SINGLE-DOMA ANTIBODIES AGAINST LAG-3 AND USES THEREOF

Description

FIELD OF THE INVENTION

[0001] The present invention relates to constructs comprising a single-domain antibody (sdAb) that specifically recognize LAG-3, and methods of making and using thereof.BACKGROUND OF THE INVENTION

[0002] Lymphocyte-activation protein 3 (LAG-3), comprised of 503 amino acids, belongs to the Ig superfamily and contains 4 extracellular Ig-like domains, designated D1 to D4. LAG3 is closely related to CD4. LAG-3 is a cell surface protein expressed on activated T cells, NK cells, B cells, and plasmacytoid dendritic cells, and plays a role in the function of these lymphocyte subsets that is important but not completely understood. The LAG-3 protein negatively regulates cellular proliferation, activation, and homeostasis of T cells. LAG-3 also helps maintain CD8 +< T cells in a tolerogenic state. The interaction between LAG-3 and its major ligand, Class II MHC, is thought to play a role in modulating dendritic cell functions. Recent preclinical studies have documented a role for LAG-3 in CD8 T cell exhaustion, and blockade of the LAG-3 / Class II MHC interaction using LAG-3 blocking antibodies or LAG-3-Ig fusion proteins is being evaluated in a number of clinical trials in cancer patients.

[0003] Programmed Cell Death Receptor 1 (PD-1) is another inhibitory immune checkpoint molecule with important negative regulation on T cell functions. T-cell responses can be attenuated by PD-1 signaling when PD-1 binds to Programmed Cell Death Ligand 1 (PD-L1) and / or Programmed Cell Death Ligand 2 (PD-L2), which regulate T-cell receptor (TCR) signaling. Blockade of the PD-1 / PD-L1 axis using antibodies targeting either PD-1 or PD-L1 has been shown to promote tumor-specific T cell immunity with significant clinical benefits to cancer patients. However, there are still huge unmet clinical needs due to resistance or relapse upon PD-1 / PD-L1 blockade.

[0004] WO 2017 / 087589 A2 discloses a single variable domain binding to cell expressing LAG-3 and bispecific anti-LAG3 x anti-PD1 single variable domain constructs. WO 2017 / 015560 A2 discloses human antibodies against LAG3. Linh T. Nguyen et al. (Nature Reviews Immunology, 2014-12-23, vol. 15, no. 1, pages 45-46) disclose a use of blocking agents such as antibodies against PD-1 and LAG-3 signalling in treating cancer. Ruea-Yea Huang et al. (Oncotarget, 2015-07-23, vol. 6, no. 29, pages 27359-27377) disclose a use of antibodies against PD-1 and LAG-3 signalling in treating cancer.BRIEF SUMMARY OF THE INVENTION

[0005] The invention is defined by the claims and any other aspects, configurations, instances or embodiments set forth herein not falling within the scope of the claims are for information only.

[0006] Any references in the description to methods of treatment (or diagnosis) refer to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method of treatment of the human (or animal) body by therapy (or for diagnosis). The present invention is defined by the independent claims. The dependent claims depict additional embodiments of the invention. The present invention relates to anti-LAG-3 constructs comprising an sdAb that specifically recognizes LAG-3 (hereinafter referred to as "anti-LAG-3 sdAb"), such as anti-LAG-3 sdAb, anti-LAG-3 HCAb (e.g., anti-LAG-3 sdAb-Fc fusion protein comprising an anti-LAG-3 sdAb fused to a crystalline fragment (Fc) of human immunoglobulin G (IgG), and multispecific (such as bispecific) antigen binding proteins comprising an anti-LAG-3 sdAb fused to, for example, other sdAbs, a full-length four-chain antibody or antigen binding fragments thereof (e.g., Fab or scFv), and methods of making and using thereof.

[0007] According to claim 1, one aspect of the present application provides an isolated anti-LAG-3 construct comprising a single-domain antibody (sdAb) specifically recognizing LAG-3, wherein the sdAb comprises a VHH domain comprising the amino acid sequence of any one of SEQ ID NOs: 304, 288, 306, 295, or 275-278. Described and not claimed per se is that sdAb comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, or a variant thereof comprising up to about 3 amino acid substitutions. In some embodiments, the isolated anti-LAG-3 construct comprises an sdAb specifically recognizing LAG-3, wherein the sdAb comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228; or a variant thereof comprising up to about 3 amino acid substitutions in the CDR regions.

[0008] Described and not claimed per se is the isolated anti-LAG-3 constructs described above, the sdAb specifically recognizing LAG-3 comprises any one of the following: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 115, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 191, or a variant thereof comprising up to about 3 amino acid substitutions; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 116, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 192, or a variant thereof comprising up to about 3 amino acid substitutions; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 117, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 193, or a variant thereof comprising up to about 3 amino acid substitutions; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 118, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 194, or a variant thereof comprising up to about 3 amino acid substitutions; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 119, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 195, or a variant thereof comprising up to about 3amino acid substitutions; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 120, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 196, or a variant thereof comprising up to about 3 amino acid substitutions; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO: 45, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 121, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 197, or a variant thereof comprising up to about 3 amino acid substitutions; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO: 46, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 122, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 198, or a variant thereof comprising up to about 3 amino acid substitutions; (9) a CDR1 comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 130, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 206, or a variant thereof comprising up to about 3 amino acid substitutions; (10) a CDR1 comprising the amino acid sequence of SEQ ID NO: 61, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 137, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 213, or a variant thereof comprising up to about 3 amino acid substitutions; (11) a CDR1 comprising the amino acid sequence of SEQ ID NO: 70, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 146, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 222, or a variant thereof comprising up to about 3 amino acid substitutions; (12) a CDR1 comprising the amino acid sequence of SEQ ID NO: 72, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 148, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 224, or a variant thereof comprising up to about 3 amino acid substitutions; or (13) a CDR1 comprising the amino acid sequence of SEQ ID NO: 73, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 149, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 225, or a variant thereof comprising up to about 3 amino acid substitutions.

[0009] Described and not claimed per se is any one of the isolated anti-LAG-3 constructs described above, the sdAb specifically recognizing LAG-3 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 39; a CDR2 comprising the amino acid sequence of SEQ ID NO: 115; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 191. Described and not claimed per se is that the sdAb specifically recognizing LAG-3 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 40; a CDR2 comprising the amino acid sequence of SEQ ID NO: 116; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 192. Described and not claimed per se is that the sdAb specifically recognizing LAG-3 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 41; a CDR2 comprising the amino acid sequence of SEQ ID NO: 117; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 193. Described and not claimed per se is that the sdAb specifically recognizing LAG-3 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 42; a CDR2 comprising the amino acid sequence of SEQ ID NO: 118; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 194. Described and not claimed per se is that the sdAb specifically recognizing LAG-3 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 43; a CDR2 comprising the amino acid sequence of SEQ ID NO: 119; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 195. Described and not claimed per se is that the sdAb specifically recognizing LAG-3 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 44; a CDR2 comprising the amino acid sequence of SEQ ID NO: 120; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 196. Described and not claimed per se is that the sdAb specifically recognizing LAG-3 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 45; a CDR2 comprising the amino acid sequence of SEQ ID NO: 121; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 197. Described and not claimed per se is that the sdAb specifically recognizing LAG-3 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 46; a CDR2 comprising the amino acid sequence of SEQ ID NO: 122; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 198. Described and not claimed per se is that the sdAb specifically recognizing LAG-3 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 54; a CDR2 comprising the amino acid sequence of SEQ ID NO: 130; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 206. Described and not claimed per se is that the sdAb specifically recognizing LAG-3 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 61; a CDR2 comprising the amino acid sequence of SEQ ID NO: 137; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 213. Described and not claimed per se is that the sdAb specifically recognizing LAG-3 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 70; a CDR2 comprising the amino acid sequence of SEQ ID NO: 146; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 222. Described and not claimed per se is that the sdAb specifically recognizing LAG-3 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 72; a CDR2 comprising the amino acid sequence of SEQ ID NO: 148; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 224. Described and not claimed per se is that the sdAb specifically recognizing LAG-3 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 73; a CDR2 comprising the amino acid sequence of SEQ ID NO: 149; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 225.

[0010] Described and not claimed per se is an isolated anti-LAG-3 construct comprising an sdAb specifically recognizing LAG-3, wherein the sdAb comprises CDR1, CDR2, and CDR3 of any one of SEQ ID NOs: 274-311.

[0011] Described and not claimed per se is any one of the isolated anti-LAG-3 constructs described above, the sdAb comprises V H H domain comprising: (1) an FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1-38, or a variant thereof comprising up to about 3 amino acid substitutions; (2) an FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 77-114, or a variant thereof comprising up to about 3 amino acid substitutions; (3) an FR3 comprising the amino acid sequence of any one of SEQ ID NOs: 153-190, or a variant thereof comprising up to about 3 amino acid substitutions; and / or (4) an FR4 comprising the amino acid sequence of any one of SEQ ID NOs: 229-266, or a variant thereof comprising up to about 3 amino acid substitutions.

[0012] Described and not claimed per se is any one of the isolated anti-LAG-3 constructs described above, the sdAb comprises a V H H domain comprising the amino acid sequence having at least about 80% sequence identify to any one of SEQ ID NOs: 274-311. Described and not claimed per se is that the sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 274, 279-287, 289- 294, 296-303, 305, and 307-311, or a variant thereof comprising up to about 3 amino acid substitutions in the V H H domain. Described and not claimed per se is that the sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 274, 279-287, 289- 294, 296-303, 305, and 307-311.

[0013] In some embodiments according to any one of the isolated anti-LAG-3 constructs described above, the K D of the binding between the sdAb and LAG-3 is about 10 -7< M to about 10 -12< M. In some embodiments, the K D of the binding between the sdAb and LAG-3 is about 10 -9< M to about 10 -11< M.

[0014] In some embodiments according to any one of the isolated anti-LAG-3 constructs described above, the sdAb cross-reacts with a LAG-3 from a non-human mammal. Described and not claimed per se is that the sdAb specifically recognizing LAG-3 is camelid, chimeric, or partially humanized.

[0015] In some embodiments according to any one of the isolated anti-LAG-3 constructs described above, the isolated anti-LAG-3 construct is a heavy chain-only antibody (HCAb) comprising the sdAb specifically recognizing LAG-3 fused to an Fc fragment. In some embodiments, the HCAb is monomeric or dimeric. In some embodiments, the Fc fragment is a human IgG1 (hIgG1) Fc, effectorless (inert) hIgG1 Fc, hIgG4 Fc, or hIgG4 Fc (S228P). In some embodiments, the sdAb is fused to the Fc fragment via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. In some embodiments, the HCAb comprises the amino acid sequence of any one of SEQ ID NOs: 313-316, 326, 333, 342, and 344. Described and not claimed per se is any one of SEQ ID NOs: 312, 317-325, 327-332, 334-341, 343, and 345-349.

[0016] In some embodiments according to any one of the isolated anti-LAG-3 constructs described above, the anti-LAG-3 construct comprises: (a) a first antigen binding portion comprising the sdAb specifically recognizing LAG-3; and (b) a second antigen binding portion that specifically recognizes a second epitope. In some embodiments, the second antigen binding portion comprises a full-length antibody, a Fab, a Fab', a (Fab') 2 , an Fv, a single chain Fv (scFv), an scFv-scFv, a minibody, a diabody, or a second sdAb. In some embodiments, the anti-LAG-3 construct is multispecific (such as bispecific). In some embodiments, the first antigen binding portion and the second antigen binding portion are fused to each other via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. In some embodiments, the N-terminus or the C-terminus of the anti-LAG-3 sdAb is fused to a peptide sequence. In some embodiments, the peptide sequence comprises the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. In some embodiments, the second antigen binding portion comprises a full-length antibody consisting of two heavy chains and two light chains. In some embodiments, the Fc fragment of the full-length antibody is human IgG1 (hIgG1) Fc, effectorless hIgG1 Fc, hIgG4 Fc, or hIgG4 Fc (S228P). In some embodiments, the N-terminus of the sdAb specifically recognizing LAG-3 is fused to the C-terminus of a heavy chain of the full-length antibody. In some embodiments, the C-terminus of the sdAb specifically recognizing LAG-3 is fused to the N-terminus of a heavy chain of the full-length antibody. In some embodiments, the N-terminus of the sdAb specifically recognizing LAG-3 is fused to the C-terminus of a light chain of the full-length antibody. In some embodiments, the C-terminus of the sdAb specifically recognizing LAG-3 is fused to the N-terminus of a light chain of the full-length antibody. In some embodiments, the second antigen binding portion specifically recognizes an immune checkpoint molecule selected from the group consisting of PD-1, 4-1BB, PD-L1, TIM-3, TIGIT, CTLA-4, VISTA, B7-1, B7-H3, CD47, OX40 and GITR. In some embodiments, the second antigen binding portion specifically recognizes PD-1. In some embodiments, the second antigen binding portion comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 356, and LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 357. In some embodiments, the second antigen binding portion comprises HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 373 and LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 374. In some embodiments, the second antigen binding portion is a full-length antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 356, and a light chain comprising the amino acid sequence of SEQ ID NO: 357. In some embodiments, the second antigen binding portion is a full-length antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 373, and a light chain comprising the amino acid sequence of SEQ ID NO: 374.

