID NO: 82)

[0275] DYGIG CTTGSDGKTYYADSVKG DQYRDCRGPYKN VHH4 / L3D4 (SEQ ID NO: 83) (SEQ ID NO: 84) (SEQ ID NO: 85)

[0276] CITWNGAEKYYAPSVKG VGAWSGRGLATRESEYQY VHH5 / L3H4 VA (SEQ ID NO: 86) (SEQ ID NO: 87)

[0277] DYAIG CISSSDGSTYYADSVKG ALLWCVSDLLGDEYDY VHH6 / L3E5 (SEQ ID NO: 88) (SEQ ID NO: 76) (SEQ ID NO: 89)

[0278] DYAIG GFTLICGTTC YTD S AKG RRRLRIEVGGGEYDY VHH7 / L3D7 (SEQ ID NO:88) (SEQ ID NO: 90) (SEQ IDNO:91)

[0279] SYAVG TITIS GGSGERYRD S VKG DLSSALFGSSWDAEYDY VHH8 / L3F11 (SEQ ID NO:78) (SEQ ID NO: 92) (SEQ ID NO:93)

[0280] SNYAMG TITIRNGSKTHYTD S VKG HRAPPMEVRSGDYEY VHH9 / L3F12 (SEQ ID NO:94) (SEQ ID NO:95) (SEQ ID NO:96)

[0281] DYAIG CIRS SDGNTYYADS VKG DRYSDCKGPYNY VHH10 / L3F1 (SEQ ID NO:88) (SEQ ID NO:97) (SEQ ID NO:98)

[0282] NYAIA C VRS SDGNTYYAD S VKG DSYSDCKGPYNY VHH11 / L3B6v2 (SEQ ID NO:99) (SEQ ID NO: 100) (SEQ ID NO: 101)

[0283] CINWSGDSKYVATSVRG VRAWSGGGLAAREDEYEY VHH12 / L3B3 VG (SEQ ID NO: 102) (SEQ ID NO: 103)

[0284] STSAGY TITCDGGTTYYTYSMKG RPALLIEIRCGEYDY VHH13 / L3A2 (SEQ ID NO: 104) (SEQ ID NO: 105) (SEQ ID NO: 106)

[0285] YSVIG CIDNSDGSPYFADSVKG PHSGRRCVGAQWRS

[0286]

[0287] VHH14 / L3F10vl (SEQ ID NO: 107) (SEQ ID NO: 108) (SEQ ID NO: 109)Attorney Docket No.: 45817-0164WO1

[0288] IAAMG AITWIS SNT YYAD S VKG NEAYS SNYYYTLTGT YP Y VHH15 / L3G8 (SEQ ID NO: 110) (SEQ ID NO: 111) (SEQ ID NO: 112)

[0289] DYAIG CIRSSDGHTYYADSVKG DRYSDCKGPYNY VHH16 / L3E7 (SEQ ID NO: 88) (SEQ ID NO: 113) (SEQ ID NO:98)

[0290] YYAVG CISAIDDSTYYADSVKG DRYPQ VF SGRLCP SD Y VHH17 / L3F4 (SEQ ID NO: 114) (SEQ ID NO: 115) (SEQ ID NO: 116)

[0291] MYAMG AITWSGGSTYYADSVKG KLTTFPILPTLYDY VHH18 / L3F12vl (SEQ ID NO: 117) (SEQ ID NO: 118) (SEQ ID NO: 119)

[0292] YYAVG AITWSGGSTYYADSVKG KLTTVSLLPTHYDY VHH19 / L3F12v2 (SEQ ID NO: 114) (SEQ ID NO: 118) (SEQ ID NO: 120)

[0293] YYAIA CLSSAGYTYYADSVKG GIHGPFLDHRLYDY VHH20 / L3G4vl (SEQ ID NO: 121) (SEQ ID NO: 122) (SEQ ID NO: 123)

[0294] YYAIA CLSSAGYTYYADSVKG GIHGPFLDQRLYDY

[0295]

[0296] VHH21 / L3G4v2 (SEQ ID NO: 121) (SEQ ID NO: 122) (SEQ ID NO: 124)Attorney Docket No.: 45817-0164WO1 In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to LAG3 (human, or human and cyno LAG3), 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 LAG3 (human or human and cyno), wherein the VHH comprises:

[0297] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:75, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:76, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:77;

[0298] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO78, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:79, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80;

[0299] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:78, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 81, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:82;

[0300] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:83, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 84, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 85;

[0301] (e) a VHH CDR1 comprising the amino acid sequence VA, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:86, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87;

[0302] (f) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:88, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:76, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 89;Attorney Docket No.: 45817-0164WO1 (g) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:88, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:90, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:91;

[0303] (h) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:92, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:93;

[0304] (i) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:94, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:95, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:96;

[0305] (j) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:88, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:97, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:98;

[0306] (k) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:99, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 100, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 101;

[0307] (l) a VHH CDR1 comprising the amino acid sequence VG, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 102, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 103;

[0308] (m) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 104, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 105, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 106;

[0309] (n) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 107, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 108, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109;Attorney Docket No.: 45817-0164WO1 (o) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 110, 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;

[0310] (p) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88, 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:98;

[0311] (q) 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: 116;

[0312] (r) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 117, 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: 119;

[0313] (s) 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: 118, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 120;

[0314] (t) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 122, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 123; or

[0315] (u) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 122, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 124.

[0316] In some cases, the VHHs bind to human LAG3. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as LAG3 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 0.1 nM to about 25 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 0.3 nM to about 5 nM. In some cases,Attorney Docket No.: 45817-0164WO1 the VHHs bind an epitope in the D1-D2 domain of LAG3. In other cases, the VHHs bind an epitope within the D3-D4 domain of LAG3.

[0317] In some instances, featured herein are single domain antibodies that specifically bind to LAG3 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to LAG3 (human, or human and cyno), which comprise: 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:116. 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: 17. In other cases, the single domain antibodies or binding molecules comprise an amino acid sequence set forth in SEQ ID NO: 17 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 LAG3. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as LAG3 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for human LAG3 of about 0.1 nM to about 5 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for human LAG3 of about 0.2 nM to about 0.8 nM. In some cases, the VHHs bind an epitope in the DI domain of LAG3. In other cases, the VHHs bind an epitope in the D2 domain of LAG3. In certain cases, the VHHs bind an epitope within the D1-D2 domain of LAG3.

[0318] Provided in Table 5 are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the clones of Table 1A according to the Chothia CDR definition. The disclosure encompasses single domain antibodies that specifically bind to LAG3 (human, or human and cyno) or binding molecules or polypeptides comprising single domainAttorney Docket No.: 45817-0164WO1 antibodies that specifically bind to LAG3 (human, or human and cyno) and which comprise the three VHH CDRs of any one of the VHHs listed in Table 5.

[0319] Table 5. Anti-LAG3 VHH CDRs Based on Chothia CDR Definition

[0320] VHH-ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0321] GFTLDYY SSSDGS PDGSRC TGARWEPD A VHH1 / L3G11 (SEQ ID NO: 125) (SEQ ID NO: 126) (SEQ ID NO:77)

[0322] GRDFSSY SMSGVY DLS S AL YGS S WDEE YDF VHH2 / L3G3v2 (SEQ ID NO: 127) (SEQ ID NO: 128) (SEQ ID NO: 80)

[0323] GRDFSSY TISGTN DLSSALFGSSWDEEYDY VHH3 / L3F10 (SEQ ID NO: 127) (SEQ ID NO: 129) (SEQ ID NO: 82)

[0324] GFTLDDY TGSDGK DQYRDCRGPYKN VHH4 / L3D4 (SEQ ID NO: 130) (SEQ ID NO: 131) (SEQ ID NO: 85)

[0325] GDTDTVA TWNGAE VGAWSGRGLATRESEYQY VHH5 / L3H4 (SEQ ID NO:33) (SEQ ID NO: 132) (SEQ ID NO: 87)

[0326] GFADDDY SSSDGS ALLWCVSDLLGDEYDY VHH6 / L3E5 (SEQ ID NO: 133) (SEQ ID NO: 126) (SEQ ID NO: 89)

[0327] GFADDDY TLICGT RRRLRIEVGGGEYDY VHH7 / L3D7 (SEQ ID NO: 133) (SEQ ID NO: 134) (SEQ ID NO:91)

[0328] GRDFSSY TISGGSG DLSSALFGSSWDAEYDY VHH8 / L3F11 (SEQ ID NO: 127) (SEQ ID NO: 135) (SEQ ID NO:93)

[0329] GRNFSSN TIRNGSK HRAPPMEVRSGDYEY VHH9 / L3F12 (SEQ ID NO: 136) (SEQ ID NO: 137) (SEQ ID NO:96)

[0330] GFTFDDY RSSDGN DRYSDCKGPYNY VHH10 / L3F1 (SEQ ID NO: 138) (SEQ ID NO: 139) (SEQ ID NO:98)

[0331] GFTFDNY RSSDGN DSYSDCKGPYNY VHH11 / L3B6v2 (SEQ ID NO: 140) (SEQ ID NO: 139) (SEQ ID NO: 101)

[0332] GDTDTVG NWSGDS VRAWSGGGLAAREDEYEY VHH12 / L3B3 (SEQ ID NO:51) (SEQ ID NO: 141) (SEQ ID NO: 103)

[0333] GRTFSST TCDGGT RPALLIEIRCGEYDY VHH13 / L3A2 (SEQ ID NO: 142) (SEQ ID NO: 143) (SEQ ID NO: 106)

[0334] GASLDYS DNSDGS PHSGRRCVGAQWRS VHH14 / L3F10vl (SEQ ID NO: 144) (SEQ ID NO: 145) (SEQ ID NO: 109)

[0335] GRTFNIA TWISSN NEAYSSNYYYTLTGTYPY VHH15 / L3G8 (SEQ ID NO: 146) (SEQ ID NO: 147) (SEQ ID NO: 112)

[0336] GFTFDDY RSSDGH DRYSDCKGPYNY VHH16 / L3E7 (SEQ ID NO: 138) (SEQ ID NO: 148) (SEQ ID NO:98)

[0337] GRTLDYY SAIDDS DRYPQVF SGRLCPSDY VHH17 / L3F4 (SEQ ID NO: 149) (SEQ ID NO: 150) (SEQ ID NO: 116)

[0338] GRTFSMY TWSGGS KLTTFPILPTLYDY

[0339]

[0340] VHH18 / L3F12V1 (SEQ ID NO: 151) (SEQ ID NO: 152) (SEQ ID NO: 119)Attorney Docket No.: 45817-0164WO1 GRTLDYY TWSGGS KLTTVSLLPTHYDY VHH19 / L3F12v2 (SEQ ID NO: 149) (SEQ ID NO: 152) (SEQ ID NO: 120)

[0341] AFTLDYY SSAGY GIHGPFLDHRLYDY VHH20 / L3G4vl (SEQ ID NO: 153) (SEQ ID NO: 154) (SEQ ID NO: 123)

[0342] AFTLDYY SSAGY GIHGPFLDQRLYDY

[0343]

[0344] VHH21 / L3G4v2 (SEQ ID NO: 153) (SEQ ID NO: 154) (SEQ ID NO: 124)

[0345] In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to LAG3 (human, or human and cyno LAG3), 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 LAG3 (human, or human and cyno), wherein the VHH comprises:

[0346] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 125, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 126, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:77;

[0347] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 127, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 128, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80;

[0348] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 127, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 129, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 82;

[0349] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 130, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:131, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 85;

[0350] (e) 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: 132, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87;Attorney Docket No.: 45817-0164WO1 (f) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 126, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 89;

[0351] (g) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:91;

[0352] (h) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 127, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO 135, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:93;

[0353] (i) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 136, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 137, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:96;

[0354] (j) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 138, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 139, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:98;

[0355] (k) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 140, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 139, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 101;

[0356] (l) 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: 141, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 103;

[0357] (m) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 142, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 143, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 106;Attorney Docket No.: 45817-0164WO1 (n) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 144, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 145, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109;

[0358] (o) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 146, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 147, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112;

[0359] (p) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 138, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 148, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:98;

[0360] (q) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 149, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 150, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 116;

[0361] (r) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 151, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 152, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 119;

[0362] (s) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 149, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 152, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 120;

[0363] (t) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 153, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 123; or

[0364] (u) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 153, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 124.

[0365] In some cases, the VHHs bind to human LAG3. In some instances, the VHHs when linked to an Ig Fc domain (e g., IgGl) act as LAG3 agonists. In certain instances, theAttorney Docket No.: 45817-0164WO1 VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 0.1 nM to about 25 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 0.3 nM to about 5 nM. In some cases, the VHHs bind an epitope in the D1-D2 domain of LAG3. In other cases, the VHHs bind an epitope within the D3-D4 domain of LAG3.

[0366] In some instances, featured herein are single domain antibodies that specifically bind to LAG3 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to LAG3 (human, or human and cyno), which comprise: a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 149, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 150, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:116. 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: 17. In other cases, the single domain antibodies or binding molecules comprise an amino acid sequence set forth in SEQ ID NO: 17 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 LAG3. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as LAG3 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for human LAG3 of about 0.1 nM to about 5 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for human LAG3 of about 0.2 nM to about 0.8 nM. In some cases, the VHHs bind an epitope in the DI domain of LAG3. In other cases, the VHHs bind an epitope in the D2 domain of LAG3. In certain cases, the VHHs bind an epitope within the D1-D2 domain of LAG3.Attorney Docket No.: 45817-0164WO1 Provided in Table 6 are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the clones of Table 1 A according to the enhanced Chothia CDR definition. The disclosure encompasses single domain antibodies that specifically bind to LAG3 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to LAG3 (human, or human and cyno) and which comprise the three VHH CDRs of any one of the VHHs listed in Table 6.