[0017] Further provided is an isolated anti-LAG-3 construct that specifically binds to LAG-3 competitively with any one of the isolated anti-LAG-3 constructs described above.

[0018] According to claim 6, further provided is a pharmaceutical composition comprising any one of the isolated anti-LAG-3 constructs described above, and a pharmaceutically acceptable carrier.

[0019] According to claim 7, another aspect of the present application provides an effective amount of any one of the pharmaceutical compositions described above for use in a method of treating an individual having a LAG-3-related disease, wherein said LAG-3-related disease is cancer, in particular colon cancer, comprising administering to the individual the effective amount of the pharmaceutical composition, and wherein anti-LAG-3 construct comprises (a) a first antigen binding portion comprising the sdAb specifically recognizing LAG-3; and (b) a second antigen binding portion that specifically recognizes a second epitope, wherein the second antigen binding portion is a full-length antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 373, and a light chain comprising the amino acid sequence of SEQ ID NO: 374. In some embodiments, the individual is a human.

[0020] According to claims 8-10, further provided is an isolated nucleic acid encoding any one of the isolated anti-LAG-3 constructs described above, a vector comprising the isolated nucleic acid, or an isolated host cell comprising the isolated nucleic acid or the vector. According to claim 12, provided an in vitro method of producing any one of isolated anti-LAG-3 constructs described above, comprising culturing the isolated host cell comprising any one of the isolated nucleic acids or vectors described above, or culturing any one of the isolated host cells described above, under conditions effective to express the encoded anti-LAG-3 construct; and obtaining the expressed anti-LAG-3 construct from the isolated host cell.

[0021] According to claim 11, also provided a kit comprising the anti-LAG-3 construct described above, the pharmaceutical composition described above, the nucleic acid described above, the vector described above, or the host cell described above.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 depicts the evaluation of immune response against LAG3-Fc or Fc alone for pre-immune serum, and post-immune serum after final boost. FIG. 2 depicts the evaluation of immune response against LAG3-Fc or Fc alone for regular antibodies (IgG1) and heavy chain antibodies (IgG2 and IgG3) in post-immune serum after final boost. Corresponding immunoglobulin fragments isolated from pre-immune serum were used as controls. FIG. 3 depicts epitope a binning test for non-humanized anti-LAG-3 sdAb-Fc fusion proteins and the benchmark antibody, BMS-986016. FIG. 4 depicts the binding of non-humanized anti-LAG-3 sdAb-Fc fusion proteins to CHO-K1 cells expressing human LAG-3, tested with FACS. BMS-986016 was used as a positive control. FIG. 5 depicts the blocking of interaction between MHC II and human LAG-3 by non-humanized anti-LAG-3 sdAb-Fc fusion proteins, as measured by flow cytometry. BMS-986016 was used as a positive control. FIG. 6 depicts the in vitro functional blockade of LAG-3 by humanized anti-LAG-3 sdAb-Fc fusion proteins, measured with a reporter assay. BMS-986016 was used as a positive control and human IgG4 isotype control as a negative control. FIG. 7 depicts a schematic structure of an exemplary BABP comprising a monospecific full-length antibody having two identical heavy chains and two identical light chains, and two identical anti-LAG-3 sdAbs, wherein the C-terminus of each anti-LAG-3 sdAb is fused to the N-terminus of one heavy chain via an optional peptide linker. An optional peptide sequence can be fused to the N-terminus of each anti-LAG-3 sdAb. Each of the two anti-LAG-3 sdAbs specifically binds a first epitope (LAG-3). The full-length antibody has two antigen binding sites, each specifically binding a second epitope. For example, the BABP can consist of four polypeptide chains with structures from the N-terminus to the C-terminus as follows: (1) V L -C L ; (2) V H H-V H -C H 1-C H 2-C H 3; (3) V H H-V H -C H 1-C H 2-C H 3; and (4) V L -C L , wherein V H and V L of polypeptide chains (1) and (2) form an antigen binding site that specifically binds a first copy of the second epitope, V H and V L of polypeptide chains (3) and (4) form an antigen binding site that specifically binds a second copy of the second epitope, and each V H H specifically binds a copy of the first epitope (LAG-3). In alternative formats, each anti-LAG-3 sdAb may be omitted, or replaced with two identical or different anti-LAG-3 sdAbs fused to each other. The monospecific full-length antibody may be replaced with a bispecific full-length antibody to further expand binding specificity. FIG. 8 depicts a schematic structure of an exemplary BABP comprising a monospecific full-length antibody having two identical heavy chains and two identical light chains, and two identical anti-LAG-3 sdAbs, wherein the N-terminus of each anti-LAG-3 sdAb is fused to the C-terminus of one heavy chain via an optional peptide linker. An optional peptide sequence can be fused to the C-terminus of each anti-LAG-3 sdAb. Each of the two anti-LAG-3 sdAbs specifically binds a first epitope (LAG-3). The full-length antibody has two antigen binding sites, each specifically binding a second epitope. For example, the BABP can consist of four polypeptide chains with structures from the N-terminus to the C-terminus as follows: (1) V L -C L ; (2) V H -C H 1-C H 2-C H 3-V H H; (3) V H -C H 1-C H 2-C H 3-V H H; and (4) V L -C L , wherein V H and V L of polypeptide chains (1) and (2) form an antigen binding site that specifically binds a first copy of the second epitope, V H and V L of polypeptide chains (3) and (4) form an antigen binding site that specifically binds a second copy of the second epitope, and each V H H specifically binds a copy of the first epitope (LAG-3). In alternative formats, each anti-LAG-3 sdAb may be omitted, or replaced with two identical or different anti-LAG-3 sdAbs fused to each other. The monospecific full-length antibody may be replaced with a bispecific full-length antibody to further expand binding specificity. FIG. 9 depicts a schematic structure of an exemplary BABP comprising a monospecific full-length antibody having two identical heavy chains and two identical light chains, and two identical anti-LAG-3 sdAbs, wherein the C-terminus of each anti-LAG-3 sdAb is fused to the N-terminus of one light chain via an optional peptide linker. An optional peptide sequence can be fused to the N-terminus of each anti-LAG-3 sdAb. Each of the two anti-LAG-3 sdAbs specifically binds a first epitope (LAG-3). The full-length antibody has two antigen binding sites, each specifically binding a second epitope. For example, the BABP can consist of four polypeptide chains with structures from the N-terminus to the C-terminus as follows: (1) V H H-V L -C L ; (2) V H -C H 1-C H 2-C H 3; (3) V H -C H 1-C H 2-C H 3; and (4) V H H-V L -C L , wherein V H and V L of polypeptide chains (1) and (2) form an antigen binding site that specifically binds a first copy of the second epitope, V H and V L of polypeptide chains (3) and (4) form an antigen binding site that specifically binds a second copy of the second epitope, and each V H H specifically binds a copy of the first epitope (LAG-3). In alternative formats, each anti-LAG-3 sdAb may be omitted, or replaced with two identical or different anti-LAG-3 sdAbs fused to each other. The monospecific full-length antibody may be replaced with a bispecific full-length antibody to further expand binding specificity. FIG. 10 depicts a schematic structure of an exemplary BABP comprising a monospecific full-length antibody having two identical heavy chains and two identical light chains, and two identical anti-LAG-3 sdAbs, wherein the N-terminus of each anti-LAG-3 sdAb is fused to the C-terminus of one light chain via an optional peptide linker. An optional peptide sequence can be fused to the C-terminus of each anti-LAG-3 sdAb. Each of the two anti-LAG-3 sdAbs specifically binds a first epitope. The full-length antibody has two antigen binding sites, each specifically binding a second epitope. For example, the BABP can consist of four polypeptide chains with structures from the N-terminus to the C-terminus as follows: (1) V L -C L -V H H; (2) V H -C H 1-C H 2-C H 3; (3) V H -C H 1-C H 2-C H 3; and (4) V L -C L -V H H, wherein V H and V L of polypeptide chains (1) and (2) form an antigen binding site that specifically binds a first copy of the second epitope, V H and V L of polypeptide chains (3) and (4) form an antigen binding site that specifically binds a second copy of the second epitope, and each V H H specifically binds a copy of the first epitope (LAG-3). In alternative formats, each anti-LAG-3 sdAb may be omitted, or replaced with two identical or different anti-LAG-3 sdAbs fused to each other. The monospecific full-length antibody may be replaced with a bispecific full-length antibody to further expand binding specificity. FIG. 11 depicts a schematic structure of an exemplary BABP comprising a monospecific full-length antibody having two identical heavy chains and two identical light chains, and four identical anti-LAG-3 sdAbs, wherein the C-terminus of each anti-LAG-3 sdAb is fused to the N-terminus of a heavy chain or a light chain of the monospecific full-length antibody via an optional peptide linker. An optional peptide sequence can be fused to the N-terminus of each anti-LAG-3 sdAb. Each anti-LAG-3 sdAb specifically binds to a first epitope (LAG-3). The full-length antibody has two antigen binding sites that each specifically binds a second epitope. For example, the BABP can consist of four polypeptide chains with structures from the N-terminus to the C-terminus as follows: (1) V H H-V L -C L ; (2) V H H-V H -C H 1-C H 2-C H 3; (3) V H H-V H -C H 1-C H 2-C H 3; and (4) V H H-V L -C L , wherein V H and V L of polypeptide chains (1) and (2) form an antigen binding site that specifically binds a first copy of the second epitope, V H and V L of polypeptide chains (3) and (4) form an antigen binding site that specifically binds a second copy of the second epitope, and each V H H specifically binds a copy of the first epitope (LAG-3). In alternative formats, each anti-LAG-3 sdAb may be omitted, or replaced with two identical or different anti-LAG-3 sdAbs fused to each other. The monospecific full-length antibody may be replaced with a bispecific full-length antibody to further expand binding specificity. FIG. 12 depicts a schematic structure of an exemplary BABP comprising a monospecific full-length antibody having two identical heavy chains and two identical light chains, and four identical anti-LAG-3 sdAbs, wherein fused to the N-terminus of each heavy chain are two identical anti-LAG-3 sdAbs, the two anti-LAG-3 sdAbs being fused to each other via an optional peptide linker, and the two anti-LAG-3 sdAbs are fused to the N-terminus of each heavy chain via an optional peptide linker. An optional peptide sequence can be fused to the N-terminus of the chimeric heavy chain. Each anti-LAG-3 sdAb specifically binds a first epitope (LAG-3). The full-length antibody has two antigen binding sites that each specifically binds a second epitope. For example, the BABP can consist of four polypeptide chains with structures from the N-terminus to the C-terminus as follows: (1) V L -C L ; (2) V H H-V H H-V H -C H 1-C H 2-C H 3; (3) V H H-V H H-V H -C H 1-C H 2-C H 3; and (4) V L -C L , wherein V H and V L of polypeptide chains (1) and (2) form an antigen binding site that specifically binds a first copy of the second epitope, V H and V L of polypeptide chains (3) and (4) form an antigen binding site that specifically binds a second copy of the second epitope, and each V H H specifically binds a copy of the first epitope (LAG-3). In alternative formats, each anti-LAG-3 sdAb may be omitted, or replaced with two identical or different anti-LAG-3 sdAbs fused to each other. The monospecific full-length antibody may be replaced with a bispecific full-length antibody to further expand binding specificity. FIG. 13 depicts a schematic structure of an exemplary BABP comprising two identical antigen-binding (Fab) fragments, two identical anti-LAG-3 sdAbs, and an Fc region, wherein the N-terminus of each anti-LAG-3 sdAb is fused to the C-terminus of the C H 1 region of the Fab fragment via an optional peptide linker and the C-terminus of each anti-LAG-3 sdAb is fused to the N-terminus of the C H 2 region of the Fc region. Each anti-LAG-3 sdAb specifically binds a first epitope (LAG-3). Each Fab fragment specifically binds a second epitope. For example, the BABP can consist of four polypeptide chains with structures from the N-terminus to the C-terminus as follows: (1) V L -C L ; (2) V H -C H 1-V H H-C H 2-C H 3; (3) V H -C H 1-V H H-C H 2-C H 3; and (4) V L -C L , wherein V H and V L of polypeptide chains (1) and (2) form an antigen binding site that specifically binds a first copy of the second epitope, V H and V L of polypeptide chains (3) and (4) form an antigen binding site that specifically binds a second copy of the second epitope, and each V H H specifically binds a copy of the first epitope (LAG-3). In alternative formats, each anti-LAG-3 sdAb may be omitted, or replaced with two identical or different anti-LAG-3 sdAbs fused to each other. In alternative formats, to expand specificity, the two Fab fragments can specifically bind different epitopes, and / or the V H H fragments can specifically bind different epitopes. FIG. 14 depicts a schematic structure of an exemplary BABP comprising two identical single chain variable fragments (scFvs), two identical anti-LAG-3 sdAbs, and an Fc region, wherein the N-terminus of each anti-LAG-3 sdAb is fused to the C-terminus of an scFv via an optional peptide linker and the C-terminus of each anti-LAG-3 sdAb is fused to the N-terminus of the Fc region. Each anti-LAG-3 sdAb specifically binds a first epitope (LAG-3). Each scFv specifically binds a second epitope. For example, the BABP can consist of two polypeptide chains each with a structure from the N-terminus to the C-terminus as follows: V L -V H -V H H-C H 2-C H 3, wherein V H and V L of each polypeptide chain form a scFv domain that specifically binds a copy of the second epitope, and each V H H specifically binds a copy of the first epitope (LAG-3). In alternative formats, the scFv domain can comprise from the N-terminus to the C-terminus: V H -V L . In alternative formats, each anti-LAG-3 sdAb may be omitted, or replaced with two identical or different anti-LAG-3 sdAbs fused to each other. Additionally, to expand specificity, the two scFvs can specifically bind different epitopes, and / or the V H H fragments can specifically bind different epitopes. FIG. 15 depicts a schematic structure of an exemplary BABP comprising two identical Fab fragments, two identical Fab-like fragments each comprising two V H H fragments, and an Fc region. In each Fab-like fragment, the V H and V L regions are each replaced by an anti-LAG-3 sdAb. Each Fab-like fragment specifically binds a first epitope (LAG-3). Each Fab fragment specifically binds a second epitope. For example, the BABP can consist of four polypeptide chains with structures from the N-terminus to the C-terminus as follows: (1) V L -C L -V H H-C L ; (2) V H -C H 1-V H H-C H 1-C H 2-C H 3; (3) V H -C H 1-V H H-C H 1-C H 2-C H 3; and (4) V L -C L -V H H-C L , wherein V H and V L of polypeptide chains (1) and (2) forms an antigen binding site that specifically binds a first copy of the second epitope, V H and V L of polypeptide chains (3) and (4) forms an antigen binding site that specifically binds a second copy of the second epitope, and each V H H specifically binds a copy of the first epitope (LAG-3). In alternative formats, to expand specificity, the two Fab fragments can specifically bind different epitopes, and / or the Fab-like fragments can specifically bind different epitopes (e.g., different epitopes from LAG-3). FIG. 16 depicts a schematic structure of an exemplary BABP comprising two identical scFvs, two identical Fab-like fragments each comprising two V H H fragments, and an Fc region. In each Fab-like fragment, the V H and V L regions are each replaced by an anti-LAG-3 sdAb. Each Fab-like fragment specifically binds a first epitope (LAG-3). Each scFv specifically binds a second epitope. For example, the BABP can consist of four polypeptide chains with structures from the N-terminus to the C-terminus as follows: (1) V H H-C L ; (2) V L -V H -V H H-C H 1-C H 2-C H 3; (3) V L -V H -V H H-C H 1-C H 2-C H 3; and (4) V H H-C L , wherein V H and V L of polypeptide chains (2) and (3) each forms an scFv that specifically binds a copy of the second epitope, and each V H H specifically binds a copy of the first epitope (LAG-3). In alternative formats, the C-terminus of the scFv may be fused to the N-terminus of the chain in the Fab-like fragment comprising V H H-C L ; and / or the scFv domain can comprise from the N-terminus to the C-terminus: V H -V L . Additionally, to expand specificity, the two scFvs can specifically bind different epitopes, and / or the V H H fragments can specifically bind different epitopes (e.g., different epitopes from LAG-3). FIGs. 17A-17B depict in vitro activities of PD-1×LAG-3 BABPs in a PD-1 cell based reporter assay (FIG. 17A) and a LAG-3 blockade reporter assay (FIG. 17B). Pembrolizumab and PD1-BM-min were used as controls for PD-1 cell based reporter assay and BMS-986016 was used as a control for LAG-3 blockade reporter assay. FIGs. 18A-18B depict in vivo efficacy of PD-1×LAG-3 BLP-4 in C57BL / 6 human PD-1 / LAG-3 double knock-in mice bearing MC38 tumor, as compared to parental elements of BLP-4 (i.e., PD1-BM-min and AS20846-Fc) and combination thereof. FIG. 18A shows average tumor volume in each treatment group. FIG. 18B shows spider plots of tumor volume of each animal. IgG4 was used as a negative control. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides novel single-domain antibodies (sdAbs) specifically recognizing LAG-3 (i.e., anti-LAG-3 sdAb), and constructs comprising the anti-LAG-3 sdAbs. The anti-LAG3 sdAbs described herein have strong binding affinity and are capable of cross-reacting with LAG-3 of a non-human mammal, such as a cynomolgus monkey. The anti-LAG3 constructs described herein are useful for treating LAG-3-related diseases such as cancer.

[0024] Accordingly, according to claim 1, one aspect of the present application provides an isolated anti-LAG-3 construct comprising an sdAb specifically recognizing LAG-3, whrein the sdAb comprises a VHH domain comprising the amino acid sequence of any one of SEQ ID NOs: 304, 288, 306, 295, or 275-278. The isolated anti-LAG-3 construct can be, for example, an anti-LAG-3 sdAb, a polypeptide comprising a plurality of anti-LAG-3 sdAbs fused together, an anti-LAG-3 sdAb-Fc fusion protein comprising an anti-LAG-3 sdAb fused to an Fc fragment (e.g., a human IgG1 Fc, effectorless IgG1 Fc, IgG2 Fc, or IgG4 Fc), or a multispecific antigen binding protein ("MABP") comprising an anti-LAG-3 sdAb fused to a full-length antibody (such as anti-PD-1 antibody) or an antigen binding fragment thereof. The anti-LAG-3 construct can be monospecific or multispecific (such as bispecific), monovalent or multivalent (such as bivalent).

[0025] According to claims 6, 7, 11 and 12, also provided are a pharmaceutical composition, and a kit comprising the anti-LAG-3 constructs described herein, a method of making thereof, and a pharmaceutical compositions for use in treating LAG-3-related cancer using the anti-LAG-3 constructs described herein.I. Definitions

[0026] The term "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0027] As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival. Also encompassed by "treatment" is a reduction of pathological consequence of cancer. The methods of the invention contemplate any one or more of these aspects of treatment.

[0028] The term "prevent," and similar words such as "prevented," "preventing" etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the recurrence of, a disease or condition, e.g., cancer. It also refers to delaying the recurrence of a disease or condition or delaying the recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also includes reducing the intensity, effect, symptoms and / or burden of a disease or condition prior to recurrence of the disease or condition.

[0029] As used herein, "delaying" the development of cancer means to defer, hinder, slow, retard, stabilize, and / or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. A method that "delays" development of cancer is a method that reduces probability of disease development in a given time frame and / or reduces the extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of individuals. Cancer development can be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT Scan), Magnetic Resonance Imaging (MRI), abdominal ultrasound, clotting tests, arteriography, or biopsy. Development may also refer to cancer progression that may be initially undetectable and includes occurrence, recurrence, and onset.

[0030] The term "effective amount" used herein refers to an amount of an agent or a combination of agents, sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and / or delay one or more of its symptoms. In reference to cancer, an effective amount comprises an amount sufficient to cause a tumor to shrink and / or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. The effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; and / or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

[0031] As used herein, an "individual" or a "subject" refers to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0032] The term "antibody" is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. The term "antibody" includes conventional 4-chain antibodies, single-domain antibodies, and antigen-binding fragments thereof.

[0033] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen-binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (V H ) followed by three constant domains (C H ) for each of the α and γ chains and four C H domains for µ and ε isotypes. Each L chain has at the N-terminus, a variable domain (V L ) followed by a constant domain at its other end. The V L is aligned with the V H and the C L is aligned with the first constant domain of the heavy chain (C H 1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a V H and V L together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (C H ), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated α, δ, ε, γ and µ, respectively. The γ and α classes are further divided into subclasses on the basis of relatively minor differences in the C H sequence and function, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.

[0034] The term "heavy chain-only antibody" or "HCAb" refers to a functional antibody, which comprises heavy chains, but lacks the light chains usually found in 4-chain antibodies. Camelid animals (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0035] The term "single-domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three complementary determining regions (CDRs). The sdAb alone is capable of binding to the antigen without pairing with a corresponding CDR-containing polypeptide. In some cases, single-domain antibodies are engineered from camelid HCAbs, and their heavy chain variable domains are referred herein as "V H Hs" (Variable domain of the heavy chain of the Heavy chain antibody). Camelid sdAb is one of the smallest known antigen-binding antibody fragments (see, e.g., Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)). A basic V H H has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3.

[0036] An "isolated" antibody (or construct) is one that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie Blue or, preferably, silver stain. Isolated antibody (or construct) includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated polypeptide, antibody, or construct will be prepared by at least one purification step.

[0037] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as "V H " and "V L ", respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites. Heavy-chain only antibodies from the Camelid species have a single heavy chain variable region, which is referred to as "V H H". V H H is thus a special type of V H .

[0038] The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions (HVRs) both in the heavy chain and light chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0039] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[0040] The terms "full-length antibody", "intact antibody", or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, full-length 4-chain antibodies include those with heavy and light chains including an Fc region. Full-length heavy-chain only antibodies include the heavy chain variable domain (such as V H H) and an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions.

[0041] An "antibody fragment" or "antigen-binding fragment" comprises a portion of an intact antibody, preferably the antigen binding and / or the variable region of the intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody (scFv) molecules; single-domain antibodies (such as V H H), and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produced two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable domain of the H chain (V H ), and the first constant domain of one heavy chain (C H 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab') 2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy-terminus of the C H 1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0042] The term "constant domain" refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the C H 1, C H 2 and C H 3 domains (collectively, C H ) of the heavy chain and the CHL (or C L ) domain of the light chain.

[0043] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0044] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0045] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the V H and V L antibody domains connected into a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the scFv to form the desired structure for antigen binding. For a review of the scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0046] The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the V H and V L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, thereby resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the V H and V L domains of the two antibodies are present on different polypeptide chains. Diabodies are described in greater detail in, for example, EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

[0047] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). "Humanized antibody" is used as a subset of "chimeric antibodies".