[0367] Table 6. Anti-LAG3 VHH CDRs Based on Enhanced Chothia CDR Definition

[0368] VHH-ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0369] GFTLDYYAIG CISSSDGSTY PDGSRCTGARWEPDA VHH1 / L3G11 (SEQ ID NO: 155) (SEQ ID NO: 156) (SEQ ID NO: 77)

[0370] GRDFSSYAVG TISMSGVYTR DL S S ALYGS S WDEEYDF VHH2 / L3G3v2 (SEQ ID NO: 157) (SEQ ID NO: 158) (SEQ ID NO: 80)

[0371] GRDFSSYAVG TITISGTNTR DLS S ALFGS SWDEEYDY VHH3 / L3F10 (SEQ ID NO: 157) (SEQ ID NO: 159) (SEQ ID NO:82)

[0372] GFTLDDYGIG CTTGSDGKTY DQYRDCRGPYKN VHH4 / L3D4 (SEQ ID NO: 160) (SEQ ID NO: 161) (SEQ ID NO:85)

[0373] GDTDTVA CITWNGAEKY VGAWSGRGLATRESEYQY VHH5 / L3H4 (SEQ ID NO:33) (SEQ ID NO: 162) (SEQ ID NO: 87)

[0374] GFADDDYAIG CISSSDGSTY ALLWCVSDLLGDEYDY VHH6 / L3E5 (SEQ ID NO: 163) (SEQ ID NO: 156) (SEQ ID NO: 89)

[0375] GFADDDYAIG GFTLICGTTC RRRLRIEVGGGEYDY VHH7 / L3D7 (SEQ ID NO: 163) (SEQ ID NO: 164) (SEQ ID NO:91)

[0376] GRDFSSYAVG TITISGGSGER DL S S ALFGS SWDAEYDY VHH8 / L3F11 (SEQ ID NO: 157) (SEQ ID NO: 165) (SEQ ID NO:93)

[0377] GRNFSSNYAMG TITIRNGSKTH HRAPPMEVRSGDYEY VHH9 / L3F12 (SEQ ID NO: 166) (SEQ ID NO: 167) (SEQ ID NO:96)

[0378] GFTFDDYAIG CIRSSDGNTY DRYSDCKGPYNY VHH10 / L3F1 (SEQ ID NO: 168) (SEQ ID NO: 169) (SEQ ID NO:98)

[0379] GFTFDNYAIA CVRSSDGNTY DSYSDCKGPYNY VHH11 / L3B6v2 (SEQ ID NO: 170) (SEQ ID NO: 171) (SEQ ID NO: 101)

[0380] GDTDTVG CINWSGDSKY VRAWSGGGLAAREDEYEY VHH12 / L3B3 (SEQ ID NO:51) (SEQ ID NO: 172) (SEQ ID NO: 103)

[0381] GRTFSSTSAGY TITCDGGTTY RP. ALLIEIRCGEYDY VHH13 / L3A2 (SEQ ID NO: 173) (SEQ ID NO: 174) (SEQ ID NO: 106)

[0382] GASLDYSVIG CIDNSDGSPY PHSGRRCVGAQWRS VHH14 / L3F10vl (SEQ ID NO: 175) (SEQ ID NO: 176) (SEQ ID NO: 109)

[0383] GRTFNIAAMG AITWISSNTY NEAYS SNYYYTLTGT YP Y VHH15 / L3G8 (SEQ ID NO: 177) (SEQ ID NO: 178) (SEQ ID NO: 112)

[0384] GFTFDDYAIG CIRSSDGHTY DRYSDCKGPYNY

[0385]

[0386] VHH16 / L3E7 (SEQ ID NO: 168) (SEQ ID NO: 179) (SEQ ID NO:98)Attorney Docket No.: 45817-0164WO1

[0387] GRTLDYYAVG CISAIDDSTY DRYPQVFSGRLCPSDY VHH17 / L3F4 (SEQ ID NO: 180) (SEQ ID NO: 181) (SEQ ID NO: 116)

[0388] GRTFSMYAMG AITWSGGSTY KLTTFPILPTLYDY VHH18 / L3F12vl (SEQ ID NO: 182) (SEQ ID NO: 183) (SEQ ID NO: 119)

[0389] GRTLDYYAVG AITWSGGSTY KLTTVSLLPTHYDY VHH19 / L3F12v2 (SEQ ID NO: 180) (SEQ ID NO: 183) (SEQ ID NO: 120)

[0390] AFTLDYYAIA CLSSAGYTY GIHGPFLDHRLYDY VHH20 / L3G4vl (SEQ ID NO: 184) (SEQ ID NO: 185) (SEQ ID NO: 123)

[0391] AFTLDYYAIA CLSSAGYTY GIHGPFLDQRLYDY

[0392]

[0393] VHH21 / L3G4v2 (SEQ ID NO: 184) (SEQ ID NO: 185) (SEQ ID NO: 124)

[0394] In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to LAG3 (human, or human and cyno LAG3), wherein the VHH comprises the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid 5 sequences of any one clone listed in Table 6. Thus, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds LAG3 (human, or human and cyno), wherein the VHH comprises:

[0395] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 155, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 156, and a w VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:77;

[0396] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 157, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 158, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80;

[0397] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 157, a 15 VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 159, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 82;

[0398] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 160, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 161, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 85;Attorney Docket No.: 45817-0164WO1 (e) 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: 162, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87;

[0399] (f) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 163, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 156, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 89;

[0400] (g) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 163, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 164, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:91;

[0401] (h) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 157, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO 165, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:93;

[0402] (i) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 166, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 167, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:96;

[0403] (j) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 168, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 169, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:98;

[0404] (k) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 170, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 171, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 101;

[0405] (l) 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: 172, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 103;Attorney Docket No.: 45817-0164WO1 (m) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 173, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 174, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 106;

[0406] (n) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 175, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 176, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109;

[0407] (o) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 177, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 178, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112;

[0408] (p) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 168, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 179, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:98;

[0409] (q) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 180, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 181, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 116;

[0410] (r) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 182, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 183, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 119;

[0411] (s) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 180, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 183, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 120;

[0412] (t) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 184, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 185, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 123; orAttorney Docket No.: 45817-0164WO1 (u) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 184, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 185, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 124.

[0413] In some cases, the VHHs bind to human LAG3. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as LAG3 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 0.1 nM to about 25 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 0.3 nM to about 5 nM. In some cases, the VHHs bind an epitope in the D1-D2 domain of LAG3. In other cases, the VHHs bind an epitope within the D3-D4 domain of LAG3.

[0414] In some instances, featured herein are single domain antibodies that specifically bind to LAG3 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to LAG3 (human, or human and cyno), which comprise: a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 180, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 181, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:116. 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: 17. In other cases, the single domain antibodies or binding molecules comprise an amino acid sequence set forth in SEQ ID NO: 17 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 LAG3. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as LAG3 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for human LAG3 of about 0.1 nM to about 5 nM. In other instances, the VHHs linked to an Ig FcAttorney Docket No.: 45817-0164WO1 domain (or in another bivalent format) have an EC50 for human LAG3 of about 0.2 nM to about 0.8 nM. In some cases, the VHHs bind an epitope in the DI domain of LAG3. In other cases, the VHHs bind an epitope in the D2 domain of LAG3. In certain cases, the VHHs bind an epitope within the D1-D2 domain of LAG3.

[0415] 5 Provided in Table 7 are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the clones of Table 1A according to the Contact CDR definition. The disclosure encompasses single domain antibodies that specifically bind to LAG3 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to LAG3 (human, or human and cyno) and which comprise the three w VHH CDRs of any one of the VHHs listed in Table 7.Attorney Docket No.: 45817-0164WO1

[0416] Table 7. Anti-LAG3 VHH CDRs Based on Contact CDR Definition

[0417] VHH-1D VHH-CDR1 VHH-CDR2 VHH-CDR3

[0418] DYYAIG GVSCISSSDGSTY ATPDGSRCTGARWEPD VHH1 / L3G11 (SEQ ID NO: 186) (SEQ ID NO: 187) (SEQ ID NO: 188)

[0419] SSYAVG FVATISMSGVYTR AADLS SALYGS SWDEEYD VHH2 / L3G3v2 (SEQ ID NO: 189) (SEQ ID NO: 190) (SEQ ID NO: 191)

[0420] SSYAVG FVATITISGTNTR GADLS SALFGS SWDEEYD VHH3 / L3F10 (SEQ ID NO: 189) (SEQ ID NO: 192) (SEQ ID NO: 193)

[0421] DDYGIG AVSCTTGSDGKTY ATDQYRDCRGPYK VHH4 / L3D4 (SEQ ID NO: 194) (SEQ ID NO: 195) (SEQ ID NO: 196)

[0422] CVTCITWNGAEKY AAVGAWSGRGLATRESEYQ VHH5 / L3H4 TVA (SEQ ID NO: 197) (SEQ ID NO: 198 )

[0423] DDYAIG GVSCISSSDGSTY AAALLWCVSDLLGDEYD VHH6 / L3E5 (SEQ ID NO: 199) (SEQ ID NO: 187) (SEQ ID NO:200)

[0424] DDYAIG SDAGFTLICGTTC AARRRLRIEVGGGEYD VHH7 / L3D7 (SEQ ID NO: 199) (SEQ ID NO:201) (SEQ ID NO:202)

[0425] SSYAVG FVATITISGGSGER GADLS SALFGS SWDAEYD VHH8 / L3F11 (SEQ ID NO: 189) (SEQ ID NO:203) (SEQ ID NO:204)

[0426] SSNYAMG SVATITIRNGSKTH AAHRAPPMEVRSGDYE VHH9 / L3F12 (SEQ ID NO:205) (SEQ ID NO:206) (SEQ ID NO:207)

[0427] DDYAIG AVSCIRSSDGNTY AKDRYSDCKGPYN VHH10 / L3F1 (SEQ ID NO: 199) (SEQ ID NO:208) (SEQ ID NO:209)

[0428] DNYAIA AVSCVRSSDGNTY AKDSYSDCKGPYN VHH11 / L3B6v2 (SEQ ID NO:210) (SEQ ID NO:211) (SEQ ID NO:212)

[0429] FVTCINWSGDSKY AAVRAWSGGGLAAREDEYE VHH12 / L3B3 TVG (SEQ ID NO:213) (SEQ ID NO:214)

[0430] SSTSAGY SVATITCDGGTTY AERPALLIEIRCGEYD VHH13 / L3A2 (SEQ ID NO:215) (SEQ ID NO:216) (SEQ ID NO:217)

[0431] DYSVIG AVSCIDNSDGSPY ATPHSGRRCVGAQWR

[0432]

[0433] VHH14 / L3F10vl (SEQ ID NO:218) (SEQ ID NO:219) (SEQ ID NO:220)Attorney Docket No.: 45817-0164WO1

[0434] NIAAMG FVAAITWISSNTY AANEAYS SNYYYTLTGT YP VHH15 / L3G8 (SEQ ID NO:221) (SEQ ID NO:222) (SEQ ID NO:223)

[0435] DDYAIG AVSCIRSSDGHTY AKDRYSDCKGPYN VHH16 / L3E7 (SEQ ID NO: 199) (SEQ ID NO:224) (SEQ ID NO:209)

[0436] DYYAVG GVSCISAIDDSTY VTDRYPQVFSGRLCPSD VHH17 / L3F4 (SEQ ID NO:225) (SEQ ID NO:226) (SEQ ID NO:227)

[0437] SMYAMG FVAAITWSGGSTY ASKLTTFPILPTLYD VHH18 / L3F12vl (SEQ ID NO:228) (SEQ ID NO:229) (SEQ ID NO:230)

[0438] DYYAVG FVAAITWSGGSTY AAKLTTVSLLPTHYD VHH19 / L3F12v2 (SEQ ID NO:225) (SEQ ID NO:229) (SEQ ID NO:231)

[0439] DYYAIA GVSCLSSAGYTY AIGIHGPFLDHRLYD VHH20 / L3G4vl (SEQ ID NO:232) (SEQ ID NO:233) (SEQ ID NO:234)

[0440] DYYAIA GVSCLSSAGYTY AIGIHGPFLDQRLYD

[0441]

[0442] VHH21 / L3G4v2 (SEQ ID NO:232) (SEQ ID NO:233) (SEQ ID NO:235)Attorney Docket No.: 45817-0164WO1 In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to LAG3 (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 LAG3 (human, or human and cyno), wherein the VHH comprises:

[0443] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 186, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 187, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 188;

[0444] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 190, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 191;

[0445] (c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 192, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 193;

[0446] (d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 194, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 195, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 196;

[0447] (e) a VHH CDR1 comprising the amino acid sequence TV A, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 197, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 198;

[0448] (f) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 187, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:200;Attorney Docket No.: 45817-0164WO1 (g) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:201, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:202;

[0449] (h) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 189, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:204;

[0450] (i) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:205, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:207;

[0451] (j) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:208, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:209;

[0452] (k) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:210, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:211, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:212;

[0453] (l) a VHH CDR1 comprising the amino acid sequence TVG, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:213, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:214;

[0454] (m) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:215, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:216, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:217;

[0455] (n) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:218, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:219, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:220;Attorney Docket No.: 45817-0164WO1 (o) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:221, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:222, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:223;

[0456] (p) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:224, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:209;

[0457] (q) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:225, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:226, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:227;

[0458] (r) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:228, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:229, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:230;

[0459] (s) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:225, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:229, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:231;

[0460] (t) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:232, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:233, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:234; or

[0461] (u) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:232, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:233, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:235.

[0462] In some cases, the VHHs bind to human LAG3. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as LAG3 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 0.1 nM to about 25 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 of about 0.3 nM to about 5 nM. In some cases,Attorney Docket No.: 45817-0164WO1 the VHHs bind an epitope in the D1-D2 domain of LAG3. In other cases, the VHHs bind an epitope within the D3-D4 domain of LAG3.

[0463] In some instances, featured herein are single domain antibodies that specifically bind to LAG3 (human, or human and cyno) or binding molecules comprising single domain antibodies that specifically bind to LAG3 (human, or human and cyno), which comprise: a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:225, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:226, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:227. 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: 17. In other cases, the single domain antibodies or binding molecules comprise an amino acid sequence set forth in SEQ ID NO: 17 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 LAG3. In some instances, the VHHs when linked to an Ig Fc domain (e.g., IgGl) act as LAG3 agonists. In certain instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for human LAG3 of about 0.1 nM to about 5 nM. In other instances, the VHHs linked to an Ig Fc domain (or in another bivalent format) have an EC50 for human LAG3 of about 0.2 nM to about 0.8 nM. In some cases, the VHHs bind an epitope in the DI domain of LAG3. In other cases, the VHHs bind an epitope in the D2 domain of LAG3. In certain cases, the VHHs bind an epitope within the D1-D2 domain of LAG3.

[0464] 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., fromAttorney Docket No.: 45817-0164WO1 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.

[0465] 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, DI 273 -DI 281 (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, 1 to 3, 1 to 2, or 1 amino acids 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 1A are substituted for humanization. In some cases, 1 to 10 amino acids in the VHH framework regions of the sequences in Table 1 A are substituted for humanization. In some cases, 1 to 5 amino acids in the VHH framework regions of the sequences in Table 1 A 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-NbBCIH0FGLA. See, e.g., Vincke et al., J. Biol. Chem., 284, 3273-3284 (2009), which is incorporated by reference herein.

[0466] Table IB provides the sequences of exemplary humanized VHHs that specifically bind to LAG3 (human LAG3, or human and cynomolgus LAG3).Attorney Docket No.: 45817-0164WO1 Encompassed by this disclosure are binding molecules comprising any of these humanized VHHs that specifically bind to LAG3 (human LAG3, or human and cynomolgus LAG3).Attorney Docket No.: 45817-0164WO1

[0467] Table IB: Amino Acid Sequences of Humanized VHHs that Bind LAG3

[0468] VHH ID VHH Amino Acid Sequence EVQLVESGGGLVQPGGSLRLSCAASGRTLDYYAVGWFRQAPGKEREGVSCISAIDDSTYYADSVKGRFTISRDNS

[0469] L3F4_hl KNTLYLQMNSLRAEDTAVYYCVTDRYPQVFSGRLCPSDYWGQGTLVTVSS (SEQ ID NO:237)

[0470] QVQLVESGGGLVQPGGSLRLSCAASGRTLDYYAVGWFRQAPGKEREGVSCISAIDDSTYYADSVKGRFTISRDNS

[0471] L3F4_h2 KNTLYLQMNSLRAEDTAVYYCVTDRYPQVFSGRLCPSDYWGQGTLVTVSS (SEQ ID NO:238)

[0472] EVQLVESGGGLVQPGGSLRLSCAASGRTLDYYAVGWFRQAPGKEREGVSCISAIDDSTYYADSVKGRFTISRDNS

[0473] L3F4 h3 KNTVYLQMNSLRAEDTAVYYCVTDRYPQVFSGRLCPSDYWGQGTLVTVSS (SEQ ID NO:239)

[0474] EVQLLESGGGLVQPGGSLRLSCAASGRTLDYYAVGWFRQAPGKEREGVSCISAIDDSTYYADSVKGRFTISRDNS

[0475] L3F4_h4 KNTLYLQMNSLRAEDTAVYYCVTDRYPQVFSGRLCPSDYWGQGTLVTVSS (SEQ ID NO:240)

[0476] QVQLVESGGGLVQPGGSLRLSCAASGRTLDYYAVGWFRQAPGKEREGVSCISAIDDSTYYADSVKGRFTISRDNS

[0477] L3F4 h5 KNTVYLQMNSLRAEDTAVYYCVTDRYPQVFSGRLCPSDYWGQGTLVTVSS (SEQ ID NO:241)

[0478] QVQLVESGGGLVQPGGSLRLSCAASGRTLDYYAVGWFRQAPGKEREGVSCISAIDDSTYYADSVKGRFTISRDNA

[0479] L3F4_h6 KNTVYLQMNSLRAEDTAVYYCVTDRYPQVFSGRLCPSDYWGQGTLVTVSS (SEQ ID NO:242)

[0480] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWFRQAPGKEREFVSCISGSGGSTYYADSVKGRFTISRDNS

[0481] L3F4 h7 KNTLYLQMNSLRAEDTAVYYCVTDRYPQVFSGRLCPSDYWGQGTLVTVSS (SEQ ID NO:243)