[0048] "Humanized" forms of non-human (e.g., llama or camelid) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an CDR (hereinafter defined) of the recipient are replaced by residues from an CDR of a non-human species (donor antibody) such as mouse, rat, rabbit, camel, llama, alpaca, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0049] A "human antibody" is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE ™< technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

[0050] The term "hypervariable region," "HVR," or "HV," when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally, single-domain antibodies comprise three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 (or CDR3) displays the most diversity of the three HVRs, and is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0051] The term "Complementarity Determining Region" or "CDR" are used to refer to hypervariable regions as defined by the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0052] A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below in Table 1. Table 1. HVR delineations. LoopKabatAbMChothiaContactL1L24-L34L24-L34L26-L32L30-L36L2L50-L56L50-L56L50-L52L46-L55L3L89-L97L89-L97L91-L96L89-L96H1H31-H35BH26-H35BH26-H32H30-H35B(Kabat Numbering)H1H31-H35H26-H35H26-H32H30-H35(Chothia Numbering)H2H50-H65H50-H58H53-H55H47-H58H3H95-H102H95-H102H96-H101H93-H101

[0053] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the V L and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the V H . The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0054] The amino acid residues of a single-domain antibody (such as V H H) are numbered according to the general numbering for V H domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to V H H domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195. According to this numbering, FR1 of a V H H comprises the amino acid residues at positions 1-30, CDR1 of a V H H comprises the amino acid residues at positions 31-35, FR2 of a V H H comprises the amino acids at positions 36-49, CDR2 of a V H H comprises the amino acid residues at positions 50-65, FR3 of a V H H comprises the amino acid residues at positions 66-94, CDR3 of a V H H comprises the amino acid residues at positions 95-102, and FR4 of a V H H comprises the amino acid residues at positions 103-113. In this respect, it should be noted that-as is well known in the art for V H domains and for V H H domains-the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering).

[0055] The expression "variable-domain residue-numbering as in Kabat" or "amino-acid-position numbering as in Kabat," and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.

[0056] Unless indicated otherwise herein, the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra. The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0057] "Framework" or "FR" residues are those variable-domain residues other than the HVR residues as herein defined.

[0058] A "human consensus framework" or "acceptor human framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin V L or V H framework sequences. Generally, the selection of human immunoglobulin V L or V H sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Examples include for the V L , the subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al., supra. Additionally, for the VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al. Alternatively, a human consensus framework can be derived from the above in which particular residues, such as when a human framework residue is selected based on its homology to the donor framework by aligning the donor framework sequence with a collection of various human framework sequences. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0059] An "affinity-matured" antibody is one with one or more alterations in one or more CDRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alteration(s). In some embodiments, an affinity-matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describes affinity maturation by V H - and V L -domain shuffling. Random mutagenesis of CDR and / or framework residues is described by, for example: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0060] As used herein, the term "specifically binds," "specifically recognizes," or is "specific for" refers to measurable and reproducible interactions such as binding between a target and an antigen binding protein (such as an sdAb), which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antigen binding protein (such as an sdAb) that specifically binds a target (which can be an epitope) is an antigen binding protein (such as an sdAb) that binds this target with greater affinity, avidity, more readily, and / or with greater duration than it binds other targets. In some embodiments, the extent of binding of an antigen binding protein (such as an sdAb) to an unrelated target is less than about 10% of the binding of the antigen binding protein (such as an sdAb) to the target as measured, e.g., by a radioimmunoassay (RIA). In some embodiments, an antigen binding protein (such as an sdAb) that specifically binds a target has a dissociation constant (K D ) of ≤10 -5< M, ≤10 -6< M, ≤10 -7< M, ≤10 -8< M, ≤10 -9< M, ≤10 -10< M, ≤10 -11< M, or ≤10 -12< M. In some embodiments, an antigen binding protein specifically binds an epitope on a protein that is conserved among the protein from different species. In some embodiments, specific binding can include, but does not require exclusive binding. Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE ™< -tests and peptide scans.

[0061] The term "specificity" refers to selective recognition of an antigen binding protein (such as an sdAb) for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term "multispecific" as used herein denotes that an antigen binding protein has polyepitopic specificity (i.e., is capable of specifically binding to two, three, or more, different epitopes on one biological molecule or is capable of specifically binding to epitopes on two, three, or more, different biological molecules). "Bispecific" as used herein denotes that an antigen binding protein has two different antigen-binding specificities. Unless otherwise indicated, the order in which the antigens bound by a bispecific antibody listed is arbitrary. That is, for example, the terms "anti-LAG-3 / PD-1," "anti-PD-1 / LAG-3," "LAG-3×PD-1" and "PD-1×LAG-3" may be used interchangeably to refer to bispecific antibodies that specifically bind to both LAG-3 and PD-1. The term "monospecific" as used herein denotes an antigen binding protein that has one or more binding sites each of which bind the same epitope of the same antigen.

[0062] The term "valent" as used herein denotes the presence of a specified number of binding sites in an antigen binding protein. A natural antibody for example or a full-length antibody has two binding sites and is bivalent. As such, the terms "trivalent", "tetravalent", "pentavalent" and "hexavalent" denote the presence of two binding site, three binding sites, four binding sites, five binding sites, and six binding sites, respectively, in an antigen binding protein.

[0063] "Antibody effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptors); and B cell activation. "Reduced or minimized" antibody effector function means that which is reduced by at least 50% (alternatively 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) from the wild type or unmodified antibody. The determination of antibody effector function is readily determinable and measurable by one of ordinary skill in the art. In a preferred embodiment, the antibody effector functions of complement binding, complement dependent cytotoxicity and antibody dependent cytotoxicity are affected. In some embodiments, effector function is eliminated through a mutation in the constant region that eliminated glycosylation, e.g., "effectorless mutation." In one aspect, the effectorless mutation is an N297A or DANA mutation (D265A+N297A) in the C H 2 region. Shields et al., J. Biol. Chem. 276 (9): 6591-6604 (2001). Alternatively, additional mutations resulting in reduced or eliminated effector function include: K322A and L234A / L235A (LALA). Alternatively, effector function can be reduced or eliminated through production techniques, such as expression in host cells that do not glycosylate (e.g., E. coli.) or in which result in an altered glycosylation pattern that is ineffective or less effective at promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5): 3466-3473 (2003).

[0064] "Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are required for killing of the target cell by this mechanism. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. Fc expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS USA 95:652-656 (1998).

[0065] The term "Fc region" or "fragment crystallizable region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0066] "Fc receptor" or "FcR" describes a receptor that binds the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See M. Daëron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

[0067] The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus. Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994). Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18: (12): 592-8 (1997); Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton et al., J. Biol. Chem. 279 (8): 6213-6 (2004); WO 2004 / 92219 (Hinton et al.). Binding to FcRn in vivo and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides having a variant Fc region are administered. WO 2004 / 42072 (Presta) describes antibody variants which improved or diminished binding to FcRs. See also, e.g., Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0068] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), may be performed. Antibody variants with altered Fc region amino acid sequences and increased or decreased C1q binding capability are described in U.S. Pat. No. 6,194,551B1 and WO99 / 51642. See, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0069] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair. Binding affinity can be indicated by K d , K off , K on , or K a . The term "K off ", as used herein, is intended to refer to the off rate constant for dissociation of an antibody (or antigen-binding domain) from the antibody / antigen complex, as determined from a kinetic selection set up, expressed in units of s -1< . The term "K on ", as used herein, is intended to refer to the on rate constant for association of an antibody (or antigen-binding domain) to the antigen to form the antibody / antigen complex, expressed in units of M -1< s -1< . The term equilibrium dissociation constant "K D " or "K d ", as used herein, refers to the dissociation constant of a particular antibody-antigen interaction, and describes the concentration of antigen required to occupy one half of all of the antibody-binding domains present in a solution of antibody molecules at equilibrium, and is equal to K off / K on , expressed in units of M. The measurement of K d presupposes that all binding agents are in solution. In the case where the antibody is tethered to a cell wall, e.g., in a yeast expression system, the corresponding equilibrium rate constant is expressed as EC50, which gives a good approximation of K d . The affinity constant, K a , is the inverse of the dissociation constant, K d , expressed in units of M -1< . The dissociation constant (K D or K d ) is used as an indicator showing affinity of antibodies to antigens. For example, easy analysis is possible by the Scatchard method using antibodies marked with a variety of marker agents, as well as by using BIACORE ™< X (made by Amersham Biosciences), which is an over-the-counter, measuring kit, or similar kit, according to the user's manual and experiment operation method attached with the kit. The K D value that can be derived using these methods is expressed in units of M (Mols). An antibody or antigen-binding fragment thereof that specifically binds to a target may have a dissociation constant (K d ) of, for example, ≤10 -5< M, ≤10 -6< M, ≤10 -7< M, ≤10 -8< M, ≤10 -9< M, ≤10 -10< M, ≤10 -11< M, or ≤10 -12< M.

[0070] Half maximal inhibitory concentration (IC 50 ) is a measure of the effectiveness of a substance (such as an antibody) in inhibiting a specific biological or biochemical function. It indicates how much of a particular drug or other substance (inhibitor, such as an antibody) is needed to inhibit a given biological process by half. The values are typically expressed as molar concentration. IC 50 is comparable to an "EC 50 " for agonist drug or other substance (such as an antibody). EC 50 also represents the plasma concentration required for obtaining 50% of a maximum effect in vivo. As used herein, an "IC 50 " is used to indicate the effective concentration of an antibody needed to neutralize 50% of the antigen bioactivity in vitro. IC 50 or EC 50 can be measured by bioassays such as inhibition of ligand binding by FACS analysis (competition binding assay), cell based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA).

[0071] "Percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN ™< (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0072] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies described herein existing naturally in cells. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0073] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0074] Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0075] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0076] The term "transfected" or "transformed" or "transduced" as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0077] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0078] The term "pharmaceutical formulation" of "pharmaceutical composition" refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. A "sterile" formulation is aseptic or free from all living microorganisms and their spores.

[0079] It is understood that embodiments of the invention described herein include "consisting" and / or "consisting essentially of" embodiments.

[0080] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0081] As used herein, reference to "not" a value or parameter generally means and describes "other than" a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

[0082] The term "about X-Y" used herein has the same meaning as "about X to about Y."

[0083] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.II. Anti-LAG-3 constructs

[0084] According to claim 1, one aspect of the present application provides isolated anti-LAG-3 constructs comprising a single-domain antibody (sdAb) that specifically recognizes LAG-3 (i.e., "anti-LAG-3 sdAb"), wherein the sdAb comprises a VHH domain comprising the amino acid sequence of any one of SEQ ID NOs: 304, 288, 306, 295, or 275-278. In some embodiments, the anti-LAG-3 construct is an anti-LAG-3 sdAb, a fusion protein, or an antigen-binding fragment thereof, which specifically binds to LAG-3 and antagonizes its ability to bind MHC class II molecules and / or mediate T cell exhaustion.(I) Anti-LAG-3 single-domain antibodies

[0085] The isolated anti-LAG-3 constructs described herein comprise an anti-LAG-3 sdAb. Inthe present invention, the anti-LAG-3 sdAb specifically recognizes human LAG-3. The complete amino acid sequence of an exemplary human LAG-3 comprises or consists of the amino acid sequence of SEQ ID NO: 350. The amino acid sequence of the extracellular domain of an exemplary human LAG-3 comprises or consists of the amino acid sequence of SEQ ID NO: 351.

[0086] The anti-LAG-3 sdAb may cross-reacts with at least one interspecies variant of the LAG-3 protein. The LAG-3 protein (or fragments thereof) may be human LAG-3 and the interspecies variant of the LAG-3 protein (or fragments thereof) may be a cynomolgus monkey variant thereof. Cross-reactivity of the anti-LAG-3 sdAbs or constructs thereof may facilitate clinical development of the anti-LAG-3 constructs, for example, by allowing more accurate and responsive dosing in animal studies.

[0087] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0088] Described and not claimed per se is that the anti-LAG-3 sdAb comprises a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, and the amino acid substitutions are in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0089] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions, wherein the amino acid substitutions are in CDR1 and / or CDR2. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0090] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0091] The sequences of the CDRs noted herein are provided in Table 11. Described and not claimed per se is that the CDRs can be combined in any combinations to generate a number of anti-LAG-3 sdAbs.

[0092] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 115, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 191, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 39; a CDR2 comprising the amino acid sequence of SEQ ID NO: 115; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 191; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 39; a CDR2 comprising the amino acid sequence of SEQ ID NO: 115; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 191. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0093] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 116, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 192, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 40; a CDR2 comprising the amino acid sequence of SEQ ID NO: 116; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 192; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 40; a CDR2 comprising the amino acid sequence of SEQ ID NO: 116; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 192. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0094] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 117, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 193, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 41; a CDR2 comprising the amino acid sequence of SEQ ID NO: 117; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 193; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 41; a CDR2 comprising the amino acid sequence of SEQ ID NO: 117; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 193. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). the anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0095] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 118, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 194, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 42; a CDR2 comprising the amino acid sequence of SEQ ID NO: 118; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 194; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. In some embodiments, the amino acid substitutions are in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 42; a CDR2 comprising the amino acid sequence of SEQ ID NO: 118; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 194. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0096] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 119, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 195, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 43; a CDR2 comprising the amino acid sequence of SEQ ID NO: 119; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 195; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. In some embodiments, the amino acid substitutions are in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 43; a CDR2 comprising the amino acid sequence of SEQ ID NO: 119; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 195. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0097] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 120, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 196, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 44; a CDR2 comprising the amino acid sequence of SEQ ID NO: 120; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 196; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 44; a CDR2 comprising the amino acid sequence of SEQ ID NO: 120; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 196. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may becamelid, chimeric, human, partially humanized, or fully humanized.