[0482] EVQLVESGGGLVQPGGSLRLSCAASGRTFDSYAMSWFRQAPGKEREFVSCISGSDDSTYYADSVKGRFTISRDNS

[0483] L3F4_h8 KNTLYLQMNSLRAEDTAVYYCVTDRYPQVFSGRLCPSDYWGQGTLVTVSS (SEQ ID NO:244)

[0484] EVQLVESGGGLVQPGGSLRLSCAASGRTLDYYAVGWFRQAPGKEREFVSCISAIDDSTYYADSVKGRFTISRDNS

[0485] L3F4_h9 KNTLYLQMNSLRAEDTAVYYCVTDRYPQVFSGRLCPSDYWGQGTLVTVSS (SEQ ID NO:245)

[0486] EVQLVESGGGLVQPGGSLRLSCAASGRTLDYYAVGWFRQAPGKERELVSCISAIDDSTYYADSVKGRFTISRDNS

[0487] L3F4_hlO KNTLYLQMNSLRAEDTAVYYCVTDRYPQVFSGRLCPSDYWGQGTLVTVSS (SEQ ID NO:246)

[0488] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWFRQAPGKEREGVSAISGSGGTYYADSVKGRFTISRDNS

[0489] L3G4v2_hl KNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRLYDYWGQGTLVTVSS (SEQ ID NO:247)

[0490] EVQLVESGGGLVQPGGSLRLSCAASGFTFDYYAMAWFRQAPGKEREGVSAISGAGYTYYADSVKGRFTISRDNS

[0491] L3G4v2 h2 KNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRLYDYWGQGTLVTVSS (SEQ ID NO:248)

[0492] EVQLVESGGGLVQPGGSLRLSCAASAFTLDYYAIAWFRQAPGKEREGVSALSSAGYTYYADSVKGRFTISRDNS

[0493]

[0494] L3G4v2_h3 KNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRLYDYWGQGTLVTVSS (SEQ ID NO:249)Attorney Docket No.: 45817-0164WO1

[0495] EVQLVESGGGLVQPGGSLRLSCAASAFTLDYYAIAWFRQAPGKEREGVSCLSSAGYTYYADSVKGRFTISRDNS

[0496] L3G4v2_h4 KNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRLYDYWGQGTLVTVSS (SEQ ID NO:250)

[0497] EVQLVESGGGLVQPGGSLRLSCAASAFTLDYYAIAWFRQAPGKEREGVSALSSAGYTYYADSVKGRFTISRDNA

[0498] L3G4v2_h5 KNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRLYDYWGQGTLVTVSS (SEQ ID NO:251)

[0499] EVQLVESGGGLVQPGGSLRLSCVASAFTLDYYAIAWFRQAPGKEREGVSALSSAGYTYYADSVKGRFTISRDNS

[0500] L3G4v2_h6 KNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRLYDYWGQGTLVTVSS (SEQ ID NO:252)

[0501] QVQLVESGGGLVQPGGSLRLSCAASAFTLDYYAIAWFRQAPGKEREGVSALSSAGYTYYADSVKGRFTISRDNS

[0502] L3G4v2_h7 KNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRLYDYWGQGTLVTVSS (SEQ ID NO:253)

[0503] EVQLVESGGGLVQPGGSLRLSCAASAFTLDYYAIAWFRQAPGKEREGVSALSSAGYTYYADSVKGRFTISRDNS

[0504] L3G4v2 h8 KNTVYLQMNSLRAEDTAVYYCAIGIHGPFLDQRLYDYWGQGTLVTVSS (SEQ ID NO:254)

[0505] EVQLVESGGGLVQPGGSLRLSCAASAFTLDYYAIAWFRQAPGKEREGVSTLSSAGYTYYADSVKGRFTISRDNS

[0506] L3G4v2_h9 KNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRLYDYWGQGTLVTVSS (SEQ ID NO:255)

[0507] L3G4v2_hl EVQLVESGGGLVQPGGSLRLSCAASAFTLDYYAIAWFRQAPGKEREFVSALSSAGYTYYADSVKGRFTISRDNS

[0508] 0 KNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRLYDYWGQGTLVTVSS (SEQ ID NO:256)

[0509] L3G4v2 h QVQLVESGGGLVQPGGSLRLSCAASAFTLDYYAIAWFRQAPGKEREGVASLSSAGYTYYADSVKGRFTISRDNS

[0510]

[0511] 7_A KNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRLYDYWGQGTLVTVSS (SEQ ID NO: 257)

[0512] L3G4v2_h7_A (SEQ ID NO: 257) is a stabilization variant of L3G4v2_h7 (SEQ ID NO: 253).Attorney Docket No.: 45817-0164WO1 In certain cases, this disclosure features a binding molecule comprising a single domain antibody that binds to human LAG3 and 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 least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs:237 to 257 (e.g., 257). These binding molecules comprising the single domain antibody that binds to human LAG3, comprise 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.: 237 to 257 (e.g., 257). These binding molecules are LAG3 agonists. In certain cases, these binding molecules bind to both human and cynomolgus LAG3. In other cases, provided is a binding molecule comprising a single domain antibody that binds to human LAG3 and comprises an amino acid sequence set forth in any one of SEQ ID NOs:237 to 257 (e.g., 257) 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 some cases, the substitutions are in the CDRs. In some cases, the substitutions are in the framework regions and the CDRs. These binding molecules comprising the single domain antibody that binds to human LAG3, comprise 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.: 237 to 257 (e.g., 257). These binding molecules are LAG3 agonists. In certain cases, these binding molecules bind to both human and cynomolgus LAG3.

[0513] Table 1C provides the sequences of exemplary affinity matured and humanized VHHs that specifically bind to LAG3 (human LAG3, or human and cynomolgus LAG3). Encompassed by this disclosure are binding molecules comprising any of these affinity matured and humanized VHHs that specifically bind to LAG3 (human LAG3, or human and cynomolgus LAG3).Attorney Docket No.: 45817-0164WO1

[0514] Table 1C: Amino Acid Sequences of Affinity Matured Humanized VHHs that Bind LAG3

[0515] VHH ID Amino Acid Sequence

[0516] QVQLVESGGGLVQPGGSLRLSCAASAFTFDYYAIAWFRQAPGKEREGVASLSSAGYTYYADSVK

[0517] L3G4_hu_v.l

[0518] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 258) QVQLVESGGGLVQPGGSLRLSCAASAFTLDYYVIAWFRQAPGKEREGVASLSSAGYTYYADSVK

[0519] L3G4_hu_v.2

[0520] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 259) QVQLVESGGGLVQPGGSLRLSCAASAFTLDYYAIAWFRQAPGKEREGVASFSSAGYTYYADSVK

[0521] L3G4_hu_v.3

[0522] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 260) QVQLVESGGGLVQPGGSLRLSCAASAFTLDYYAIAWFRQAPGKEREGVASLSSAGYTYYADSVK

[0523] L3G4_hu_v.4

[0524] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAISIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 261) QVQLVESGGGLVQPGGSLRLSCAASAFTFDYYAIAWFRQAPGKEREGVAVLSSAGYTYYADSVK

[0525] L3G4_hu_v.5

[0526] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 262) QVQLVESGGGLVQPGGSLRLSCAASAFTFDYYAIAWFRQAPGKEREGVASWSSAGYTYYADSVK

[0527] L3G4_hu_v.6

[0528] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 263) QVQLVESGGGLVQPGGSLRLSCAASAFTFDYYAIAWFRQAPGKEREGVASYSSAGYTYYADSVK

[0529] L3G4_hu_v.7

[0530] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 264) QVQLVESGGGLVQPGGSLRLSCAASAFTFDYYAIAWFRQAPGKEREGVASLSSPGYTYYADSVK

[0531] L3G4_hu_v.8

[0532] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 265) QVQLVESGGGLVQPGGSLRLSCAASAFPLDYYAIAWFRQAPGKEREGVASWSSAGYTYYADSVK

[0533] L3G4_hu_v.9

[0534] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 266) QVQLVESGGGLVQPGGSLRLSCAASAFTLDYYVIAWFRQAPGKEREGVASYSSAGYTYYADSVK

[0535] L3G4_hu_v.lO

[0536] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDIRLYDYWGQGTLVTVSS (SEQ ID NO: 267) QVQLVESGGGLVQPGGSLRLSCAASAFTFDYYAIAWFRQAPGKEREGVAVLSSAGYTYYADSVK

[0537] L3G4_hu_v.l2

[0538] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 268) QVQLVESGGGLVQPGGSLRLSCAASAFTFDYYAIAWFRQAPGKEREGVAVLSSAGYTYYADSVK

[0539] L3G4_hu_v.l3

[0540] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAISIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 269)

[0541]

[0542] L3G4_hu_v.ll QVQLVESGGGLVQPGGSLRLSCAASAFTFDYYAIAWFRQAPGKEREGVAVLSSAGYTYYADSVKAttorney Docket No.: 45817-0164WO1

[0543] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDIRIYDYWGQGTLVTVSS (SEQ ID NO: 270) QVQLVESGGGLVQPGGSLRLSCAASAFTLDYYVIAWFRQAPGKEREGVASYSSAGYTYYADSVK

[0544] L3G4_hu_v.l4

[0545] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAISIHGPFLDIRLYDYWGQGTLVTVSS (SEQ ID NO: 271) QVQLVESGGGLVQPGGSLRLSCAASAFTFDYYVIAWFRQAPGKEREGVASYSSAGYTYYADSVK

[0546] L3G4_hu_v.l5

[0547] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAISIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 272) QVQLVESGGGLVQPGGSLRLSCAASAFPLDYYVIAWFRQAPGKEREGVALLSSAGYTYYADSVK

[0548] L3G4_hu_v.l6

[0549] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAISIHGPFLDIRLYDYWGQGTLVTVSS (SEQ ID NO: 273) QVQLVESGGGLVQPGGSLRLSCAASAFTFDYYAIAWFRQAPGKEREGVASLSSAGYTYFADSVK

[0550] L3G4_hu_v.l7

[0551] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 274) QVQLVESGGGLVQPGGSLRLSCAASAFTLDYYALAWFRQAPGKEREGVASLSSAGYTYFADSVK

[0552] L3G4_hu_v.l8

[0553] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 275) QVQLVESGGGLVQPGGSLRLSCAASAFTLDYWAIAWFRQAPGKEREGVASLSSAGYTYFADSVK

[0554] L3G4_hu_v.l9

[0555] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 276) QVQLVESGGGLVQPGGSLRLSCAASAFTLDYYAIAWFRQAPGKEREGVASFSSAGYTYFADSVK

[0556] L3G4_hu_v.2O

[0557] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 277) QVQLVESGGGLVQPGGSLRLSCAASAFTLDYYAIAWFRQAPGKEREGVASLSSAGYTYFADSVK

[0558] L3G4_hu_v.22

[0559] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 278) QVQLVESGGGLVQPGGSLRLSCAASAFTLDYYAIAWFRQAPGKEREGVASLSSAGYTYFADSVK

[0560] L3G4_hu_v.23

[0561] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDKRIYDYWGQGTLVTVSS (SEQ ID NO: 279) QVQLVESGGGLVQPGGSLRLSCAASAFTLDYWAIAWFRQAPGKEREGVASFSSAGYTYFADSVK

[0562] L3G4_hu_v.24

[0563] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 280) QVQLVESGGGLVQPGGSLRLSCAASAFTLDYYAIAWFRQAPGKEREGVASFSSAGYTYFADSVK

[0564] L3G4_hu_v.25

[0565]

[0566] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCALGIHGPFLDQRIYDYWGQGTLVTVSS (SEQ ID NO: 281)Attorney Docket No.: 45817-0164WO1 In certain cases, this disclosure features a binding molecule comprising a single domain antibody that binds to human LAG3 and 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 least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs:258 to 281 (e.g., 264, 281). These binding molecules comprising the single domain antibody that binds to human LAG3, comprise 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.: 258 to 281 (e.g., 264, 281). These binding molecules are LAG3 agonists. In certain cases, these binding molecules bind to both human and cynomolgus LAG3. In other cases, provided is a binding molecule comprising a single domain antibody that binds to human LAG3 and comprises an amino acid sequence set forth in any one of SEQ ID NOs: 258 to 281 (e.g., 264, 281) 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 some cases, the substitutions are in the CDRs. In some cases, the substitutions are in the framework regions and the CDRs. When substitutions are made in the CDRs, they are generally not made at the paratope residues identified in FIG. 17. These binding molecules are LAG3 agonists. In certain cases, these binding molecules bind to both human and cynomolgus LAG3.

[0567] Provided in Table 3A are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the affinity matured antibodies of Table 1C according to the IMGT definition. The disclosure encompasses polypeptides, single domain antibodies, and binding molecules that specifically bind to LAG3 (human, or human and cyno) and which comprise the three VHH CDRs of any one of the VHHs listed in Table 3A.

[0568] Table 3A: Anti-LAG3 VHH CDRs Based on IMGT CDR Definition

[0569] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0570] L3G4_hu_v.1 AFTFDYYA LSSAGYT AIGIHGPFLDQRIYDY

[0571]

[0572] Attorney Docket No.: 45817-0164WO1 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 293) 292) 72)

[0573] AFTLDYYV LSSAGYT AIGIHGPFLDQRIYDY

[0574] (SEQ ID NO: (SEQ ID NO:

[0575] L3G4_hu_v.2 294) 72) (SEQ ID NO: 293)

[0576] FSSAGYT AFTLDYYA AIGIHGPFLDQRIYDY

[0577] (SEQ ID NO:

[0578] L3G4_hu_v.3 (SEQ ID NO: 71) 295) (SEQ ID NO: 293)

[0579] LSSAGYT AFTLDYYA AISIHGPFLDQRIYDY

[0580] (SEQ ID NO:

[0581] L3G4_hu_v.4 (SEQ ID NO: 71) 72) (SEQ ID NO: 296)

[0582] AFTFDYYA LSSAGYT AIGIHGPFLDQRIYDY

[0583] (SEQ ID NO: (SEQ ID NO:

[0584] L3G4_hu_v.5 292) 72) (SEQ ID NO: 293)

[0585] AFTFDYYA WSSAGYT AIGIHGPFLDQRIYDY

[0586] (SEQ ID NO: (SEQ ID NO:

[0587] L3G4_hu_v.6 292) 297) (SEQ ID NO: 293)

[0588] AFTFDYYA YSSAGYT AIGIHGPFLDQRIYDY

[0589] (SEQ ID NO: (SEQ ID NO:

[0590] L3G4_hu_v.7 292) 298) (SEQ ID NO: 293)

[0591] AFTFDYYA LSSPGYT AIGIHGPFLDQRIYDY

[0592] (SEQ ID NO: (SEQ ID NO:

[0593] L3G4 hu v.8 292) 299) (SEQ ID NO: 293)

[0594]

[0595] Attorney Docket No.: 45817-0164WO1 AFPLDYYA WSSAGYT AIGIHGPFLDQRIYDY

[0596] (SEQ ID NO: (SEQ ID NO:

[0597] L3G4_hu_v.9 300) 297) (SEQ ID NO: 293)

[0598] AFTLDYYV YSSAGYT AIGIHGPFLDIRLYDY

[0599] (SEQ ID NO: (SEQ ID NO:

[0600] L3G4_hu_v.lO 294) 298) (SEQ ID NO: 301)

[0601] AFTFDYYA LSSAGYT AIGIHGPFLDIRIYDY

[0602] (SEQ ID NO: (SEQ ID NO:

[0603] L3G4_hu_v.11 292) 72) (SEQ ID NO: 302)

[0604] AFTFDYYA LSSAGYT ALGIHGPFLDQRIYDY

[0605] (SEQ ID NO: (SEQ ID NO:

[0606] L3G4_hu_v.l2 292) 72) (SEQ ID NO: 303)

[0607] AFTFDYYA LSSAGYT AISIHGPFLDQRIYDY

[0608] (SEQ ID NO: (SEQ ID NO:

[0609] L3G4_hu_v.l3 292) 72) (SEQ ID NO: 296)

[0610] AFTLDYYV YSSAGYT AISIHGPFLDIRLYDY

[0611] (SEQ ID NO: (SEQ ID NO:

[0612] L3G4_hu_v.l4 294) 298) (SEQ ID NO: 304)

[0613] AFTFDYYV YSSAGYT AISIHGPFLDQRIYDY

[0614] (SEQ ID NO: (SEQ ID NO:

[0615] L3G4_hu_v.l5 305) 298) (SEQ ID NO: 296)

[0616] AISIHGPFLDIRLYDY

[0617] L3G4_hu_v.l6 AFPLDYYV LSSAGYT (SEQ ID NO: 304)

[0618]

[0619] Attorney Docket No.: 45817-0164WO1 (SEQ ID NO: (SEQ ID NO:

[0620] 306) 72)

[0621] AFTFDYYA LSSAGYT AIGIHGPFLDQRIYDY

[0622] (SEQ ID NO: (SEQ ID NO:

[0623] L3G4_hu_v.l7 292) 72) (SEQ ID NO: 293)

[0624] LSSAGYT AFTLDYYA AIGIHGPFLDQRIYDY

[0625] (SEQ ID NO:

[0626] L3G4_hu_v.18 (SEQ ID NO: 71) 72) (SEQ ID NO: 293)

[0627] AFTLDYWA LSSAGYT AIGIHGPFLDQRIYDY

[0628] (SEQ ID NO: (SEQ ID NO:

[0629] L3G4_hu_v.l9 307) 72) (SEQ ID NO: 293)

[0630] FSSAGYT AFTLDYYA AIGIHGPFLDQRIYDY

[0631] (SEQ ID NO:

[0632] L3G4_hu_v.2O (SEQ ID NO: 71) 295) (SEQ ID NO: 293)

[0633] LSSAGYT AFTLDYYA ALGIHGPFLDQRIYDY

[0634] (SEQ ID NO:

[0635] L3G4_hu_v.22 (SEQ ID NO: 71) 72) (SEQ ID NO: 303)

[0636] LSSAGYT AFTLDYYA AIGIHGPFLDKRIYDY

[0637] (SEQ ID NO:

[0638] L3G4_hu_v.23 (SEQ ID NO: 71) 72) (SEQ ID NO: 308)

[0639] AFTLDYWA FSSAGYT AIGIHGPFLDQRIYDY

[0640] (SEQ ID NO: (SEQ ID NO:

[0641] L3G4 hu v.24 307) 295) (SEQ ID NO: 293)

[0642]

[0643] Attorney Docket No.: 45817-0164WO1

[0644] FSSAGYT AFTLDYYA ALGIHGPFLDQRIYDY

[0645] (SEQ ID NO:

[0646] L3G4 _hu_v.25 (SEQ ID NO: 71) 295) (SEQ ID NO: 303)

[0647]

[0648] The disclosure also encompasses polypeptides, single domain antibodies, and binding molecules that specifically bind to LAG3 (human, or human and cyno) and which comprise the three VHH CDRs with 0, 1, 2, 3, or 4 amino acid substitutions in one, two, or all three of the VHH-CDRs of any one of the VHHs listed in Table 3A. For example, in some instances, the disclosure encompasses polypeptides, single domain antibodies, and binding molecules that specifically bind to LAG3 (human, or human and cyno) and which comprise the three VHH CDRs: VHH-CDR1:

[0649] AF(T / P)(L / F)D(Y / W)(A / V); VHH-CDR2: (L / F / W / Y)SS(A / P)GYT (SEQ ID NO: 284); and VHH-CDR3: A(I / L)(G / S)IHGPFLD(Q / I / K)R(I / L)YDY (SEQ ID NO: 285).

[0650] In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to LAG3 (human, or human and cyno LAG3), wherein the VHH comprises:

[0651] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:292, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:298, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:293; or

[0652] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:71, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:295, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:303.

[0653] Provided in Table 4A are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the affinity matured antibodies of Table 1C according to the Kabat definition. The disclosure encompasses polypeptides, single domain antibodies, and binding molecules that specifically bind to LAG3 (human, or human and cyno) and which comprise the three VHH CDRs of any one of the VHHs listed in Table 4A.Attorney Docket No.: 45817-0164WO1 Table 4A: Anti-LAG3 VHH CDRs Based on Kabat CDR Definition

[0654] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0655] YYAIA SLSSAGYTYYADSVKG GIHGPFLDQRIYDY

[0656] (SEQ ID NO:

[0657] L3G4_hu_v.1 121) (SEQ ID NO: 309) (SEQ ID NO: 310)

[0658] YYVIA SLSSAGYTYYADSVKG GIHGPFLDQRIYDY

[0659] (SEQ ID NO:

[0660] L3G4_hu_v.2 311) (SEQ ID NO: 309) (SEQ ID NO: 310)

[0661] YYAIA SLSSAGYTYYADSVKG GIHGPFLDQRIYDY

[0662] (SEQ ID NO:

[0663] L3G4_hu_v.3 121) (SEQ ID NO: 312) (SEQ ID NO: 310)

[0664] YYAIA SLSSAGYTYYADSVKG SIHGPFLDQRIYDY

[0665] (SEQ ID NO:

[0666] L3G4_hu_v.4 121) (SEQ ID NO: 309) (SEQ ID NO: 313)

[0667] YYAIA VLSSAGYTYYADSVKG GIHGPFLDQRIYDY

[0668] (SEQ ID NO:

[0669] L3G4_hu_v.5 121) (SEQ ID NO: 314) (SEQ ID NO: 310)

[0670] YYAIA SWSSAGYTYYADSVKG GIHGPFLDQRIYDY

[0671] (SEQ ID NO:

[0672] L3G4_hu_v.6 121) (SEQ ID NO: 315) (SEQ ID NO: 310)

[0673] YYAIA SYSSAGYTYYADSVKG GIHGPFLDQRIYDY

[0674] (SEQ ID NO:

[0675] L3G4 hu v.7 121) (SEQ ID NO: 316) (SEQ ID NO: 310)

[0676]

[0677] Attorney Docket No.: 45817-0164WO1 YYAIA SLSSPGYTYYADSVKG GIHGPFLDQRIYDY

[0678] (SEQ ID NO:

[0679] L3G4_hu_v.8 121) (SEQ ID NO: 317) (SEQ ID NO: 310)

[0680] YYAIA SWSSAGYTYYADSVKG GIHGPFLDQRIYDY

[0681] (SEQ ID NO:

[0682] L3G4_hu_v.9 121) (SEQ ID NO: 315) (SEQ ID NO: 310)

[0683] YYVIA SYSSAGYTYYADSVKG GIHGPFLDIRLYDY

[0684] (SEQ ID NO:

[0685] L3G4_hu_v.lO 311) (SEQ ID NO: 316) (SEQ ID NO: 318)

[0686] YYAIA VLSSAGYTYYADSVKG GIHGPFLDIRIYDY

[0687] (SEQ ID NO:

[0688] L3G4_hu_v.11 121) (SEQ ID NO: 314) (SEQ ID NO: 319)

[0689] YYAIA VLSSAGYTYYADSVKG GIHGPFLDQRIYDY

[0690] (SEQ ID NO:

[0691] L3G4_hu_v.l2 121) (SEQ ID NO: 314) (SEQ ID NO: 310)

[0692] YYAIA VLSSAGYTYYADSVKG SIHGPFLDQRIYDY

[0693] (SEQ ID NO:

[0694] L3G4_hu_v.l3 121) (SEQ ID NO: 314) (SEQ ID NO: 313)

[0695] YYVIA SYSSAGYTYYADSVKG SIHGPFLDIRLYDY

[0696] (SEQ ID NO:

[0697] L3G4_hu_v.l4 311) (SEQ ID NO: 316) (SEQ ID NO: 320)

[0698] SYSSAGYTYYADSVKG SIHGPFLDQRIYDY

[0699] L3G4_hu_v.l5 YYVIA (SEQ ID NO: 316) (SEQ ID NO: 313)

[0700]

[0701] Attorney Docket No.: 45817-0164WO1 (SEQ ID NO:

[0702] 311)

[0703] YYVIA LLSSAGYTYYADSVKG SIHGPFLDIRLYDY

[0704] (SEQ ID NO:

[0705] L3G4_hu_v.l6 311) (SEQ ID NO: 321) (SEQ ID NO: 320)

[0706] YYAIA SLSSAGYTYFADSVKG GIHGPFLDQRIYDY

[0707] (SEQ ID NO:

[0708] L3G4_hu_v.l7 121) (SEQ ID NO: 322) (SEQ ID NO: 310)

[0709] YYALA SLSSAGYTYFADSVKG GIHGPFLDQRIYDY

[0710] (SEQ ID NO:

[0711] L3G4_hu_v.l8 323) (SEQ ID NO: 322) (SEQ ID NO: 310)

[0712] YWAIA SLSSAGYTYFADSVKG GIHGPFLDQRIYDY

[0713] (SEQ ID NO:

[0714] L3G4_hu_v.l9 324) (SEQ ID NO: 322) (SEQ ID NO: 310)

[0715] YYAIA SFSSAGYTYFADSVKG GIHGPFLDQRIYDY

[0716] (SEQ ID NO:

[0717] L3G4_hu_v.2O 121) (SEQ ID NO: 325) (SEQ ID NO: 310)

[0718] YYAIA SLSSAGYTYFADSVKG GIHGPFLDQRIYDY

[0719] (SEQ ID NO:

[0720] L3G4_hu_v.22 121) (SEQ ID NO: 322) (SEQ ID NO: 310)

[0721] YYAIA SLSSAGYTYFADSVKG GIHGPFLDKRIYDY

[0722] (SEQ ID NO:

[0723] L3G4 hu v.23 121) (SEQ ID NO: 322) (SEQ ID NO: 326)

[0724]

[0725] Attorney Docket No.: 45817-0164WO1 YWAIA SFSSAGYTYFADSVKG GIHGPFLDQRIYDY

[0726] (SEQ ID NO:

[0727] L3G4_hu_v.24 324) (SEQ ID NO: 325) (SEQ ID NO: 310)

[0728] YYAIA SFSSAGYTYFADSVKG GIHGPFLDQRIYDY

[0729] (SEQ ID NO:

[0730] L3G4_hu_v.25 121) (SEQ ID NO: 325) (SEQ ID NO: 310)

[0731]

[0732] In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to LAG3 (human, or human and cyno LAG3), wherein the VHH comprises:

[0733] 5 (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:316, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:310; or

[0734] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:325, and a w VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:310.

[0735] Provided in Table 5A are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the affinity matured antibodies of Table 1C according to the Chothia definition. The disclosure encompasses polypeptides, single domain antibodies, and binding molecules that specifically bind to LAG3 (human, or human and cyno) and 15 which comprise the three VHH CDRs of any one of the VHHs listed in Table 5A.

[0736] Table 5A: Anti-LAG3 VHH CDRs Based on Chothia CDR Definition

[0737] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0738] L3G4_hu_v.1 AFTFDYY SSAGY GIHGPFLDQRIYDY

[0739]

[0740] Attorney Docket No.: 45817-0164WO1 (SEQ ID NO: 327) (SEQ ID NO: (SEQ ID NO: 310)

[0741] 154)

[0742] SSAGY AFTLDYY GIHGPFLDQRIYDY

[0743] (SEQ ID NO:

[0744] L3G4_hu_v.2 (SEQ ID NO: 153) 154) (SEQ ID NO: 310)

[0745] SSAGY AFTLDYY GIHGPFLDQRIYDY

[0746] (SEQ ID NO:

[0747] L3G4_hu_v.3 (SEQ ID NO: 153) 154) (SEQ ID NO: 310)

[0748] SSAGY AFTLDYY SIHGPFLDQRIYDY

[0749] (SEQ ID NO:

[0750] L3G4_hu_v.4 (SEQ ID NO: 153) 154) (SEQ ID NO: 313)

[0751] SSAGY AFTFDYY GIHGPFLDQRIYDY

[0752] (SEQ ID NO:

[0753] L3G4_hu_v.5 (SEQ ID NO: 327) 154) (SEQ ID NO: 310)

[0754] SSAGY AFTFDYY GIHGPFLDQRIYDY

[0755] (SEQ ID NO:

[0756] L3G4_hu_v.6 (SEQ ID NO: 327) 154) (SEQ ID NO: 310)

[0757] SSAGY AFTFDYY GIHGPFLDQRIYDY

[0758] (SEQ ID NO:

[0759] L3G4_hu_v.7 (SEQ ID NO: 327) 154) (SEQ ID NO: 310)

[0760] SSPGY AFTFDYY GIHGPFLDQRIYDY

[0761] (SEQ ID NO:

[0762] L3G4 hu v.8 (SEQ ID NO: 327) 328) (SEQ ID NO: 310)

[0763]

[0764] Attorney Docket No.: 45817-0164WO1

[0765] SSAGY AFPLDYY GIHGPFLDQRIYDY

[0766] (SEQ ID NO:

[0767] L3G4_hu_v.9 (SEQ ID NO: 329) 154) (SEQ ID NO: 310)

[0768] SSAGY AFTLDYY GIHGPFLDIRLYDY

[0769] (SEQ ID NO:

[0770] L3G4_hu_v.lO (SEQ ID NO: 153) 154) (SEQ ID NO: 318)

[0771] SSAGY AFTFDYY GIHGPFLDIRIYDY

[0772] (SEQ ID NO:

[0773] L3G4_hu_v.11 (SEQ ID NO: 327) 154) (SEQ ID NO: 319)

[0774] SSAGY AFTFDYY GIHGPFLDQRIYDY

[0775] (SEQ ID NO:

[0776] L3G4_hu_v.l2 (SEQ ID NO: 327) 154) (SEQ ID NO: 310)

[0777] SSAGY AFTFDYY SIHGPFLDQRIYDY

[0778] (SEQ ID NO:

[0779] L3G4_hu_v.l3 (SEQ ID NO: 327) 154) (SEQ ID NO: 313)

[0780] SSAGY AFTLDYY SIHGPFLDIRLYDY

[0781] (SEQ ID NO:

[0782] L3G4_hu_v.l4 (SEQ ID NO: 153) 154) (SEQ ID NO: 320)

[0783] SSAGY AFTFDYY SIHGPFLDQRIYDY

[0784] (SEQ ID NO:

[0785] L3G4_hu_v.l5 (SEQ ID NO: 327) 154) (SEQ ID NO: 313)

[0786] AFPLDYY SIHGPFLDIRLYDY

[0787] L3G4_hu_v.l6 (SEQ ID NO: 329) SSAGY (SEQ ID NO: 320)

[0788]

[0789] Attorney Docket No.: 45817-0164WO1

[0790] (SEQ ID NO:

[0791] 154)

[0792] SSAGY AFTFDYY GIHGPFLDQRIYDY

[0793] (SEQ ID NO:

[0794] L3G4_hu_v.l7 (SEQ ID NO: 327) 154) (SEQ ID NO: 310)

[0795] SSAGY AFTLDYY GIHGPFLDQRIYDY

[0796] (SEQ ID NO:

[0797] L3G4_hu_v.18 (SEQ ID NO: 153) 154) (SEQ ID NO: 310)

[0798] SSAGY AFTLDYW GIHGPFLDQRIYDY

[0799] (SEQ ID NO:

[0800] L3G4_hu_v.l9 (SEQ ID NO: 330) 154) (SEQ ID NO: 310)

[0801] SSAGY AFTLDYY GIHGPFLDQRIYDY

[0802] (SEQ ID NO:

[0803] L3G4_hu_v.2O (SEQ ID NO: 153) 154) (SEQ ID NO: 310)

[0804] SSAGY AFTLDYY GIHGPFLDQRIYDY

[0805] (SEQ ID NO:

[0806] L3G4_hu_v.22 (SEQ ID NO: 153) 154) (SEQ ID NO: 310)

[0807] SSAGY AFTLDYY GIHGPFLDKRIYDY

[0808] (SEQ ID NO:

[0809] L3G4_hu_v.23 (SEQ ID NO: 153) 154) (SEQ ID NO: 326)

[0810] SSAGY AFTLDYW GIHGPFLDQRIYDY

[0811] (SEQ ID NO:

[0812] L3G4 hu v.24 (SEQ ID NO: 330) 154) (SEQ ID NO: 310)

[0813]

[0814] Attorney Docket No.: 45817-0164WO1

[0815] SSAGY AFTLDYY GIHGPFLDQRIYDY

[0816] (SEQ ID NO:

[0817] L3G4 _hu_v.25 (SEQ ID NO: 153) 154) (SEQ ID NO: 310)

[0818]

[0819] In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to LAG3 (human, or human and cyno LAG3), wherein the VHH comprises:

[0820] 5 (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 327, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:310; or

[0821] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 153, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154, and a w VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:310.