[0098] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 45, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 121, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 197, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 45; a CDR2 comprising the amino acid sequence of SEQ ID NO: 121; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 197; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 45; a CDR2 comprising the amino acid sequence of SEQ ID NO: 121; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 197. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may beabout 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0099] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 46, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 122, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 198, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 46; a CDR2 comprising the amino acid sequence of SEQ ID NO: 122; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 198; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions are in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 46; a CDR2 comprising the amino acid sequence of SEQ ID NO: 122; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 198. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0100] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 130, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 206, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 54; a CDR2 comprising the amino acid sequence of SEQ ID NO: 130; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 206; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 54; a CDR2 comprising the amino acid sequence of SEQ ID NO: 130; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 206. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may beabout 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0101] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 61, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 137, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 213, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 61; a CDR2 comprising the amino acid sequence of SEQ ID NO: 137; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 213; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 61; a CDR2 comprising the amino acid sequence of SEQ ID NO: 137; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 213. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0102] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 70, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 146, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 222, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 70; a CDR2 comprising the amino acid sequence of SEQ ID NO: 146; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 222; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regionsThe amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 70; a CDR2 comprising the amino acid sequence of SEQ ID NO: 146; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 222. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0103] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 72, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 148, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 224, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 72; a CDR2 comprising the amino acid sequence of SEQ ID NO: 148; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 224; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 72; a CDR2 comprising the amino acid sequence of SEQ ID NO: 148; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 224. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may becamelid, chimeric, human, partially humanized, or fully humanized.

[0104] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 73, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 149, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 225, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 73; a CDR2 comprising the amino acid sequence of SEQ ID NO: 149; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 225; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 73; a CDR2 comprising the amino acid sequence of SEQ ID NO: 149; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 225. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0105] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 53, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0106] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 60, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0107] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 69, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may becamelid, chimeric, human, partially humanized, or fully humanized.

[0108] Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223; or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions in the CDR regions. The amino acid substitutions may be in CDR1 and / or CDR2. Described and not claimed per se is an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0109] Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, the amino acid sequence of any one of SEQ ID NOs: 115-152, and the amino acid sequence of any one of SEQ ID NOs: 191-228. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 39, 115 and 191. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 40, 116 and 192. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 41, 117 and 193. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 42, 118 and 194. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 43, 119 and 195. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 44, 120 and 196. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 45, 121 and 197. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 46, 122 and 198. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 54, 130 and 206. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequence of SEQ ID NO: 61, 137, 213. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 70, 146 and 222. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 72, 148 and 224. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 73, 149 and 225. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 53, 129 and 205. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 60, 136 and 212. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 69, 145 and 221. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the sequences of SEQ ID NOs: 71, 147 and 223. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0110] Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 274. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 275. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 276. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 277. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 278. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 279. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 280. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 281. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 282. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 289. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 296. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 305. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 307. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 308. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 288. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 295. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 304. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising one, two or three CDRs of the amino acid sequence of SEQ ID NO: 306. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H comprising CDR1, CDR2, and CDR3 of the amino acid sequence of any one of SEQ ID NOs: 274-306. The K D of the binding between the anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.

[0111] Described and not claimed per se is that the anti-LAG-3 sdAbs described herein may comprise any suitable sequences for the FR region. Described and not claimed per se is that the anti-LAG-3 sdAb comprises a FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1-38, or a variant thereof comprising up to about 3 (such as about any one of 1, 2, 3, or more) amino acid substitutions. Described and not claimed per se is that the anti-LAG-3 sdAb comprises a FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 77-114, or a variant thereof comprising up to about 3 (such as about any one of 1, 2, 3, or more) amino acid substitutions. Described and not claimed per se is that the anti-LAG-3 sdAb comprises a FR3 comprising the amino acid sequence of any one of SEQ ID NOs: 153-190, or a variant thereof comprising up to about 3 (such as about any one of 1, 2, 3, or more) amino acid substitutions. Described and not claimed per se is that the anti-LAG-3 sdAb comprises a FR4 comprising the amino acid sequence of any one of SEQ ID NOs: 229-266, or a variant thereof comprising up to about 3 (such as about any one of 1, 2, 3, or more) amino acid substitutions. Described and not claimed per se is that the anti-LAG-3 sdAb comprises a FR1, a FR2, a FR3, and a FR4 of any one of the anti-LAG-3 sdAbs of Table 11.

[0112] Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 274, 279-287, 289- 294, 296-303, 305, and 307-311, or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to any one of SEQ ID NOs: 289-324. Described and not claimed per se is an anti-LAG-3 sdAb comprising a V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 274, 279-287, 289- 294, 296-303, 305, and 307-311, or a variant thereof comprising up to about 10 (such as about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions in the V H H domain. Described and not claimed per se is that the anti-LAG-3 sdAb comprising the V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 274, 279-287, 289- 294, 296-303, 305, and 307-311, or a variant thereof comprising up to about 3 (such as about any one of 1, 2, or 3) amino acid substitutions in the CDR1, and / or the CDR2, and / or the CDR3. Described and not claimed per se is that the anti-LAG-3 sdAb comprising the V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 274, 279-287, 289- 294, 296-303, 305, and 307-311, or a variant thereof comprising up to about 3 (such as about any one of 1, 2, or 3) amino acid substitutions are in the FR1, and / or the FR2, and / or the FR3, and / or the FR4. Described and not claimed per se is that the anti-LAG-3 sdAb comprising the V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 274, 279-287, 289- 294, 296-303, 305, and 307-311, or a variant thereof comprising amino acid substitutions in both CDRs and FRs. According to claim 1, provided is an anti-LAG-3 sdAb comprising a V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 304, 288, 306, 295, or 275-278. In some embodiments, the K D of the binding between the anti-LAG-3 sdAb and LAG-3 is about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). In some embodiments, the anti-LAG-3 sdAb cross-reacts with a LAG-3 from a non-human mammal.

[0113] In some embodiments, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of SEQ ID NO: 288. Described and not claimed per se is a variant having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 288. Described and not claimed per se is that the anti-LAG-3 sdAb comprises one or more amino acid substitutions selected from the group consisting of: (1) at amino acid position 11 (e.g., S or L); (2) at amino acid position 14 (e.g., A or P); (3) at amino acid position 44 (e.g., E or G); (4) at amino acid position 45 (e.g., R or L); (5) at amino acid position 49 (e.g., A or S); (6) at amino acid position 71 (e.g., K or R); (7) at amino acid position 74 (e.g., A or S); (8) at amino acid position 83 (e.g., D or N); (9) at amino acid position 86 (e.g., R or K); (10) at amino acid position 87 (e.g., A or P); (11) at amino acid position 92 (e.g., M or V); (12) at amino acid position 119 (e.g., Q or L); wherein the amino acid positions are based on SEQ ID NO: 288.

[0114] In some embodiments, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of SEQ ID NO: 295. Described and not claimed per se is a variant having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 295. Described and not claimed per se is that the anti-LAG-3 sdAb comprises one or more amino acid substitutions selected from the group consisting of: (1) at amino acid position 1 (e.g., Q or E); (2) at amino acid position 5 (e.g., A or V); (3) at amino acid position 11 (e.g., S or L); (4) at amino acid position 14 (e.g., A or P); (5) at amino acid position 44 (e.g., E or G); (6) at amino acid position 45 (e.g., R or L); (7) at amino acid position 71 (e.g., K or R); (8) at amino acid position 74 (e.g., A or S); (9) at amino acid position 86 (e.g., R or K); (10) at amino acid position 87 (e.g., A or P); (11) at amino acid position 92 (e.g., M or V); or (12) at amino acid position 117 (e.g., Q or L); wherein the amino acid positions are based on SEQ ID NO: 295.

[0115] In some embodiments, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of SEQ ID NO: 304. Described and not claimed per se is a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 304. Described and not claimed per se is that the anti-LAG-3 sdAb comprises one or more amino acid substitutions selected from the group consisting of: (1) at amino acid position 1 (e.g., Q or E); (2) at amino acid position 3 (e.g., Q or H); (3) at amino acid position 5 (e.g., M or V); (4) at amino acid position 11 (e.g., S or L); (5) at amino acid position 14 (e.g., V or P); (6) at amino acid position 44 (e.g., E or G); (7) at amino acid position 45 (e.g., R or L); (8) at amino acid position 49 (e.g., A or S); (8) at amino acid position 71 (e.g., K or R); (9) at amino acid position 74 (e.g., A or S); (10) at amino acid position 86 (e.g., R or K); (11) at amino acid position 87 (e.g., A or P); (12) at amino acid position 92 (e.g., M or V); (13) at amino acid position 94 (e.g., F or Y); or (14) at amino acid position 117 (e.g., Q, L, or I); wherein the amino acid positions are based on SEQ ID NO: 304.

[0116] In some embodiments, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of SEQ ID NO: 306. Described and not claimed per se is a variant having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to SEQ ID NO: 306. Described and not claimed per se is that the anti-LAG-3 sdAb comprises one or more amino acid substitutions selected from the group consisting of: (1) at amino acid position 5 (e.g., A or V); (2) at amino acid position 11 (e.g., S or L); (3) at amino acid position 14 (e.g., A or P); (4) at amino acid position 44 (e.g., E or G); (5) at amino acid position 45 (e.g., R or L); (6) at amino acid position 49 (e.g., A or S); (7) at amino acid position 71 (e.g., K or R); (8) at amino acid position 74 (e.g., A or S); (9) at amino acid position 86 (e.g., R or S); (10) at amino acid position 87 (e.g., A or P); (11) at amino acid position 92 (e.g., M or V); or (12) at amino acid position 121 (e.g., Q or L); wherein the amino acid positions are based on SEQ ID NO: 306.

[0117] Described and not claimed per se is an anti-LAG-3 sdAb or anti-LAG-3 construct comprising an anti-LAG-3 sdAb that specifically binds to LAG-3 competitively with any one of the anti-LAG-3 sdAbs described herein. Competitive binding may be determined using an ELISA assay. Described and not claimed per se is an anti-LAG-3 sdAb (or an anti-LAG-3 construct comprising an anti-LAG-3 sdAb) that specifically binds to LAG-3 competitively with an anti-LAG-3 sdAb comprising the amino acid sequence of any one of SEQ ID NOs: 274-311. Described and not claimed per se is an anti-LAG-3 sdAb (or an anti-LAG-3 construct comprising an anti-LAG-3 sdAb) that specifically binds to LAG-3 competitively with an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228. Described and not claimed per se is an anti-LAG-3 sdAb (or an anti-LAG-3 construct comprising an anti-LAG-3 sdAb) that specifically binds to LAG-3 competitively with any one of the anti-LAG-3 sdAbs of Table 11. The K D of the binding between the competing anti-LAG-3 sdAb and LAG-3 may be about 10 -7< M to about 10 -12< M (such as about 10 -8< M to about 10 -12< M, or about 10 -9< M to about 10 -11< M). The anti-LAG-3 sdAb may cross-react with a LAG-3 from a non-human mammal. The competing anti-LAG-3 sdAb may be camelid, chimeric, human, partially humanized, or fully humanized.Single-domain antibodies

[0118] In some embodiments, the anti-LAG-3 construct is a single-domain antibody. Described and not claimed per se is that exemplary sdAbs include, but are not limited to, heavy chain variable domains from heavy-chain only antibodies (e.g., V H H (Variable domain of the heavy chain of the Heavy chain antibody) in Camelidae or V NAR (Variable domain of the shark New Antigen Receptor) in cartilaginous fish), binding molecules naturally devoid of light chains, single domains (such as V H or V L ) derived from conventional 4-chain antibodies, humanized heavy-chain only antibodies, human single-domain antibodies produced by transgenic mice or rats expressing human heavy chain segments, and engineered domains and single domain scaffolds other than those derived from antibodies. Described and not claimed per se is that the sdAbs may be derived from any species including, but not limited to mouse, rat, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine. Described and not claimed per se is that sdAbs contemplated herein may also include naturally occurring sdAb molecules from species other than Camelidae and sharks.