[0822] Provided in Table 6A are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the affinity matured antibodies of Table 1C according to the enhanced Chothia definition. The disclosure encompasses polypeptides, single domain antibodies, and binding molecules that specifically bind to LAG3 (human, or human and cyno) and 15 which comprise the three VHH CDRs of any one of the VHHs listed in Table 6A.

[0823] Table 6A: Anti-LAG3 VHH CDRs Based on Enhanced Chothia CDR Definition VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0824] SLSSAGYTY AFTFDYYAIA GIHGPFLDQRIYDY

[0825] (SEQ ID NO:

[0826] L3G4 hu v.1 (SEQ ID NO: 331) 332) (SEQ ID NO: 310) L3G4_hu_v.2 AFTLDYYVIA SLSSAGYTY GIHGPFLDQRIYDY

[0827]

[0828] Attorney Docket No.: 45817-0164WO1 (SEQ ID NO: 333) (SEQ ID NO: (SEQ ID NO: 310)

[0829] 332)

[0830] SFSSAGYTY AFTLDYYAIA GIHGPFLDQRIYDY

[0831] (SEQ ID NO:

[0832] L3G4_hu_v.3 (SEQ ID NO: 184) 334) (SEQ ID NO: 310)

[0833] SLSSAGYTY AFTLDYYAIA SIHGPFLDQRIYDY

[0834] (SEQ ID NO:

[0835] L3G4_hu_v.4 (SEQ ID NO: 184) 332) (SEQ ID NO: 313)

[0836] VLSSAGYTY AFTFDYYAIA GIHGPFLDQRIYDY

[0837] (SEQ ID NO:

[0838] L3G4_hu_v.5 (SEQ ID NO: 331) 335) (SEQ ID NO: 310)

[0839] SWSSAGYTY AFTFDYYAIA GIHGPFLDQRIYDY

[0840] (SEQ ID NO:

[0841] L3G4_hu_v.6 (SEQ ID NO: 331) 336) (SEQ ID NO: 310)

[0842] SYSSAGYTY AFTFDYYAIA GIHGPFLDQRIYDY

[0843] (SEQ ID NO:

[0844] L3G4 hu v.7 (SEQ ID NO: 331) 337) (SEQ ID NO: 310)

[0845] SLSSPGYTY AFTFDYYAIA GIHGPFLDQRIYDY

[0846] (SEQ ID NO:

[0847] L3G4_hu_v.8 (SEQ ID NO: 331) 338) (SEQ ID NO: 310)

[0848] SWSSAGYTY AFPLDYYAIA GIHGPFLDQRIYDY

[0849] (SEQ ID NO:

[0850] L3G4 hu v.9 (SEQ ID NO: 339) 336) (SEQ ID NO: 310)

[0851]

[0852] Attorney Docket No.: 45817-0164WO1

[0853] SYSSAGYTY AFTLDYYVIA GIHGPFLDIRLYDY

[0854] (SEQ ID NO:

[0855] L3G4_hu_v.lO (SEQ ID NO: 333) 337) (SEQ ID NO: 318)

[0856] VLSSAGYTY AFTFDYYAIA GIHGPFLDIRIYDY

[0857] (SEQ ID NO:

[0858] L3G4_hu_v.11 (SEQ ID NO: 331) 335) (SEQ ID NO: 319)

[0859] VLSSAGYTY AFTFDYYAIA GIHGPFLDQRIYDY

[0860] (SEQ ID NO:

[0861] L3G4_hu_v.l2 (SEQ ID NO: 331) 335) (SEQ ID NO: 310)

[0862] VLSSAGYTY AFTFDYYAIA SIHGPFLDQRIYDY

[0863] (SEQ ID NO:

[0864] L3G4_hu_v.l3 (SEQ ID NO: 331) 335) (SEQ ID NO: 313)

[0865] SYSSAGYTY AFTLDYYVIA SIHGPFLDIRLYDY

[0866] (SEQ ID NO:

[0867] L3G4_hu_v.l4 (SEQ ID NO: 333) 337) (SEQ ID NO: 320)

[0868] SYSSAGYTY AFTFDYYVIA SIHGPFLDQRIYDY

[0869] (SEQ ID NO:

[0870] L3G4_hu_v.l5 (SEQ ID NO: 340) 337) (SEQ ID NO: 313)

[0871] LLSSAGYTY AFPLDYYVIA SIHGPFLDIRLYDY

[0872] (SEQ ID NO:

[0873] L3G4_hu_v.l6 (SEQ ID NO: 341) 342) (SEQ ID NO: 320)

[0874] AFTFDYYAIA GIHGPFLDQRIYDY

[0875] L3G4_hu_v.l7 (SEQ ID NO: 331) SLSSAGYTY (SEQ ID NO: 310)

[0876]

[0877] Attorney Docket No.: 45817-0164WO1

[0878] (SEQ ID NO:

[0879] 332)

[0880] SLSSAGYTY AFTLDYYALA GIHGPFLDQRIYDY

[0881] (SEQ ID NO:

[0882] L3G4_hu_v.l8 (SEQ ID NO: 343) 332) (SEQ ID NO: 310)

[0883] SLSSAGYTY AFTLDYWAIA GIHGPFLDQRIYDY

[0884] (SEQ ID NO:

[0885] L3G4_hu_v.l9 (SEQ ID NO: 344) 332) (SEQ ID NO: 310)

[0886] SFSSAGYTY AFTLDYYAIA GIHGPFLDQRIYDY

[0887] (SEQ ID NO:

[0888] L3G4_hu_v.2O (SEQ ID NO: 184) 334) (SEQ ID NO: 310)

[0889] SLSSAGYTY AFTLDYYAIA GIHGPFLDQRIYDY

[0890] (SEQ ID NO:

[0891] L3G4_hu_v.22 (SEQ ID NO: 184) 332) (SEQ ID NO: 310)

[0892] SLSSAGYTY AFTLDYYAIA GIHGPFLDKRIYDY

[0893] (SEQ ID NO:

[0894] L3G4_hu_v.23 (SEQ ID NO: 184) 332) (SEQ ID NO: 326)

[0895] SFSSAGYTY AFTLDYWAIA GIHGPFLDQRIYDY

[0896] (SEQ ID NO:

[0897] L3G4_hu_v.24 (SEQ ID NO: 344) 334) (SEQ ID NO: 310)

[0898] SFSSAGYTY AFTLDYYAIA GIHGPFLDQRIYDY

[0899] (SEQ ID NO:

[0900] L3G4 hu v.25 (SEQ ID NO: 184) 334) (SEQ ID NO: 310)

[0901]

[0902] Attorney Docket No.: 45817-0164WO1 In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to LAG3 (human, or human and cyno LAG3), wherein the VHH comprises:

[0903] (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:331, a 5 VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:337, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:310; or

[0904] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 184, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:334, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:310.

[0905] w Provided in Table 7A are the VHH CDR1, VHH CDR2, and VHH CDR3 amino acid sequences of the affinity matured antibodies of Table 1C according to the Contact definition. The disclosure encompasses polypeptides, single domain antibodies, and binding molecules that specifically bind to LAG3 (human, or human and cyno) and which comprise the three VHH CDRs of any one of the VHHs listed in Table 7A.

[0906] 15 Table 7A: Anti-LAG3 VHH CDRs Based on Contact CDR Definition

[0907] VHH ID VHH-CDR1 VHH-CDR2 VHH-CDR3

[0908] DYYAIA

[0909] GVASLSSAGYTY AIGIHGPFLDQRIYD

[0910] (SEQ ID NO:

[0911] L3G4_hu_v.1 232) (SEQ ID NO: 345) (SEQ ID NO: 346)

[0912] DYYVIA GVASLSSAGYTY AIGIHGPFLDQRIYD

[0913] (SEQ ID NO:

[0914] L3G4_hu_v.2 347) (SEQ ID NO: 345) (SEQ ID NO: 346)

[0915] DYYAIA GVASFSSAGYTY AIGIHGPFLDQRIYD

[0916] (SEQ ID NO:

[0917] L3G4_hu_v.3 232) (SEQ ID NO: 348) (SEQ ID NO: 346)

[0918]

[0919] Attorney Docket No.: 45817-0164WO1 DYYAIA GVASLSSAGYTY AISIHGPFLDQRIYD

[0920] (SEQ ID NO:

[0921] L3G4_hu_v.4 232) (SEQ ID NO: 345) (SEQ ID NO: 349)

[0922] DYYAIA GVAVLSSAGYTY AIGIHGPFLDQRIYD

[0923] (SEQ ID NO:

[0924] L3G4_hu_v.5 232) (SEQ ID NO: 350) (SEQ ID NO: 346)

[0925] DYYAIA GVASWSSAGYTY AIGIHGPFLDQRIYD

[0926] (SEQ ID NO:

[0927] L3G4_hu_v.6 232) (SEQ ID NO: 351) (SEQ ID NO: 346)

[0928] DYYAIA GVASYSSAGYTY AIGIHGPFLDQRIYD

[0929] (SEQ ID NO:

[0930] L3G4 hu v.7 232) (SEQ ID NO: 352) (SEQ ID NO: 346)

[0931] DYYAIA GVASLSSPGYTY AIGIHGPFLDQRIYD

[0932] (SEQ ID NO:

[0933] L3G4_hu_v.8 232) (SEQ ID NO: 353) (SEQ ID NO: 346)

[0934] DYYAIA GVASWSSAGYTY AIGIHGPFLDQRIYD

[0935] (SEQ ID NO:

[0936] L3G4_hu_v.9 232) (SEQ ID NO: 351) (SEQ ID NO: 346)

[0937] DYYVIA GVASYSSAGYTY AIGIHGPFLDIRLYD

[0938] (SEQ ID NO:

[0939] L3G4_hu_v.lO 347) (SEQ ID NO: 352) (SEQ ID NO: 354)

[0940] GVAVLSSAGYTY AIGIHGPFLDIRIYD

[0941] L3G4_hu_v.ll DYYAIA (SEQ ID NO: 350) (SEQ ID NO: 355)

[0942]

[0943] Attorney Docket No.: 45817-0164WO1 (SEQ ID NO:

[0944] 232)

[0945] DYYAIA GVAVLSSAGYTY ALGIHGPFLDQRIYD

[0946] (SEQ ID NO:

[0947] L3G4_hu_v.l2 232) (SEQ ID NO: 350) (SEQ ID NO: 356)

[0948] DYYAIA GVAVLSSAGYTY AISIHGPFLDQRIYD

[0949] (SEQ ID NO:

[0950] L3G4_hu_v.l3 232) (SEQ ID NO: 350) (SEQ ID NO: 349)

[0951] DYYVIA GVASYSSAGYTY AISIHGPFLDIRLYD

[0952] (SEQ ID NO:

[0953] L3G4_hu_v.l4 347) (SEQ ID NO: 352) (SEQ ID NO: 357)

[0954] DYYVIA GVASYSSAGYTY AISIHGPFLDQRIYD

[0955] (SEQ ID NO:

[0956] L3G4_hu_v.l5 347) (SEQ ID NO: 352) (SEQ ID NO: 349)

[0957] DYYVIA GVALLSSAGYTY AISIHGPFLDIRLYD

[0958] (SEQ ID NO:

[0959] L3G4_hu_v.16 347) (SEQ ID NO: 358) (SEQ ID NO: 357)

[0960] DYYAIA GVASLSSAGYTY AIGIHGPFLDQRIYD

[0961] (SEQ ID NO:

[0962] L3G4_hu_v.l7 232) (SEQ ID NO: 345) (SEQ ID NO: 346)

[0963] DYYALA GVASLSSAGYTY AIGIHGPFLDQRIYD

[0964] (SEQ ID NO:

[0965] L3G4 hu v.18 359) (SEQ ID NO: 345) (SEQ ID NO: 346)

[0966]

[0967] Attorney Docket No.: 45817-0164WO1 DYWAIA GVASLSSAGYTY AIGIHGPFLDQRIYD

[0968] (SEQ ID NO:

[0969] L3G4_hu_v.l9 360) (SEQ ID NO: 345) (SEQ ID NO: 346)

[0970] DYYAIA GVASFSSAGYTY AIGIHGPFLDQRIYD

[0971] (SEQ ID NO:

[0972] L3G4_hu_v.2O 232) (SEQ ID NO: 348) (SEQ ID NO: 346)

[0973] DYYAIA GVASLSSAGYTY ALGIHGPFLDQRIYD

[0974] (SEQ ID NO:

[0975] L3G4_hu_v.22 232) (SEQ ID NO: 345) (SEQ ID NO: 356)

[0976] DYYAIA GVASLSSAGYTY AIGIHGPFLDKRIYD

[0977] (SEQ ID NO:

[0978] L3G4_hu_v.23 232) (SEQ ID NO: 345) (SEQ ID NO: 361)

[0979] DYWAIA GVASFSSAGYTY AIGIHGPFLDQRIYD

[0980] (SEQ ID NO:

[0981] L3G4_hu_v.24 360) (SEQ ID NO: 348) (SEQ ID NO: 346)

[0982] DYYAIA GVASFSSAGYTY ALGIHGPFLDQRIYD

[0983] (SEQ ID NO:

[0984] L3G4 _hu_v.25 232) (SEQ ID NO: 348) (SEQ ID NO: 356)

[0985]

[0986] In some instances, the disclosure provides a binding molecule or polypeptide comprising a VHH that specifically binds to LAG3 (human, or human and cyno LAG3), wherein the VHH comprises:Attorney Docket No.: 45817-0164WO1 (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:232, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:352, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:346; or

[0987] (b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:232, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:348, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:356.

[0988] The disclosure also features a polypeptide comprising a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds to human LAG3, the VHH comprising VHH complementarity determining region (CDR) 1, VHH CDR2, and VHH CDR3, wherein the VHH binds to amino acids 174, 182, 184-186, 188 (with its associated glycan), 196, 220-223, and 225-229 of human LAG3 (numbering according to SEQ ID NO: 465).

[0989] 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-Ig 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 region 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).

[0990] 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 IgG4PAA (SEQ ID NO:469). 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 IgG4PAA (SEQ ID NO:469). In someAttorney Docket No.: 45817-0164WO1 instances, the human Ig Fc region is modified to e.g., alter (increase or decrease) the effector function and / or to promote heterodimerization.

[0991] Bispecific Constructs

[0992] Other binding molecules are also encompassed by this disclosure. For example, the LAG3 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 LAG3 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.

[0993] 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: 290), (GGS)n (SEQ ID NO: 291), (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)a (SEQ ID NO:474)

[0994] In some instances, the bispecific construct comprises a first VHH of this disclosure (that specifically binds to LAG3) linked to a first human 1g hinge-Fc region (e.g., from IgGl, IgG2, IgG3, IgG4, or IgA) and a second VHH (that binds to a different epitope of LAG3 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, TgG2, IgG3, IgG4, orAttorney Docket No.: 45817-0164WO1 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 of IgG4PAA set forth in SEQ ID NO:468 or 469. 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 or 469 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 etal., 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).

[0995] Multispecific Constructs

[0996] 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 VHHs 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 bispecificAttorney Docket No.: 45817-0164WO1 construct. In all of the above, at least one antigen binding domain specifically binds to LAG3 (human, or human and cyno).

[0997] Conjugates

[0998] 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)s (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.

[0999] 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, 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 byAttorney Docket No.: 45817-0164WO1 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 other cases, the dimerization module is the hinge-Fc domain of IgG4PAA (e.g., SEQ ID NO:469). 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).

[1000] Methods of Making and Pharmaceutical Compositions

[1001] 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.

[1002] 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.

[1003] 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, aHEK293T cell, or aNIH3T3 cell.

[1004] 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. InAttorney Docket No.: 45817-0164WO1 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.

[1005] 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.

[1006] mRNA Constructs

[1007] 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.

[1008] The disclosure features an mRNA comprising an ORF encoding any of the polypeptides described above.

[1009] 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. In other instances, the polynucleotide comprises a nucleic acid encoding a signal sequence at the 5’ end of the ORF.

[1010] The disclosure features compositions including an mRNA encoding a polypeptide described herein (e.g., an anti-LAG3 VHH, a polypeptide comprising an anti-LAG3 VHH, a bivalent anti-LAG3 VHH-human IgGl (i.e., the VHH is linked at its C-terminal to a hinge-Fc domain of human IgGl), a bivalent anti-LAG3 VHH-IgG4PAA (i.e., the VHH is linked at its C-terminal to a hinge-Fc domain of human IgG4PAA), a bispecificAttorney Docket No.: 45817-0164WO1 antibody comprising an anti-LAG3 VHH, or a multispecific antibody comprising an anti-LAG3 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.

[1011] mRNA: 5 ’-cap

[1012] 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.

[1013] 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-m ethylated. 5 '-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[1014] 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 guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate

[1015] nucleotides.Attorney Docket No.: 45817-0164WO1 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.

[1016] 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.

[1017] 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.

[1018] For example, the Anti -Reverse Cap Analog (ARC A) 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).

[1019] 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).

[1020] 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 capAttorney Docket No.: 45817-0164WO1 analogs described in US Patent No. US 8,519,110, the contents of which are herein incorporated by reference in its entirety.

[1021] In some instances, the cap analog is aN7-(4-chlorophenoxyethyl) substituted dicnucleotide 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.

[1022] While cap analogs allow for the concomitant capping of a nucleic acid molecule in an in 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.

[1023] 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 anN7 methylation and the 5 '-terminal nucleotide of the mRNA 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 structuresAttorney Docket No.: 45817-0164WO1 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.

[1024] 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.

[1025] 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.

[1026] Untranslated Regions (UTRs)

[1027] 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).

[1028] 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) intramolecularAttorney Docket No.: 45817-0164WO1 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).

[1029] 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., (2002) Meeh Dev 110(1 -2):97-l 12). Studies have shown that naturally-occurring, cis-acting 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).Attorney Docket No.: 45817-0164WO1 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 aberrant translation products. In some instances, the desired translational regulatory activity is a combination of one or more of the foregoing translational regulatory activities.

[1030] Accordingly, the present disclosure provides a polynucleotide, e.g., an mRNA, comprising an RNA element that comprises a sequence and / or an RNA secondaryAttorney Docket No.: 45817-0164WO1 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.

[1031] 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.

[1032] 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 RNA 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,Attorney Docket No.: 45817-0164WO1 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.

[1033] 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.

[1034] 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, about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine.

[1035] 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, precedingAttorney Docket No.: 45817-0164WO1 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 1, 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.

[1036] 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, wherein n = 1 to 10 (SEQ ID NO: 362), 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, wherein n = 1, 2, 3, 4 or 5 (SEQ ID NO: 286). 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, 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, wherein n = 4 (SEQ ID NO: 364). In some instances, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 5 (SEQ ID NO: 365).Attorney Docket No.: 45817-0164WO1 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.

[1037] 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.

[1038] 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’Attorney Docket No.: 45817-0164WO1 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.

[1039] 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.

[1040] 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 sequences 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 translation (IVT).

[1041] 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 sequenceAttorney Docket No.: 45817-0164WO1 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:

[1042] 5 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA (SEQ ID NO:

[1043] 479).

[1044] 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

[1045] w sequence shown in Table 8:

[1046] GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA (SEQ ID NO:

[1047] 479).

[1048] In some instances, the 5’ UTR comprises the following sequence set forth in Table 8:

[1049] 15 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGC CGCCACC (SEQ ID NO: 236)

[1050] Table 8

[1051] 5' UTRs 5' UTR Sequence GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAG

[1052] Standard

[1053] AAAUAUAAGAGCCACC (SEQ ID NO:481) GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAG AAAUAUAAGACCCCGGCGCCGCCACC (SEQ ID VI -UTR

[1054] NO: 480) GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAG AAAUAUAAGACCCCGGCGCCACC (SEQ ID NO:

[1055] V2-UTR

[1056] 482)

[1057] GC-Rich RNA Elements Sequence

[1058]

[1059] Attorney Docket No.: 45817-0164WO1 K0 (Traditional Kozak consensus) [GCCA / GCC]

[1060] EK [GCCGCC]

[1061] VI [CCCCGGCGCC] (SEQ ID NO:476) V2 [CCCCGGC]

[1062] (CCG)n, where n=1-10 [CCG]n(SEQ ID NO: 362)

[1063] (GCC)n, where n=1-10 [GCC]n(SEQ ID NO: 363)

[1064]

[1065] 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 stable RNA secondary structure comprising a sequence of nucleotides, or 5 derivatives or analogs thereof, linked in an order which forms a hairpin or a stem-loop.

[1066] In one embodiment, the stable RNA secondary structure is upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is 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 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 is located about 15-30, about 15-20, about 15-25, about 10-15, or about 5-10 nucleotides 15 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.Attorney Docket No.: 45817-0164WO1 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.

[1067] In another embodiment, the sequence of the GC-rich RNA element is comprised exclusively of guanine (G) and cytosine (C) nucleobases.

[1068] 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.

[1069] 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. InAttorney Docket No.: 45817-0164WO1 some instances, the polynucleotide comprises two or more 3'UTRs or functional fragments thereof, each of which have the same or different nucleotide sequences.

[1070] In some instances, the 5'UTR or functional fragment thereof, 3' UTR or functional fragment thereof, or any combination thereof is sequence optimized.

[1071] 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.

[1072] 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.

[1073] 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.

[1074] 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 as 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.,Attorney Docket No.: 45817-0164WO1 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).

[1075] 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.

[1076] 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 5UTR.

[1077] 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.

[1078] Exemplary UTRs of the application include, but are not limited to, one or more 5 TR and / or 3UTR derived from the nucleic acid sequence of: a globin, such as an a-or 0-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 albumin?); aHSD17B4 (hydroxysteroid (17-0) dehydrogenase); a virus (e.g., a tobacco etch virus (TEV), a Venezuelan equine encephalitis virus (VEEV), a 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., Hsp 70); a translation initiation factor (e.g., elF4G); a glucose transporter (e.g., hGLUTl (human glucose transporter 1)); an actin (e.g., human a or 0 actin); a GAPDH; a tubulin; a histone; a citric acid cycle enzyme; a topoisomeraseAttorney Docket No.: 45817-0164WO1 (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 P subunit of mitochondrial H+-ATP synthase); a growth hormone e (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 (CollA2), collagen type I, alpha 1 (Coll Al), collagen type VI, alpha 2 (Col6A2), collagen type VI, alpha 1 (C0I6AI)); a ribophorin (e.g., ribophorin I (RPNI)); 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).

[1079] 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- ) 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 (DENV) 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.

[1080] 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 barley yellow dwarf virus (BYDV-PAV) 3'UTR; an elongation factor 1 al (EEF1A1) 3'UTR; a manganese superoxide dismutase (MnSOD) 3'UTR; a P subunit of mitochondrial H(+)-ATP synthase (p-mRNA) 3 TR; a GLUT1 3'UTR; a MEF2A 3'UTR; a P-Fl-ATPase 3'UTR; functional fragments thereof and combinations thereof.Attorney Docket No.: 45817-0164WO1 Wild-type UTRs derived from any gene or mRNA can be incorporated into the polynucleotides of the present disclosure. Tn 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.

[1081] 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.

[1082] 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.

[1083] 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).

[1084] 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:

[1085] 5' UTR-001 (Upstream UTR) (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:486);Attorney Docket No.: 45817-0164WO1 5' UTR-002 (Upstream UTR) (GGGAGAUCAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:487);

[1086] 5’ UTR-004 (Upstream UTR) (GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAGCCACC) (SEQ ID NO:488);

[1087] 5' UTR-005 (Upstream UTR) (GGGAGAUCAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:489);

[1088] 5' UTR-007 (Upstream UTR) (GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAGCCACC) (SEQ ID NO:490);

[1089] 5’ UTR-008 (Upstream UTR) (GGGAAUUAACAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:491);

[1090] 5' UTR-009 (Upstream UTR) (GGGAAAUUAGACAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:492);

[1091] 5' UTR-010, Upstream (GGGAAAUAAGAGAGUAAAGAACAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:493);

[1092] 5' UTR-011 (Upstream UTR) (GGGAAAAAAGAGAGAAAAGAAGACUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:494);

[1093] 5' UTR-012 (Upstream UTR) (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAUAUAUAAGAGCCACC) (SEQ ID NO:495);Attorney Docket No.: 45817-0164WO1 5' UTR-013 (Upstream UTR) (GGGAAAUAAGAGACAAAACAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:496);

[1094] 5’ UTR-014 (Upstream UTR) (GGGAAAUUAGAGAGUAAAGAACAGUAAGUAGAAUUAAAAGAGCCACC) (SEQ ID NO:497);

[1095] 5' UTR-015 (Upstream UTR) (GGGAAAUAAGAGAGAAUAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:498);

[1096] 5' UTR-016 (Upstream UTR) (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAAUUAAGAGCCACC) (SEQ ID NO:499);

[1097] 5’ UTR-017 (Upstream UTR); or (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUUUAAGAGCCACC) (SEQ ID NO:500);

[1098] 5' UTR-018 (Upstream UTR) 5' UTR (UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGG AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC) (SEQ ID NO:501)

[1099] In some instances, the 3' UTR comprises:

[1100] 3 UTR:

[1101] UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGG GCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUG GUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:502)

[1102] 142-3p 3' UTR (UTR including miR142-3p binding site)

[1103] UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCAttorney Docket No.: 45817-0164WO1 CAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGC ACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:503);

[1104] 142-3p 3' UTR (UTR including miR142-3p binding site) UGAUAAUAGGCUGGAGCCUCGGUGGCUCCAUAAAGUAGGAAACACUACA CAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGC ACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:504); or

[1105] 142-3p 3' UTR (UTR including miR142-3p binding site) UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUCCAUAA AGUAGGAAACACUACAUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGC ACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:505);

[1106] 142-3p 3' UTR (UTR including miR142-3p binding site) UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCU CCCCCCAGUCCAUAAAGUAGGAAACACUACACCCCUCCUCCCCUUCCUGC ACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:506);

[1107] 142-3p 3' UTR (UTR including miR142-3p binding site) UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCUCCAUAAAGUAGGAAACACUACACUGC ACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:507);

[1108] 142-3p 3' UTR (UTR including miR142-3p binding site) UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAAttorney Docket No.: 45817-0164WO1 GGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:508)

[1109] 142-3p 3' UTR (UTR including miR142-3p binding site) UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGA AUAAAGUUCCAUAAAGUAGGAAACACUACACUGAGUGGGCGGC (SEQ ID NO:509);

[1110] 3’UTR-018 GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAG GGUGUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG (SEQ ID NO:510)

[1111] 3' UTR (miR142 and miR126 binding sites variant 1)

[1112] UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGC CAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGC ACCCGUACCCCCCGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUAA AGUCUGAGUGGGCGGC (SEQ ID NO:511)

[1113] 3' UTR (miR142 and miR126 binding sites variant 2)

[1114] UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGC CUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGC ACCCGUACCCCCCGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUAA AGUCUGAGUGGGCGGC) (SEQ ID NO:512); or

[1115] 3’UTR (miR142-3p binding site variant 3)

[1116] UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCU CCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAAttorney Docket No.: 45817-0164WO1 GGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:513)

[1117] 3’UTR (miR142-3p binding site variant 3, DNA sequence)

[1118] TGATAATAGGCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCC CCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCTCCATAAAGTAGGA AACACTACAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (SEQ ID NO:514)

[1119] 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.

[1120] 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).

[1121] 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.

[1122] Incorporation of intronic sequences can increase protein production as well as polynucleotide expression levels. In some instances, the polynucleotide of the present 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,Attorney Docket No.: 45817-0164WO1 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.

[1123] 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.

[1124] 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.

[1125] In some instances, a 5'UTR and / or 3UTR 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.

[1126] 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.

[1127] 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 a 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 sequencesAttorney Docket No.: 45817-0164WO1 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.

[1128] 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 3'UTR 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.

[1129] 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).

[1130] 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).

[1131] mRNA: Coding region

[1132] Provided herein are mRNA that encode the coding regions of the VHHs, polypeptides, bispecific antibodies, and multispecific antibodies, of the disclosure. InAttorney Docket No.: 45817-0164WO1 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-LAG3 VHH. For example, the polynucleotides can comprise a codon optimized ORF encoding a polypeptide comprising an anti-LAG3 VHH.

[1133] 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.: 45817-0164WO1 Modified Nucleic Acids

[1134] 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.: 45817-0164WO1 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-methylaminornethyl-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.

[1135] Nucleosides Containing Modified Sugars

[1136] 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.

[1137] Exemplary substitutions at the 2'-position include, but are not limited to, H, halo, optionally substituted Ci-6 alkyl; optionally substituted Ci-6 alkoxy; optionally substituted Cg- io aryl oxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted Ce- 10 aryl oxy; optionally substituted Ce-ioaryl-Ci-6 alkoxy, 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.: 45817-0164WO1 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

[1138] 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.

[1139] Alterations on the Nucleobase

[1140] 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.

[1141] 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.: 45817-0164WO1 synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more alternative or alternative nucleosides).

[1142] 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.

[1143] 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.

[1144] 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.

[1145] 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.: 45817-0164WO1 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), l-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1^), 5-methyl-2-thio-uridine (m’s2U), l-methyl-4-thio-pseudouridine (m's4\| / ), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (

[1146]

[1147] m3\| / ), 2-thio-l-methyl-pseudouridine, 1-m ethyl- 1-deaza-pseudouri dine, 2-thio-l -methyl- 1-deaza-pseudouri dine, 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 (\| / m ), 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.

[1148] In one instance, the nucleic acid is modified to contain 1 -methylpseudouridine (m1^) in lieu of uridine at each instance.Attorney Docket No.: 45817-0164WO1 In some instances, the alternative nucleobase is an alternative cytosine.

[1149] 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 (FC), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hnrC, 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 (kiC), a-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 (FCm), N4, N4,2'-O-trimethyl-cytidine (m42Cm), 1 -thio-cytidine, 2’-F-ara-cytidine, 2’-F-cytidine, and 2’-OH-ara-cytidine.

[1150] 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 (m'A), 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.: 45817-0164WO1 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.

[1151] 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 (o yW), 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 (Gl), 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), l-methyl-2'-O-methyl-guanosine (m'Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), l,2'-O-dimethyl-inosine (m'lm), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1 -thio-guanosine, O6-methyl-guanosine, 2’-F-ara-guanosine, and 2’-F-guanosine.

[1152] 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.: 45817-0164WO1 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)).

[1153] 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.: 45817-0164WO1 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.

[1154] 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.: 45817-0164WO1 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.

[1155] 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.: 45817-0164WO1 the disclosure contain 5-methoxy-uridine, uridine, 5-methoxy-cytidine, and cytidine as the only uridines and cytidines. Tn 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.

[1156] 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.: 45817-0164WO1 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.