[0119] In some embodiments, the sdAb is derived from a naturally occurring single-domain antigen binding molecule known as heavy chain antibody devoid of light chains (also referred herein as "heavy chain-only antibodies", or "HCAb"). Such single domain molecules are disclosed in WO 94 / 04678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448, for example. For clarity reasons, the variable domain derived from a heavy chain molecule naturally devoid of light chain is known herein as a V H H to distinguish it from the conventional V H of four chain immunoglobulins.

[0120] Described and not claimed per se is that the sdAb is derived from a variable region of the immunoglobulin found in cartilaginous fish. For example, the sdAb can be derived from the immunoglobulin isotype known as Novel Antigen Receptor (NAR) found in the serum of shark. Methods of producing single domain molecules derived from a variable region of NAR ("IgNARs") are described in WO 03 / 014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

[0121] Described and not claimed per se is that the sdAb is recombinant, CDR-grafted, camelized, de-immunized and / or in vitro generated (e.g., selected by phage display). Described and not claimed per se is that the amino acid sequence of the framework regions may be altered by "camelization" of specific amino acid residues in the framework regions. Camelization refers to the replacing or substitution of one or more amino acid residues in the amino acid sequence of a (naturally occurring) V H domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a V H H domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein. Such "camelizing" substitutions are preferably inserted at amino acid positions that form and / or are present at the V H -V L interface, and / or at the so-called Camelidae hallmark residues, as defined herein (see for example WO 94 / 04678, Davies and Riechmann FEBS Letters 339: 285-290, 1994; Davies and Riechmann Protein Engineering 9 (6): 531-537, 1996; Riechmann J. Mol. Biol. 259: 957-969, 1996; and Riechmann and Muyldermans J. Immunol. Meth. 231: 25-38, 1999).

[0122] In some embodiments, the sdAb is a human sdAb produced by transgenic mice or rats expressing human heavy chain segments. See, e.g., US20090307787A1, U.S. Pat. No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794. In some embodiments, the sdAb is affinity-matured.

[0123] Described and not claimed per se is that naturally occurring V H H domains against a particular antigen or target, can be obtained from (naïve or immune) libraries of Camelid V H H sequences. Such methods may or may not involve screening such a library using said antigen or target, or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Such libraries and techniques are for example described in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020 and WO 03 / 035694. Alternatively, improved synthetic or semi-synthetic libraries derived from (naïve or immune) V H H libraries may be used, such as V H H libraries obtained from (naïve or immune) V H H libraries by techniques such as random mutagenesis and / or CDR shuffling, as for example described in WO 00 / 43507.

[0124] In some embodiments, the sdAbs are generated from conventional 4-chain antibodies. See, for example, EP 0 368 684, Ward et al. (Nature 1989 Oct. 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; WO 06 / 030220; and WO 06 / 003388.

[0125] Because of the unique properties of sdAbs, using V H H domains as single antigen-binding proteins or as antigen-binding domains (i.e. as part of a larger protein or polypeptide) offers a number of significant advantages over the conventional V H and V L , scFv and conventional antibody fragments (such as Fab or (Fab') 2 ): 1) only a single domain is required to bind an antigen with high affinity, so there is no need to have a second domain, nor to assure that these two domains are present in the correct spatial conformation and configuration (e.g. no need to pair the heavy chain and light chain during folding, no need to use a specially designed linker such as for scFv); 2) V H H domains and other sdAbs can be expressed from a single gene and require no post-translational folding or modifications; 3) V H H domains and other sdAbs can be easily engineered into multivalent and / or multispecific formats (such as those described in the present application); 4) V H H domains and other sdAbs are highly soluble and do not have a tendency to aggregate (as with the mouse-derived "dAbs" described by Ward et al., Nature. 1989 Oct 12;341(6242):544-6); 5) V H H domains and other sdAbs are highly stable against heat, pH, proteases and other denaturing agents or conditions; 6) V H H domains and other sdAbs are easy and relatively cheap to prepare (even on a large production scale), such as using microbial fermentation, because there is no need to use mammalian expression systems (required by production of, for example, conventional antibody fragments); 7) V H H domains and other sdAbs are relatively small (approximately 15 kDa, or 10 times smaller than a conventional IgG) compared to conventional 4-chain antibodies and antigen-binding fragments thereof, thus have high(er) tissue penetration ability, such as for solid tumors and other dense tissues; and 8) V H H domains and other sdAbs can exhibit so-called "cavity-binding properties" (due to their extended CDR3 loop compared to that of conventional V H domains) and can therefore access targets and epitopes not accessible to conventional 4-chain antibodies and antigen-binding fragments thereof, for example, it has been shown that V H H domains and other sdAbs can inhibit enzymes (see for example WO1997049805; Transue et al., Proteins. 1998 Sep 1;32(4):515-22; Lauwereys et al., EMBO J. 1998 Jul 1;17(13):3512-20).LAG-3

[0126] Lymphocyte-activation protein 3 (LAG-3), comprised of 503 amino acids, belongs to the Ig superfamily and contains 4 extracellular Ig-like domains, designated D1 to D4. LAG-3 is a cell surface protein expressed on activated T cells, NK cells, B cells, and plasmacytoid dendritic cells, and plays a role in the function of these lymphocyte subsets that is important but not completely understood.

[0127] The terms "lymphocyte-activation protein 3", "LAG-3", "LAG-3 antigen", and "LAG-3 epitope" are used interchangeably, and include variants, isoforms, species homologs of human LAG-3, and analogs having at least one common epitope with LAG-3.

[0128] An exemplary amino acid sequence of human LAG-3 is disclosed at Genbank Accession Number P18627. In some embodiments, a human LAG-3 may be at least about 90%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the human LAG-3 of Genbank Accession Number P18627. In some embodiments, a human LAG-3 sequence has no more than about 10 amino acid differences from the human LAG-3 of Genbank Accession Number P18627. In some embodiments, the human LAG-3 may display no more than 5, 4, 3, 2, or 1 amino acid difference from the human LAG-3 of Genbank Accession Number P18627. In some embodiments, a human LAG-3 sequence may differ from the human LAG-3 of Genbank Accession Number P18627 by having, for example, conserved mutations or mutations in non-conserved regions and the LAG-3 has substantially the same biological function as the human LAG-3 of Genbank Accession Number P18627.

[0129] The anti-LAG-3 sdAb described herein may specifically recognizes a LAG-3 polypeptide having at least about 90% amino acid sequence identity to the human LAG-3 of Genbank Accession Number P18627. The anti-LAG-3 sdAb described herein may specifically recognizes a LAG-3 polypeptide comprising an amino acid sequence of SEQ ID NO: 350 or 351.

[0130] The anti-LAG-3 sdAb may cross-react with LAG-3 from species other than human, or other proteins which are structurally related to human LAG-3 (e.g., human LAG-3 homologs). The anti-LAG-3 sdAb may be completely specific for human LAG-3 and not exhibit species or other types of cross-reactivity. The anti-LAG-3 sdAb may specifically recognize a soluble isoform of human LAG-3. The anti-LAG-3 sdAb may specifically recognize a membrane-bound isoform of human LAG-3 (e.g., SEQ ID NO: 350).

[0131] The anti-LAG-3 sdAb described herein may specifically recognize the extracellular domain (ECD) of LAG-3. The anti-LAG-3 sdAb may specifically recognize the N-terminal portion of the LAG-3 ECD. The anti-LAG-3 sdAb may specifically recognizes the C-terminal portion of the LAG-3 ECD. The anti-LAG-3 sdAb may specifically recognize the middle portion of the LAG-3 ECD. The ECD of LAG-3 specifically recognized by the anti-LAG-3 sdAb may be at least about 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the ECD of the human LAG-3 of Genbank Accession Number P18627. The ECD of LAG-3 specifically recognized by the anti-LAG-3 sdAb may be 100% identical in amino acid sequence to the ECD of the human LAG-3 of Genbank Accession Number P18627. the anti-LAG-3 sdAb may specifically recognize a LAG-3 polypeptide comprising an amino acid sequence of SEQ ID NO: 351.Antibody affinity

[0132] Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE ™< -tests and peptide scans.

[0133] In some embodiments, the K D of the binding between the anti-LAG-3 sdAb and LAG-3 is about 10 -7< M to about 10 -12< M, about 10 -7< M to about 10 -8< M, about 10 -8< M to about 10 -9< M, about 10 -9< M to about 10 -10< M, about 10 -10< M to about 10 -11< M, about 10 -11< M to about 10 -12< M, about 10 -7< M to about 10 -12< M, about 10 -8< M to about 10 -12< M, about 10 -9< M to about 10 -12< M, about 10 -10< M to about 10 -12< M, about 10 -7< M to about 10 -11< M, about 10 -8< M to about 10 -11< M, about 10 -9< M to about 10 -11< M, about 10 -7< M to about 10 -10< M, about 10 -8< M to about 10 -10< M, or about 10 -7< M to about 10 -9< M. In some embodiments, the K d of the binding between the anti-LAG-3 sdAb and LAG-3 is stronger than about any one of 10 -7< M, 10 -8< M, 10 -9< M, 10 -10< M, 10 -11< M, or 10 -12< M. In some embodiments, the LAG-3 is human LAG-3. The LAG-3 may be cynomolgus monkey LAG-3. The LAG-3 may be the extracellular domain of LAG-3.

[0134] In some embodiments, the K on of the binding between the anti-LAG-3 sdAb and LAG-3 is about 10 3< M -1< s -1< to about 10 8< M -1< s -1< , about 10 3< M -1< s -1< to about 10 4< M -1< s -1< , about 10 4< M -1< s -1< to about 10 5< M -1< s -1< , about 10 5< M -1< s -1< to about 10 6< M -1< s -1< , about 10 6< M -1< s -1< to about 10 7< M -1< s -1< , or about 10 7< M -1< s -1< to about 10 8< M -1< s -1< . In some embodiments, the K on of the binding between the anti-LAG-3 sdAb and LAG-3 is about 10 3< M -1< s -1< to about 10 5< M -1< s -1< , about 10 4< M -1< s -1< to about 10 6< M -1< s -1< , about 10 5< M -1< s -1< to about 10 7< M -1< s -1< , about 10 6< M -1< s -1< to about 10 8< M -1< s -1< , about 10 4< M -1< s -1< to about 10 7< M -1< s -1< , or about 10 5< M -1< s -1< to about 10 8< M -1< s -1< . In some embodiments, the K on of the binding between the anti-LAG-3 sdAb and LAG-3 is no more than about any one of 10 3< M -1< s -1< , 10 4< M -1< s -1< , 10 5< M -1< s -1< , 10 6< M -1< s -1< , 10 7< M -1< s -1< or 10 8< M -1< s -1< .

[0135] In some embodiments, the K off of the binding between the anti-LAG-3 sdAb and LAG-3 is about 1 s -1< to about 10 -6< s -1< , about 1 s -1< to about 10 -2< s -1< , about 10 -2< s -1< to about 10 -3< s -1< , about 10 -3< s -1< to about 10 -4< s -1< , about 10 -4< s -1< to about 10 -5< s -1< , about 10 -5< s -1< to about 10 -6< s -1< , about 1 s -1< to about 10 -5< s -1< , about 10 -2< s -1< to about 10 -6< s -1< , about 10 -3< s -1< to about 10 -6< s -1< , about 10 -4< s -1< to about 10 -6< s -1< , about 10 -2< s -1< to about 10 -5< s -1< , or about 10 -3< s -1< to about 10 -5< s -1< . In some embodiments, the K off of the binding between the anti-LAG-3 sdAb and LAG-3 is at least about any one of 1 s -1< , 10 -2< s -1< , 10 -3< s -1< , 10 -4< s -1< , 10 -5< s -1< or 10 -6< s -1< .