[1157] 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.: 45817-0164WO1 Table 9 - Exemplary Uracil-containing Nucleosides

[1158] Nucleoside Name

[1159] 5-methoxy -uridine

[1160] 1 -Methyl -pseudo-uridine

[1161] Pseudouridine

[1162] 5-methyl-uridine

[1163] 5-bromo-uridine

[1164] 2-thio-uridine

[1165] 4-thiouridine

[1166] 2'-O-methyluridine

[1167] 5-methyl-2 -thiouridine

[1168] 5,2'-O-dimethyluridine

[1169] 5-aminomethyl-2-thiouridine

[1170] 5 -( 1 -Propyny l)ara-uridine

[1171] 2'-O-Methyl-5-(l-propynyl)uridine

[1172] 5-Vinylarauridine

[1173] (Z)-5-(2-Bromo-vinyl)ara-uridine

[1174] (E)-5-(2-Bromo-vinyl)ara-uridine

[1175] (Z)-5-(2-Bromo-vinyl)uridine

[1176] (E)-5-(2-Bromo-vinyl)uridine

[1177] 5-Cyanouridine

[1178] 5-Formyluridine

[1179] 5 -Di m ethyl am i n ouri dine

[1180] 5-Trideuteromethyl-6-deuterouridine

[1181] 5-(2-Furanyl)uridine

[1182] 5 -Phenyl ethy ny luri dine

[1183] 4'-Carbocyclic uridine

[1184] 4'-Ethynyluridine

[1185] 4'-Azidouridine

[1186] 2'-Deoxy-2',2'-difluorouridine

[1187] 2'-Deoxy-2'-b-fluorouridine

[1188] 2'-Deoxy-2'-b-chlorouridine

[1189] 2'-Deoxy-2'-b-bromouridine

[1190] 2'-Deoxy-2'-b-iodouridine

[1191] 5'-Homo-uridine

[1192] 2'-Deoxy-2'-a-mercaptouridine

[1193] 2'-Deoxy-2'-a-thiomethoxyuridine

[1194] 2'-Deoxy-2'-a-azidouridine

[1195]

[1196] 2'-Deoxy-2'-a-aminouridineAttorney Docket No.: 45817-0164WO1

[1197] Nucleoside Name

[1198] 2'-Deoxy-2'-b-mercaptouridine

[1199] 2'-Deoxy-2'-b-thiomethoxyuridine

[1200] 2'-Deoxy-2'-b-azidouridine

[1201] 2'-Deoxy-2'-b-aminouridine

[1202] 2'-b-Trifluoromethyluridine

[1203] 2'-a-Trifluoromethyluri dine

[1204] 2' -b -Ethy ny luri dine

[1205] 2'-a-Ethynyluridine

[1206] 1 -ethyl -p seudo-uri dine

[1207] 1 -propyl-pseudo-uridine

[1208] 1 -zso-propyl -pseudo-uridine

[1209] l-(2,2,2-trifluoroethyl)-pseudo-uridine

[1210] 1 -cyclopropyl-pseudo-uridine

[1211] 1-cyclopropylmethyl-pseudo-uridine

[1212] 1 -phenyl-pseudo-uridine

[1213] 1 -benzyl-pseudo-uridine

[1214] 1 -aminomethyl-pseudo-uridine

[1215] pseudo-uridine-l-2-ethanoic acid

[1216] 1 -(3 -amino-3 -carboxypropyl )pseudo-uri dine

[1217] l-methyl-3-(3-amino-3-carboxypropyl)pseudo-uridine

[1218] 6-methyl-pseudo-uridine

[1219] 6-trifluoromethyl-pseudo-uridine

[1220] 6-methoxy-pseudo-uridine

[1221] 6-phenyl-pseudo-uridine

[1222] 6-iodo-pseudo-uridine

[1223] 6-bromo-pseudo-uridine

[1224] 6-chloro-pseudo-uridine

[1225] 6-fluoro-pseudo-uridine

[1226] 4-Thio-pseudo-uridine

[1227] 2-Thio-pseudo-uridine

[1228] Alpha-thio-pseudo-uridine

[1229] 1 -Me-alpha-thio-pseudo-uridine

[1230] 1 -butyl-pseudo-uridine

[1231] 1 -tert-butyl -pseudo-uridine

[1232] 1 -pentyl-pseudo-uridine

[1233] 1 -hexyl-pseudo-uridine

[1234] 1 -trifluoromethyl-pseudo-uridine

[1235] 1 -cyclobutyl-pseudo-uridine

[1236]

[1237] 1-cyclopentyl-pseudo-uridineAttorney Docket No.: 45817-0164WO1

[1238] Nucleoside Name

[1239] 1-cyclohexyl-pseudo-uridine

[1240] 1-cycloheptyl-pseudo-uridine

[1241] 1 -cyclooctyl-pseudo-uridine

[1242] 1-cyclobutylmethyl-pseudo-uridine

[1243] 1-cyclopentylmethyl-pseudo-uridine

[1244] 1 -cy cl ohexyl methyl -p seu do-uri dine

[1245] 1-cycloheptylmethyl-pseudo-uridine

[1246] 1-cyclooctylmethyl-pseudo-uridine

[1247] 1 - / ?-tolyl-pseudo-uridine

[1248] l-(2,4,6-trimethyl-phenyl)pseudo-uridine

[1249] l-(4-methoxy-phenyl)pseudo-uridine

[1250] l-(4-amino-phenyl)pseudo-uridine

[1251] 1 (4-nitro-phenyl)pseudo-uridine

[1252] pseudo-uridine-Nl-p-benzoic acid

[1253] l-(4-methyl-benzyl)pseudo-uridine

[1254] l-(2,4,6-trimethyl-benzyl)pseudo-uridine

[1255] l-(4-methoxy-benzyl)pseudo-uridine

[1256] 1 -(4-amino-benzyl)pseudo-uridine

[1257] l-(4-nitro-benzyl)pseudo-uridine

[1258] pseudo-uridine-N 1 -methyl-p-benzoic acid

[1259] 1 -(2-amino-ethyl)pseudo-uridine

[1260] l-(3-amino-propyl)pseudo-uridine

[1261] l-(4-amino-butyl)pseudo-uridine

[1262] l-(5-amino-pentyl)pseudo-uridine

[1263] 1 -(6-amino-hexyl)pseudo-uridine

[1264] pseudo-uridine-N 1 -3-propionic acid

[1265] pseudo-uridine-Nl-4-butanoic acid

[1266] pseudo-uridine-Nl-5-pentanoic acid

[1267] pseudo-uridine-Nl-6-hexanoic acid

[1268] pseudo-uridine-Nl-7-heptanoic acid

[1269] l-(2-amino-2-carboxyethyl)pseudo-uridine

[1270] l-(4-amino-4-carboxybutyl)pseudo-uridine

[1271] 3-alkyl-pseudo-uridine

[1272] 6-ethy 1 -p seudo-uri dine

[1273] 6-propyl-pseudo-uridine

[1274] 6-i so-propy 1 -p seudo-uri dine

[1275] 6-buty 1 -p seudo-uri dine

[1276] 6-tert-butyl-pseudo-uridine

[1277]

[1278] 6-(2,2,2-trifluoroethyl)-pseudo-uridineAttorney Docket No.: 45817-0164WO1

[1279] Nucleoside Name

[1280] 6-ethoxy-pseudo-uridine

[1281] 6-trifluoromethoxy -pseudo-uridine

[1282] 6-phenyl-pseudo-uridine

[1283] 6-(substituted-phenyl)-pseudo-uridine

[1284] 6-cyano-pseudo-uridine

[1285] 6-azido-pseudo-uridine

[1286] 6-amino-pseudo-uridine

[1287] 6-ethylcarboxylate-pseudo-uridine

[1288] 6-hydroxy -pseudo-uridine

[1289] 6-methylamino-pseudo-uridine

[1290] 6-dimethylamino-pseudo-uridine

[1291] 6-hydroxyamino-pseudo-uridine

[1292] 6-formyl-pseudo-uridine

[1293] 6-(4-morpholino)-pseudo-uridine

[1294] 6-(4-thiomorpholino)-pseudo-uridine

[1295] 1 -me-4-thio-pseudo-uridine

[1296] 1 -me-2-thio-pseudo-uridine

[1297] 1, 6-dimethyl -pseudo-uri dine

[1298] l-methyl-6-trifluoromethyl-pseudo-uridine

[1299] 1 -methyl-6-ethyl-pseudo-uridine

[1300] 1 -methyl-6-propyl-pseudo-uridine

[1301] 1 -methyl -6-i so-propyl -pseudo-uri di ne

[1302] 1 -methyl-6-butyl-pseudo-uridine

[1303] l-methyl-6-tert-butyl-pseudo-uridine

[1304] l-methyl-6-(2,2,2-trifluoroethyl)pseudo-uridine

[1305] 1 -methyl -6-i odo-pseudo-uridine

[1306] 1 -methyl-6-bromo-pseudo-uridine

[1307] l-methyl-6-chloro-pseudo-uridine

[1308] 1 -methyl-6-fluoro-pseudo-uridine

[1309] l-methyl-6-methoxy-pseudo-uridine

[1310] 1 -methyl-6-ethoxy -pseudo-uri dine

[1311] l-methyl-6-trifluorom ethoxy -pseudo-uri dine

[1312] 1 -methyl-6-phenyl-pseudo-uridine

[1313] l-methyl-6-(substituted phenyl)pseudo-uridine

[1314] l-methyl-6-cyano-pseudo-uridine

[1315] 1 -methyl-6-azido-pseudo-uridine

[1316] 1 -methyl-6-amino-pseudo-uridine

[1317] l-methyl-6-ethylcarboxylate-pseudo-uridine

[1318]

[1319] l-methyl-6-hydroxy-pseudo-uridineAttorney Docket No.: 45817-0164WO1

[1320] Nucleoside Name

[1321] l-methyl-6-methylamino-pseudo-uridine

[1322] l-methyl-6-dimethylamino-pseudo-uridine

[1323] l-methyl-6-hydroxyamino-pseudo-uridine

[1324] 1 -methyl-6-formyl-pseudo-uridine

[1325] l-methyl-6-(4-morpholino)-pseudo-uridine

[1326] l-methyl-6-(4-thiomorpholino)-pseudo-uridine

[1327] 1 -alkyl-6-vinyl-pseudo-uridine

[1328] l-alkyl-6-allyl-pseudo-uridine

[1329] l-alkyl-6-homoallyl-pseudo-uridine

[1330] l-alkyl-6-ethynyl-pseudo-uridine

[1331] 1 -alkyl -6-(2-propynyl )-p seudo-uri dine

[1332] 1 -alkyl-6-( 1 -propynyl)-pseudo-uridine

[1333] 1 -Hydroxymethylpseudouridine

[1334] 1 -(2-Hydroxyethyl)pseudouridine

[1335] 1 -Methoxymethylpseudouridine

[1336] 1 -(2 -Methoxy ethyl)pseudouri dine

[1337] l-(2,2-Diethoxyethyl)pseudouridine

[1338] (±)l-(2-Hydroxypropyl)pseudouridine

[1339] (2R)-l-(2-Hydroxypropyl)pseudouridine

[1340] (2S)-l-(2-Hydroxypropyl)pseudouridine

[1341] 1-Cyanomethylpseudouridine

[1342] 1 -Morpholinomethylpseudouridine

[1343] 1-Thiomorpholinomethylpseudouridine

[1344] 1 -Benzyloxymethylpseudouridine

[1345] l-(2,2,3,3,3-Pentafluoropropyl)pseudouridine

[1346] 1 -Thi omethoxy methyl p seudouri dine

[1347] 1-Methanesulfonylmethylpseudouridine

[1348] 1 -Vinylpseudouridine

[1349] 1 -Allylpseudouridine

[1350] 1 -Homoallylpseudouridine

[1351] 1 -Propargylpseudouridine

[1352] 1 -(4-Fluorobenzyl)pseudouridine

[1353] 1 -(4-Chlorobenzyl)pseudouridine

[1354] 1 -(4-Bromobenzyl)pseudouridine

[1355] 1 -(4-Iodobenzyl)pseudouridine

[1356] l-(4-Methylbenzyl)pseudouridine

[1357] l-(4-Trifluoromethylbenzyl)pseudouridine

[1358] l-(4-Methoxybenzyl)pseudouridine

[1359]

[1360] l-(4-Trifluoromethoxybenzyl)pseudouridineAttorney Docket No.: 45817-0164WO1

[1361] Nucleoside Name

[1362] l-(4-Thiomethoxybenzyl)pseudouridine

[1363] l-(4-Methanesulfonylbenzyl)pseudouridine

[1364] Pseudouridine l-(4-methylbenzoic acid)

[1365] Pseudouridine l-(4-m ethylbenzenesulfonic acid)

[1366] l-(2,4,6-Trimethylbenzyl)pseudouridine

[1367] 1 -(4-Nitrobenzyl)pseudouridine

[1368] 1 -(4-Azidobenzyl)pseudouridine

[1369] l-(3,4-Dimethoxybenzyl)pseudouridine

[1370] l-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine

[1371] 1 -Acetylpseudouridine

[1372] 1 -Tri fluoroacetylpseudouridine

[1373] 1 -Benzoylpseudouridine

[1374] 1 -Pivaloylpseudouridine

[1375] l-(3-Cyclopropyl-prop-2-ynyl)pseudouridine

[1376] Pseudouridine 1-methylphosphonic acid diethyl ester Pseudouridine 1-methylphosphonic acid

[1377] Pseudouridine l-[3-(2-ethoxy)]propionic acid

[1378] 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

[1379] l-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl } pseudouridine l-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine 1 -Biotinylpseudouridine

[1380] 1 -Biotinyl-PEG2 -pseudouridine

[1381] 5-Oxyacetic acid-methyl ester-uridine

[1382] 3-Methyl-pseudo-uridine

[1383] 5-trifluoromethyl-uridine

[1384] 5-methyl-amino-methyl-uridine

[1385] 5-carboxy-methyl-amino-methyl-uridine

[1386] 5 -carb oxy m ethyl aminomethyl -2 ’ -OMe-uri dine

[1387] 5 -carb oxy m ethyl ami nomethyl -2-thi o-uri di ne

[1388] 5-methylaminomethyl-2-thio-uridine

[1389] 5-methoxy-carbonyl-methyl-uridine

[1390] 5-methoxy-carbonyl-methyl-2’-OMe-uridine

[1391] 5-oxyacetic acid-uridine

[1392] 3 -(3 -amino-3 -carboxypropyl )-uridine

[1393]

[1394] 5-(carboxyhydroxymethyl)uridine methyl esterAttorney Docket No.: 45817-0164WO1

[1395] Nucleoside Name

[1396] 5-(carboxyhydroxymethyl)uridine

[1397] 2’-OMe-pseudo-uridine

[1398] 2 ’ - Azi do-2 ’ -deoxy -uri di ne

[1399] 2 ’ - Amino-2 ’ -deoxy -uri dine

[1400] 2 ’-F- 5 -Methyl -2’ -deoxy -uridine

[1401] 5-i odo-2’ -fluoro-deoxyuri dine

[1402] 2’ -bromo-deoxyuridine

[1403] 2,2’-anhydro-uridine

[1404] 2’-Azido-deoxyuridine

[1405] 5-Methoxycarbonylmethyl-2 -thiouridine

[1406] 5 -Methyl ami nomethyl -2-thi ouri dine

[1407] 5-Carbamoylmethyluridine

[1408] 5-Carbamoylmethyl-2'-O-methyluridine

[1409] 1 -Methyl -3 -(3 -amino-3 -carboxypropyl) pseudouridine

[1410] 5-Methylaminomethyl-2-selenouridine

[1411] 5-Carboxymethyluridine

[1412] 5 -Methyl dihy drouri di ne

[1413] 5-Taurinomethyluridine

[1414] 5-Taurinomethyl-2-thiouridine

[1415] 5-(Ao-Pentenylaminomethyl)uridine

[1416] 5-(Ao-Pentenylaminomethyl)- 2-thiouridine

[1417] 5-( / .w-Pentenylami nomethyl )-2'-O-methyluri dine

[1418] 2'-O-Methylpseudouridine

[1419] 2-Thio-2'-O-methyluridine

[1420] 3,2'-O-Dimethyluridine

[1421] 5-Methoxy-carbonylmethyl-uridine

[1422] 5-hydroxy-uridine

[1423]