[0136] In some embodiments, the EC 50 of the anti-LAG-3 sdAb is less than 10 nM in an amplified luminescent proximity homogeneous assay (AlphaLISA). In some embodiments, the EC 50 of the anti-LAG-3 sdAb is less than 500 nM in an inhibition of ligand binding by FACS analysis (competition binding assay), or cell based cytokine release assay. In some embodiments, the EC 50 of the anti-LAG-3 sdAb is less than 1 nM (such as about 0.001 nM to about 0.01 nM, about 0.01 nM to about 0.1 nM, about 0.1 nM to about 1 nM, etc.), about 1nM to about 10nM, about 1nM to about 5nM, about 5 nM to about 10 nM, about 10 nM to about 50 nM, about 50 nM to about 100 nM, about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, or about 400 nM to about 500 nM. In some embodiments, the EC 50 of the binding between the anti-LAG-3 sdAb and LAG-3 is no more than about any one of 0.1nM, 1nM, 5nM, 10nM, 50nM, 10nM, 100nM, 200nM, or 500nM.Chimeric or humanized antibodies

[0137] In some embodiments, the anti-LAG-3 sdAb provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a camelid species, such as llama) and a human constant region. In some embodiments, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0138] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0139] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall' Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" approach to FR shuffling).

[0140] Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0141] In some embodiments, the anti-LAG-3 sdAbs are modified, such as humanized, without diminishing the native affinity of the domain for antigen and while reducing its immunogenicity with respect to a heterologous species. For example, the amino acid residues of the antibody variable domain (V H H) of an llama antibody can be determined, and one or more of the Camelid amino acids, for example, in the framework regions, are replaced by their human counterpart as found in the human consensus sequence, without that polypeptide losing its typical character, i.e. the humanization does not significantly affect the antigen binding capacity of the resulting polypeptide. Humanization of Camelid single-domain antibodies requires the introduction and mutagenesis of a limited amount of amino acids in a single polypeptide chain. This is in contrast to humanization of scFv, Fab', (Fab') 2 and IgG, which requires the introduction of amino acid changes in two chains, the light and the heavy chain and the preservation of the assembly of both chains.

[0142] Described and not claimed per se is that sdAbs comprising a V H H domain can be humanized to have human-like sequences. The FR regions of the V H H domain used herein can comprise at least about any one of 50%, 60%, 70%, 80%, 90%, 95% or more of amino acid sequence homology to human VH framework regions. One exemplary class of humanized V H H domains is characterized in that the V H Hs carry an amino acid from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine, or glutamine at position 45, such as, for example, L45 and a tryptophan at position 103, according to the Kabat numbering. As such, polypeptides belonging to this class show a high amino acid sequence homology to human VH framework regions and said polypeptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanization.

[0143] Another exemplary class of humanized Camelid single-domain antibodies has been described in WO 03 / 035694 and contains hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensating this loss in hydrophilicity by the charged arginine residue on position 103 that substitutes the conserved tryptophan residue present in V H from double-chain antibodies. As such, peptides belonging to these two classes show a high amino acid sequence homology to human V H framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanization.Human antibodies

[0144] In some embodiments, the anti-LAG-3 sdAb provided herein is a human antibody (known as human domain antibody, or human DAb). Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001), Lonberg, Curr. Opin. Immunol. 20:450-459 (2008), and Chen, Mol. Immunol. 47(4):912-21 (2010). Transgenic mice or rats capable of producing fully human single-domain antibodies (or DAb) are known in the art. See, e.g., US20090307787A1, U.S. Pat. No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.

[0145] Human antibodies (e.g., human DAbs) may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE ™< technology; U.S. Patent No. 5,770,429 describing HUMAB ®< technology; U.S. Patent No. 7,041,870 describing K-M MOUSE ®< technology, and U.S. Patent Application Publication No. US 2007 / 0061900, describing VELOCIMOUSE ®< technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

[0146] Human antibodies (e.g., human DAbs) can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0147] Human antibodies (e.g., human DAbs) may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0148] One technique for obtaining V H H sequences directed against a particular antigen or target involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e. so as to raise an immune response and / or heavy chain antibodies directed against said antigen or target), obtaining a suitable biological sample from said transgenic mammal that contains (nucleic acid sequences encoding) said V H H sequences (such as a blood sample, serum sample or sample of B-cells), and then generating V H H sequences directed against said antigen or target, starting from said sample, using any suitable technique known per se (such as any of the methods described herein or a hybridoma technique). For example, for this purpose, the heavy chain antibody-expressing mice and the further methods and techniques described in WO 02 / 085945, WO 04 / 049794 and WO 06 / 008548 and Janssens et al., Proc. Natl. Acad. Sci. USA. 2006 Oct. 10; 103(41):15130-5 can be used. For example, such heavy chain antibody expressing mice can express heavy chain antibodies with any suitable (single) variable domain, such as (single) variable domains from natural sources (e.g. human (single) variable domains, Camelid (single) variable domains or shark (single) variable domains), as well as for example synthetic or semi-synthetic (single) variable domains.Library-derived antibodies

[0149] Antibodies of the present application may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004). Methods for constructing single-domain antibody libraries have been described, for example, see U.S. Pat. NO. 7371849.

[0150] In certain phage display methods, repertoires of V H and V L genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Repertoires of V H H genes can be similarly cloned by PCR, recombined randomly in phage libraries, and screened for antigen-binding phage. Phage typically display antibody fragments, either as scFv fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0151] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.Biological activities

[0152] The biological activity of anti-LAG-3 sdAb described herein can be determined by measuring its half maximal effective concentration (EC 50 ), which is a measure of the effectiveness of an antibody in binding to its target, or half maximal inhibitory concentration (IC 50 ), which is a measure of the effectiveness of an antibody in inhibiting a specific biological or biochemical function (such as inhibiting the binding between LAG-3 and MHC class II molecules). For example, here EC 50 can be used to indicate the effective concentration of an anti-LAG-3 sdAb needed to bind 50% LAG-3 on cell surface, IC 50 can be used to indicate the effective concentration of anti-LAG-3 sdAb needed to neutralize 50% of LAG-3 bioactivity in vitro. EC 50 also represents the plasma concentration required for obtaining 50% of a maximum effect in vivo. EC 50 or IC 50 can be measured by assays known in the art, for example, bioassays such as FACS binding analysis, inhibition of ligand binding by FACS analysis (competition binding assay), cell-based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA).

[0153] For example, the blockade of ligand binding can be studied using flow cytometry (also see Example 1). CHO cells expressing human LAG-3 can be dissociated from adherent culture flasks and mixed with varying concentrations of anti-LAG-3 sdAb for test, and a constant concentration of labeled-MHC class II protein. An anti-LAG-3 antibody positive control can be employed, such as BMS-986016 (Bristol-Myers Squibb). The mixture is equilibrated for 30 minutes at room temperature, washed three times with FACS buffer (PBS containing 1% BSA). Then, an antibody specifically recognizing the labeled MHC class II of constant concentration is added and incubated for 15 minutes at room temperature. Cells are washed with FACS buffer and analyzed by flow cytometry. Data can be analyzed with Prism (GraphPad Software, San Diego, CA) using non-linear regression to calculate IC 50 . The results from the competition assay can demonstrate the ability of anti-LAG-3 sdAbs in inhibiting the interaction between MHC class II and LAG-3.

[0154] The biological activity of anti-LAG-3 sdAb can be tested using a LAG-3-blockade assay via a luciferase reporter (also see Example 4). LAG-3 blockade reporter assay was performed using Promega LAG-3 blockade reporter assay kit (Promega, Cat#CS194819), according to the vendor's protocol. Briefly, Thaw-and-Use MHC-II APC Cells (including TCR Activating Antigen) can be plated overnight and then incubated with a serial dilution of anti-LAG-3 antibodies or anti-LAG-3 sdAb-Fc fusion proteins, followed by addition of Thaw-and-Use LAG-3 Effector cells. After 6 hours of induction at 37°C and 5% CO 2 , BIO-GLO ™< Luciferase Assay Reagent can be added, and luminescence can be determined. The results can demonstrate the ability of anti-LAG-3 sdAbs in inhibiting the interaction between MHC class II and LAG-3.

[0155] In some embodiments, the anti-LAG-3 sdAb blocks or antagonizes signals transduced by the LAG-3 receptor. In some embodiments, the anti-LAG-3 sdAb can bind to an epitope on LAG-3 so as to inhibit LAG-3 from interacting with MHC class II molecules. In some embodiments, the anti- LAG-3 sdAb can reduce the binding of LAG-3 to MHC class II molecules by at least about any one of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99% or 99.9%.(II) Fusion constructs

[0156] The present application further provides anti-LAG-3 fusion constructs comprising any one of the anti-LAG-3 sdAbs described herein and a second polypeptide, such as a second antibody or antigen binding fragment thereof, or an Fc fragment of an immunoglobulin. In some embodiments, the anti-LAG-3 construct comprises two or more polypeptides other than the anti-LAG-3 sdAb. The additional polypeptide(s) may or may not change or otherwise influence the biological properties of the anti-LAG-3 sdAb, and may or may not add further functionality to the anti-LAG-3 sdAb. In some embodiments, the second polypeptide confers one or more desired properties or functionalities to the anti- LAG-3 sdAb.

[0157] In some embodiments, the anti-LAG-3 construct comprises a second antibody or antigen binding fragment thereof (such as sdAb, scFv, Fab, full-length antibody, etc.) that specifically recognizes a second epitope. In some embodiments, the second epitope is from LAG-3. In some embodiments, the second epitope is not from LAG-3. In some embodiments, the second antibody specifically recognizes the same epitope on LAG-3 as the anti-LAG-3 sdAb described herein. In some embodiments, the second antibody specifically recognizes a different epitope on LAG-3 as the anti-LAG-3 sdAb described herein.

[0158] In some embodiments, there is provided an anti-LAG-3 construct comprising a plurality (such as 2, 3, 4, or more) of anti-LAG-3 sdAbs described herein. In some embodiments, the plurality of the anti-LAG-3 sdAbs are fused to each other via a linker (such as a peptide linker). The plurality of the anti-LAG-3 sdAbs can be the same or different.

[0159] In some embodiments, the anti-LAG-3 construct comprises a second polypeptide that enhances the half-life, solubility, and / or absorption, reduces immunogenicity or toxicity, eliminates or attenuates undesirable side effects, confers other advantageous properties to and / or reduces other undesired properties of the anti-LAG-3 construct, compared to the anti-LAG-3 sdAb alone. Some non-limiting examples of such polypeptides include serum proteins, such as human serum albumin (HSA; see e.g. WO 00 / 27435) or haptenic molecules (e.g. haptens that are recognized by circulating antibodies, see e.g. WO 98 / 22141). It was shown that linking fragments of immunoglobulins (such as V H domains) to serum albumin or fragments thereof may increase antibody half-life (see e.g. WO 00 / 27435 and WO 01 / 077137). Thus, in some embodiments, the anti-LAG-3 construct comprises an anti-LAG-3 sdAb fused to serum albumin or a fragment thereof, optionally via a suitable linker (such as peptide linker). In some embodiments, the serum albumin comprises at least domain III (see PCT / EP2007 / 002817). The anti-LAG-3 sdAb-HSA fusion protein can be of any suitable format, such as (sdAb) n -HSA (n is an integer of at least 1), sdAb-HSA-sdAb, etc.Anti-LAG-3 heavy chain-only antibody (HCAb)

[0160] In some embodiments, the anti-LAG-3 construct is a heavy chain-only antibody (HCAb) comprising an anti-LAG-3 sdAb described herein. In some embodiments, the anti-LAG-3 sdAb is fused to one or more C H 2 and / or C H 3 domains, e.g., an Fc fragment. In some embodiments, C H 2 and / or C H 3 domains are derived from human immunoglobulins. In some embodiments, the anti-LAG-3 sdAb is fused to the C H 2 and / or C H 3 domains via a peptide linker. The C H 2 and / or C H 3 domains may increase the half-life of the anti-LAG-3 construct in vivo.

[0161] Thus, in some embodiments, there is provided an isolated anti-LAG-3 HCAb comprising an anti-LAG-3 sdAb described herein fused to an Fc fragment of an immunoglobulin, such as IgA, IgD, IgE, IgG, or IgM. In some embodiments, the anti-LAG-3 HCAb comprises an Fc fragment of IgG, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc fragment is a human Fc, such as human IgG1 (hIgG1) Fc, hIgG2 Fc, or hIgG4 Fc. In some embodiments, the Fc fragment is effectorless, with reduced, minimized, or eliminated antibody effector functions such as ADCC, CDC, and / or ADCP (antibody-dependent cellular phagocytosis). In some embodiments, the effectorless Fc comprises an N297A or DANA mutation (D265A+N297A) in the C H 2 region. In some embodiments, the effectorless Fc comprises K322A and L234A / L235A (LALA) mutations. In some embodiments, the Fc fragment is an effectorless IgG1 Fc, such as effectorless hIgG1 Fc. In some embodiments, the Fc fragment is a human IgG4 Fc (S228P). In some embodiments, the anti-LAG-3 HCAb is monomeric. In some embodiments, the anti-LAG-3 HCAb is dimeric. In some embodiments, the anti-LAG-3 HCAb is multispecific and multivalent (such as bispecific and bivalent), e.g., comprising two or more different anti-LAG-3 sdAbs described herein. In some embodiments, the anti-LAG-3 HCAb is monospecific and multivalent (e.g., bivalent), e.g., comprising two or more copies of the same anti-LAG-3 sdAb.