[1424] 5-Isopentenyl-aminomethyl-uridine

[1425] 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.: 45817-0164WO1 Table 10 - Exemplary Cytosine Containing Nucleosides

[1426] Nucleoside Name

[1427] a-thio-cytidine

[1428] Pseudoisocytidine

[1429] pyrrolo-cytidine

[1430] 5-methyl-cytidine

[1431] N4-acetyl -cytidine

[1432] 5-Bromo-cytidine

[1433] 5-Trifluoromethyl-cytidine

[1434] 5-Hydroxymethyl-cytidine

[1435] 5-Iodo-cytidine

[1436] 5-Ethyl-cytidine

[1437] 5-Methoxy-cytidine

[1438] 5 -Ethy ny 1 -cy ti dine

[1439] 5-Fluoro-cytidine

[1440] 5-Phenyl-cytidine

[1441] N4-Bz-cytidine

[1442] N4-Methyl-cytidine

[1443] 5-Pseudo-iso-cytidine

[1444] 5-Formyl-cytidine

[1445] 5-Aminoallyl-cytidine

[1446] 2'-O-methylcytidine

[1447] 2'-O-M ethy 1 -5 -( 1 -propynyl )cyti dine

[1448] 5-( 1 -Propynyl)ara-cytidine

[1449] 5-Ethynylara-cytidine

[1450] 5 -Ethy ny 1 cy ti dine

[1451] 5-Cyanocytidine

[1452] 5-(2-Chloro-phenyl)-2 -thiocytidine

[1453] 5-(4-Amino-phenyl)-2 -thiocytidine

[1454] N4,2'-O-Dimethylcytidine

[1455] 3 ' -Ethy ny 1 cy ti dine

[1456] 4'-Carbocyclic cytidine

[1457] 4'-Ethynylcytidine

[1458] 4'-Azidocytidine

[1459] 2'-Deoxy-2',2'-difluorocytidine

[1460] 2'-Deoxy-2'-b-fluorocytidine

[1461] 2'-Deoxy-2'-b-chlorocytidine

[1462] 2'-Deoxy-2'-b-bromocytidine

[1463]

[1464] 2'-Deoxy-2'-b-iodocytidineAttorney Docket No.: 45817-0164WO1

[1465] Nucleoside Name

[1466] 5'-Homo-cytidine

[1467] 2'-Deoxy-2'-a-mercaptocytidine

[1468] 2'-Deoxy-2'-a-thiomethoxy cytidine

[1469] 2'-Deoxy-2'-a-azidocytidine

[1470] 2'-Deoxy-2'-a-aminocytidine

[1471] 2'-Deoxy-2'-b-mercaptocytidine

[1472] 2'-Deoxy-2'-b-thiomethoxy cytidine

[1473] 2'-Deoxy-2'-b-azidocytidine

[1474] 2'-Deoxy-2'-b-aminocytidine

[1475] 2'-b-Tri fluoromethylcytidine

[1476] 2'-a-Tri fluoromethylcytidine

[1477] 2'-b-Ethynylcytidine

[1478] 2'-a-Ethynylcytidine

[1479] (E)-5-(2-Bromo-vinyl)cytidine

[1480] 2 ’ - Azi do-2 ’ -deoxy-cy ti di ne

[1481] 2 ’ -Amino-2 ’ -deoxy-cy ti dine

[1482] 5-aminoallyl-cytidine

[1483] 2,2’ -anhydro-cytidine

[1484] N4-amino-cytidine

[1485] 2’-O-Methyl-N4-acetyl-cytidine

[1486] 2’-fluoro-N4-acetyl-cytidine

[1487] 2’-fluor-N4-Bz-cytidine

[1488] 2’-O-methyl-N4-Bz-cytidine

[1489] N4,2'-O-Dimethylcytidine

[1490]

[1491] 5-Formyl-2'-O-methylcytidine

[1492] Alterations on the Internucleoside Linkage

[1493] The alternative nucleotides, which may be incorporated into a polynucleotide molecule, can be altered on the internucleoside linkage (e.g., phosphate backbone).

[1494] 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.

[1495] The alternative nucleosides and nucleotides can include the wholesale replacement of an unaltered phosphate moiety with another intemucleoside linkage asAttorney Docket No.: 45817-0164WO1 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).

[1496] 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 (a), beta (P) or gamma (y) position) can be replaced with a sulfur (thio) and a methoxy.

[1497] 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.

[1498] In specific instances, an alternative nucleoside includes an alpha-thio-nucleoside (e.g., 5'-O-(l-thiophosphate)-adenosine, 5'-O-(l-thiophosphate)-cytidine (a-thio-cytidine), 5'-O-(l-thiophosphate)-guanosine, 5'-O-(l-thiophosphate)-uridine, or 5'-O-(l-thi ophosphate)-p seudouri di ne).

[1499] 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.: 45817-0164WO1 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 intemucleoside linkage. These combinations can include any one or more alterations described herein.

[1500] mRNA: Poly A tail

[1501] 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: 283).

[1502] 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.

[1503] 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 Atail 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.

[1504] 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 Atail); incorporation of stabilizing sequences after the poly A tail (e.g., aAttorney Docket No.: 45817-0164WO1 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.

[1505] 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.

[1506] 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.: 45817-0164WO1 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.

[1507] 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.

[1508] 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.

[1509] 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.: 45817-0164WO1 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.

[1510] 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.

[1511] In some instances, a poly A tail may also be used in the present disclosure to protect against 3 ’-5’ exonuclease digestion.

[1512] 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: 366).

[1513] 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.: 45817-0164WO1 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.

[1514] 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.

[1515] 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.

[1516] 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).

[1517] 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.

[1518] Synthesis of Polynucleotides

[1519] 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.: 45817-0164WO1 (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.

[1520] 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).

[1521] 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.

[1522] 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.

[1523] 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.: 45817-0164WO1 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.

[1524] Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art.

[1525] 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.

[1526] 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.

[1527] 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.: 45817-0164WO1 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.

[1528] When referring to percentage incorporation by an alternative nucleoside, nucleotide, or intemucleoside 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.

[1529] Nanoparticles

[1530] 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.: 45817-0164WO1 reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.

[1531] In some instances, the present application provides pharmaceutical compositions comprising: (a) a delivery agent (e.g., 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 within the delivery agent.

[1532] Lipid Nanoparticles

[1533] 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).

[1534] 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-dehydroepisterol, 7-dehydrositosterol, 20a,22R-dihydroxycholesterol, dinosterol, epibrassicasterol, episterol, ergosterol, ergosterol, fecosterol, fucosterol, fungisterol,Attorney Docket No.: 45817-0164WO1 ganoderenic acid, ganoderic acid, ganoderiol, ganodermadiol, 7a-hydroxycholesterol, 22R-hydroxy cholesterol, 27-hydroxy cholesterol, 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.

[1535] 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;

[1536] PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016000129;

[1537] PCT / US2016 / 014280; PCT / US2016 / 014280; PCT / US2017 / 038426;

[1538] PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 52117;

[1539] PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575 and PCT / US2016 / 069491 all of which are incorporated by reference herein in their entirety.

[1540] 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.

[1541] 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.: 45817-0164WO1 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.

[1542] 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.

[1543] Ionizable Amino Lipids

[1544] 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):

[1545]

[1546] (I) or its N-oxide, or a salt or isomer thereof,

[1547] wherein R’ais R’branched; wherein

[1548] RaaRa7

[1549] R

[1550]

[1551] ’ branched js; RapRas. denotes a point of attachment; wherein Ra“, Ra|3, Ray, and RaSare each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl;

[1552] R2and R3are each independently selected from the group consisting of Ci-i4 alkyl and

[1553] C2-14 alkenyl;

[1554] R4is selected from the group consisting of -(CH2)n0H, wherein n is selected from O. zP

[1555] Rio N \ / X

[1556] the group consisting of 1, 2, 3, 4, and 5, and

[1557]

[1558] H n2Attorney Docket No.: 45817-0164WO1

[1559] wherein

[1560]

[1561] denotes a point of attachment; wherein

[1562] R10is N(R)?; each R is independently selected from the group consisting of Ci-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[1563] each R5is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[1564] each R6is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[1565] M and M’ are each independently selected from the group consisting of -C(O)O-and

[1566] -OC(O)-;

[1567] R’ is a C1-12 alkyl or C2-12 alkenyl;

[1568] 1 is selected from the group consisting of 1, 2, 3, 4, and 5; and

[1569] m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[1570] Ra«RaY

[1571] In some instances, in Formula (I), R’ais R’branched; R

[1572]

[1573] ,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.

[1574] In some instances, in Formula (I), R’ais R’branched; R’branched js

[1575] Ra" Ray

[1576]

[1577] 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.

[1578] In some instances of the compounds of Formula (I), R’ais R’brancbed;

[1579] Ra“ Ray

[1580] R’ branched js

[1581]

[1582] RapRao. denotes apoint of attachment; Ra“ is C2-12Attorney Docket No.: 45817-0164WO1 alkyl; Ra|3, Ray, and RaSare each H; R2and R3are each C1-14 alkyl; R4is

[1583] p 10 N in -

[1584]

[1585] 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.

[1586] In some instances of the compounds of Formula (I), R’ais R,branched;

[1587] Ra" Ray

[1588] R’ branched jspapRas. =s denotes a point of attachment; Raa, Ra^, and RaSare

[1589]

[1590] 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.

[1591] In some instances, the compound of Formula (I) is selected from:

[1592] HO

[1593]

[1594] Attorney Docket No.: 45817-0164WO1 HO

[1595]

[1596] In some instances, the compound of Formula (I) is:

[1597] 0

[1598]

[1599] (Compound 1-2).

[1600] In some instances, the compound of Formula (I) is:

[1601]

[1602] (Compound 1-3).

[1603] In one instance, the ionizable amino lipid is or comprises Compound 1-1.

[1604] Phospholipids

[1605] 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.

[1606] 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.

[1607] 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.: 45817-0164WO1 arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[1608] 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.

[1609] 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).

[1610] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.

[1611] 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), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipal...

Claims

Attorney Docket No.: 45817-0164WO1 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 LAG3, 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.: 281, 264, 257, 21, 1 to 20 or 237-256, 258-263, or 265-280; 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 LAG3 is human or human and cynomolgus LAG3; and / or(iii) the VHH comprises VHH CDR1, VHH CDR2, and VHH CDR3 of any one of the amino acid sequences set forth in SEQ ID NO.: 281, 264, 257, or 21; and / or(iv) the VHH comprises VHH CDR1, VHH CDR2, and VHH CDR3 of the amino acid sequence set forth in SEQ ID NO.: 281 or 264; and / or(v) the VHH-CDR1 comprises the sequence AF(T / P)(L / F)D(Y / W)(A / V), the VHH-CDR2 comprises the sequence set forth in SEQ ID NO: 284; and VHH-CDR3 comprises the sequence set forth in SEQ ID NO: 285; and / or(vi) the VHH binds to an epitope within the D2 domain of LAG3.

2. A polypeptide comprising a variable domain of the heavy chain of a heavy chain-only antibody (VHH) that specifically binds to human LAG3, the VHH comprising VHH complementarity determining region (CDR) 1, VHH CDR2, and VHH CDR3, wherein the VHH binds to amino acids 174, 182, 184-186, 188 (with its associated glycan), 196, 220-223, and 225-229 of human LAG3 (numbering according to SEQ ID NO: 465), optionally wherein the VHH-CDR1 comprises the sequence AF(T / P)(L / F)D(Y / W)(A / V), the VHH-CDR2 comprises the sequence set forth in SEQ ID NO: 284; and VHH-CDR3 comprises the sequence set forth in SEQ ID NO: 285.Attorney Docket No.: 45817-0164WO1 3. 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.

4. The polypeptide of any one of claims 1 to 3, wherein the VHH in bivalent format is a LAG3 agonist.

5. The polypeptide of any one of claims 1 to 4, wherein the VHH in bivalent format binds to human LAG3 with an EC50 of about 0.1 nM to about 25 nM, optionally wherein the VHH in bivalent format binds to human LAG3 with an EC50 of about 0.2 nM to about 5 nM.

6. The polypeptide of any one of claims 1 to 5, further comprising a second VHH that specifically binds to a T cell surface antigen, NK cell antigen, or a tumor associated antigen.

7. The polypeptide of any one of claims 1 to 6, wherein the VHH comprises:(a) the VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:71, the VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:295, and the VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:303;(b) the VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:292, the VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:298, and the VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:293;(c) the VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, the VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:325, and the VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:310;Attorney Docket No.: 45817-0164WO1 (d) the VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 121, the VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:316 and the VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:310;(e) the VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 153, the VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154, and the VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:310;(f) the VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:327, the VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154 and the VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:310;(g) the VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 184, the VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:334, and the VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:310;(h) the VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:331, the VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:337 and the VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:310;(i) the VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:232, the VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:348, and the VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:356; or(j) the VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:232, the VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:352 and the VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:346.

8. The polypeptide of any one of claims 1 to 6, wherein the VHH comprises a VHH CDR1, a VHH CDR2, and VHH CDR3 of any one VHH of Tables 3A, 4A, 5A, 6A or 7A, or wherein the VHH comprises:Attorney Docket No.: 45817-0164WO1 (a) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:71, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:72, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:74;(b) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:22, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:23, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:24;(c) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25, 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;(d) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25, 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;(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;(f) 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:35;(g) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:36, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:23, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:37;(h) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:36, 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:39;Attorney Docket No.: 45817-0164WO1 (i) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:25, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:40, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:41;(j) 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:44;(k) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:45, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:46, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:47;(l) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:48, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:49, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50;(m) 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;(n) 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;(o) 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;(p) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:60, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:61, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:62;Attorney Docket No.: 45817-0164WO1 (q) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:45, 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:47;(r) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:64, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:65, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:66;(s) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:67, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:68, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:69;(t) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:64, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:68, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:70; or(u) a VHH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:71, a VHH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:72, and a VHH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:73.

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 21; 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 any one of the amino acid sequences set forth in SEQ ID NO.: 10, 11, 16, 17 and 20; further 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.: 17.

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 LAG3 than theAttorney Docket No.: 45817-0164WO1 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.

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.

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 bi specific 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 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 polypeptide of any one of claims 1 to 9, or the multispecific antibody of claim 10, further optionally wherein administration is intravenous or subcutaneous; yet further optionally wherein the VHH comprises VHH CDR1, VHH CDR2, and VHH CDR3 of any one of the amino acid sequences set forth in any one of SEQ ID NOs.: 281, 264, 257, or 21; and also optionally wherein the VHH comprises VHH CDR1, VHH CDR2, and VHH CDR3 of the amino acid sequence set forth in SEQ ID NO.: 281 or 264.

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:Attorney Docket No.: 45817-0164WO1 (i) a 5' UTR;(ii) an open reading frame (ORF) comprising a nucleic acid encoding a polypeptide of any one of claims 1 to 9, or the multispecific antibody of claim 10;(iii) a stop codon; and(iv) a 3' UTR, optionally wherein the polynucleotide encodes a nucleic acid encoding signal sequence, wherein the nucleic acid encoding the signal sequence is positioned after the 5’UTR and before the ORF.

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 17 to 19, 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 18 to 22, 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.Attorney Docket No.: 45817-0164WO1 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 areN 1 -methylpseudouracil s.

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, and (iv) guanosine or a modified guanosine, optionally wherein the modified uridine is 1 -methylpseudouridine.

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, and 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 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.

29. A lipid nanoparticle that comprises 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 2000Attorney Docket No.: 45817-0164WO1 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.

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 any one of the amino acid sequences set forth in any one of SEQ ID NOs.: 264, 281, 257, or 21; and also optionally wherein the VHH comprises VHH CDR1, VHH CDR2, and VHH CDR3 of the amino acid sequences set forth in SEQ ID NO.:281 or 264.

31. A method of treating a LAG3 -expressing or dependent cancer 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 sequence set forth in any one of SEQ ID NOs.: 2, 3, 5, 7, 8, 9, 12, 13, 18, or 19; optionally wherein the cancer is a solid tumor or a hematological malignancy.

32. 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 18 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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