[0162] In some embodiments, the anti-LAG-3 sdAb and the Fc fragment are fused to each other via a peptide linker. In some embodiments, the peptide linker is a human IgG1 hinge (SEQ ID NO: 352). In some embodiments, the peptide linker is a mutated human IgG1 hinge (SEQ ID NO: 353). In some embodiments, the peptide linker is a human IgG4 hinge. In some embodiments, the peptide linker is a hIgG2 hinge.

[0163] Described and not claimed per se is an isolated anti-LAG-3 HCAb comprising an sdAb specifically recognizing LAG-3, wherein the anti-LAG-3 sdAb comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and wherein the anti-LAG-3 sdAb is fused to an Fc fragment of an immunoglobulin. Described and not claimed per se is an anti-LAG-3 HCAb comprising an sdAb specifically recognizing LAG-3, wherein the sdAb comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221; or (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223, and wherein the anti-LAG-3 sdAb is fused to an Fc fragment of an immunoglobulin. In some embodiments, there is provided an isolated anti-LAG-3 HCAb comprising an sdAb specifically recognizing LAG-3, wherein the sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 304, 288, 306, 295, or 275-278. Described and not claimed per se is a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to any one of SEQ ID NOs: 274-311, and wherein the anti-LAG-3 sdAb is fused to an Fc fragment of an immunoglobulin. In some embodiments, there is provided an isolated anti-LAG-3 HCAb comprising an sdAb specifically recognizing LAG-3, wherein the sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 304, 288, 306, 295, or 275-278, and wherein the anti-LAG-3 sdAb is fused to an Fc fragment of an immunoglobulin. In some embodiments, the anti-LAG-3 sdAb is fused to the Fc fragment via a peptide linker. In some embodiments, the anti-LAG-3 HCAb is monomeric. In some embodiments, the Fc fragment is a human IgG1 Fc, effectorless human IgG1 Fc, hIgG2 Fc, human IgG4 Fc, or hIgG4 Fc (S228P).

[0164] Described and not claimed per se is an isolated anti-LAG-3 HCAb comprising two sdAbs specifically recognizing LAG-3, wherein each anti-LAG-3 sdAb comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and wherein the C-terminus of each anti-LAG-3 sdAb is fused to the N-terminus of an Fc fragment of an immunoglobulin. Described and not claimed per se is an isolated anti-LAG-3 HCAb comprising two sdAbs specifically recognizing LAG-3, wherein each anti-LAG-3 sdAb comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221; or (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223, and wherein the C-terminus of each anti-LAG-3 sdAb is fused to the N-terminus of an Fc fragment of an immunoglobulin. The two anti-LAG-3 sdAbs may be the same. The two anti-LAG-3 sdAbs may be different. The anti-LAG-3 sdAbs may be fused to the Fc fragment via a peptide linker. The Fc fragment may be a human IgG1 Fc, effectorless human IgG1 Fc, hIgG2 Fc, human IgG4 Fc, or hIgG4 Fc (S228P).

[0165] In some embodiments, there is provided an isolated anti-LAG-3 HCAb comprising the amino acid sequence of any one of SEQ ID NOs:313-316, 326, 333, 342, 344. Described and not claimed per se is a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identify to any one of SEQ ID NOs: 312-349. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 313-316, 326, 333, 342, 344.

[0166] Also provided are isolated anti-LAG-3 HCAbs that specifically binds to LAG-3 competitively with any one of the isolated anti-LAG-3 HCAbs, anti-LAG-3 sdAbs, or anti-LAG-3 constructs described herein.Multivalent and / or multispecific constructs

[0167] The present application provides multivalent and multispecific anti-LAG-3 constructs. Multispecific anti-LAG-3 constructs are also referred herein as "anti-LAG-3 multispecific antigen binding proteins (MABPs)." In some embodiments, the anti-LAG-3 constructs are bispecific, which are also referred herein as "anti-LAG-3 bispecific antigen binding proteins (BABPs)."

[0168] Described and not claimed per se is an isolated anti-LAG-3 construct (e.g., MABP or BABP) comprising: (a) a first antigen binding portion comprising an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and (b) a second antigen binding portion that specifically binds a second epitope (e.g., immune checkpoint molecule, such as PD-1), wherein the first antigen binding portion and the second antigen binding portion are fused to each other. Described and not claimed per se is an isolated anti-LAG-3 construct (e.g., MABP or BABP) comprising: (a) a first antigen binding portion comprising an anti-LAG-3 sdAb comprising: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221; or (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223, and (b) a second antigen binding portion that specifically binds a second epitope (e.g., immune checkpoint molecule, such as PD-1), wherein the first antigen binding portion and the second antigen binding portion are fused to each other. The second antigen binding portion may specifically recognizes an immune checkpoint molecule, such as PD-1, 4-1BB, PD-L1, TIM-3, TIGIT, CTLA-4, VISTA, B7-1, B7-H3, CD47, OX40 or GITR. The second antigen binding portion may comprise a heavy chain comprising the V H and a light chain comprising the V L . The first antigen binding portion may be fused to the second antigen binding portion at the N-terminus of the heavy chain, the N-terminus of the light chain, the N-terminus of the Fc region, the C-terminus of the heavy chain, or the C-terminus of the light chain. The second antigen binding portion may comprise a Fab or an scFv. The first antigen binding portion may be fused to the second antigen binding portion at the C-terminus of the Fab or scFv. The second antigen binding portion may comprise a full-length 4-chain antibody. The first antigen binding portion may be fused to the second antigen binding portion via a peptide linker. The peptide linker may be no more than about 30 (such as no more than about any one of 25, 20, or 15) amino acids long. The peptide linker may comprise the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. The N-terminus or the C-terminus of the anti-LAG-3 sdAb may be fused to a peptide sequence. The peptide sequence may comprise the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. The second antigen binding fragment may comprise an Fc region, such as an IgG1 Fc, effectorless IgG1 Fc, IgG2 Fc, IgG4 Fc, or IgG4 Fc (S228P).

[0169] In some embodiments, the anti-LAG-3 construct is an anti-LAG-3 MABP comprising a second antigen binding portion that specifically recognizes an immune checkpoint molecule. As used herein, the term "immune checkpoint molecules" refer molecules in the immune system that generally act to maintain self-tolerance or modulate the duration and amplitude of physiological immune responses to minimize collateral tissue damage. Immune checkpoint inhibitors can inhibit an immune system checkpoint by stimulating the activity of a stimulatory checkpoint molecule, or inhibiting the activity of an inhibitory checkpoint molecule in the pathway. Stimulatory checkpoint molecules are molecules, such as proteins, that stimulate or positively regulate the immune system. Inhibitory checkpoint molecules are molecules, such as proteins, that inhibit or negatively regulate the immune system. Immune system checkpoint molecules include, but are not limited to, cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death 1 protein (PD-1), programmed cell death 1 ligand 1 (PD-L1), programmed cell death 1 ligand 2 (PD-L2), lymphocyte activation gene 3 (LAG3), B7-1, B7-H3, T cell membrane protein 3 (TIM3), B- and T-lymphocyte attenuator (BTLA), T cell immunoreceptor with Ig and ITIM domains (TIGIT), V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation (VISTA), tumor necrosis factor receptor superfamily member 4 (TNFRSF4, also known as CD134 or OX40), tumor necrosis factor receptor superfamily member 9 (TNFRSF9, also known as 4-1BB or CD137), cluster of differentiation 47 (CD47 or IAP) and glucocorticoid-induced tumor necrosis factor receptor (GITR). Any of the known antibodies against the immune checkpoint molecules may be used in an anti-LAG-3 MABP.

[0170] Described and not claimed per se is an isolated anti-LAG-3 construct (e.g., MABP or BABP) comprising: (a) a first antigen binding portion comprising an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and (b) a second antigen binding portion that specifically binds an immune checkpoint molecule (e.g., PD-1), wherein the first antigen binding portion and the second antigen binding portion are fused to each other. Described and not claimed per se is an isolated anti-LAG-3 construct (e.g., MABP or BABP) comprising: (a) a first antigen binding portion comprising an anti-LAG-3 sdAb comprising: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221; or (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223, and (b) a second antigen binding portion that specifically binds an immune checkpoint molecule (e.g., PD-1), wherein the first antigen binding portion and the second antigen binding portion are fused to each other. The second antigen binding portion may comprise a heavy chain comprising the V H and a light chain comprising the V L . The first antigen binding portion may be fused to the second antigen binding portion at the N-terminus of the heavy chain, the N-terminus of the light chain, the N-terminus of the Fc region, the C-terminus of the heavy chain, or the C-terminus of the light chain. the second antigen binding portion may comprise a Fab or an scFv. The first antigen binding portion may be fused to the second antigen binding portion at the C-terminus of the Fab or scFv. the second antigen binding portion may comprise a full-length 4-chain antibody. The first antigen binding portion may be fused to the second antigen binding portion chemically. The first antigen binding portion may be fused to the second antigen binding portion via a peptide linker. The peptide linker may be no more than about 30 (such as no more than about any one of 25, 20, or 15) amino acids long. The peptide linker may comprise the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. The N-terminus or the C-terminus of the anti-LAG-3 sdAb may be fused to a peptide sequence. The peptide sequence may comprise the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. The second antigen binding fragment may comprise an Fc region, such as an IgG1 Fc, effectorless IgG1 Fc, IgG2 Fc, IgG4 Fc, or IgG4 Fc (S228P).

[0171] In some embodiments, the isolated anti-LAG-3 construct comprises an anti-LAG-3 sdAb described herein fused to a second antibody or antigen binding fragment thereof. In some embodiments, the anti-LAG-3 construct comprises two or more antibodies or antigen binding fragments thereof fused to the anti-LAG-3 sdAb. In some embodiments, the second antibody specifically recognizes an antigen other than LAG-3, or a second epitope on LAG-3. In some embodiments, the second antibody is a full-length antibody, a Fab, a Fab', a (Fab') 2 , an Fv, an scFv, an scFv-scFv, a minibody, a diabody, or an sdAb. In some embodiments, the second antibody comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ).

[0172] In some embodiments, the anti-LAG-3 construct is monospecific. In some embodiments, the anti-LAG-3 construct is multispecific (such as bispecific). Multispecific molecules are molecules that have binding specificities for at least two different epitopes (e.g., bispecific antibodies have binding specificities for two epitopes). Multispecific molecules with more than two valencies and / or specificities are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).

[0173] In some embodiments, the anti-LAG-3 construct is monovalent. In some embodiments, the anti-LAG-3 construct is multivalent (e.g., bivalent) and monospecific. In some embodiments, the anti-LAG-3 construct comprises an anti-LAG-3 sdAb described herein and a second antibody (such as a full-length antibody, sdAb, or an antigen binding fragment comprising a V H and a V L ) specifically recognizing the same LAG-3 epitope as the anti-LAG-3 sdAb. In some embodiments, the second antibody comprises the same CDRs and / or the same V H H as the anti-LAG-3 sdAb. For example, the anti-LAG-3 construct may comprise two or more anti-LAG-3 sdAbs described herein, wherein the two or more anti-LAG-3 sdAbs are the same. In some embodiments, the two or more anti-LAG-3 sdAbs are fused to each other via a peptide linker. The monospecific or multispecific anti-LAG-3 construct comprising two or more anti-LAG-3 sdAbs may have increase avidity compared to that of a single anti-LAG-3 sdAb described herein.

[0174] In some embodiments, the anti-LAG-3 construct is multivalent and multispecific (e.g., bispecific). In some embodiments, the anti-LAG-3 construct comprises an anti-LAG-3 sdAb described herein and a second antibody (such as a full-length antibody, sdAb, or an antigen binding fragment comprising a V H and a V L ) specifically recognizing a second antigen other than LAG-3, or a different LAG-3 epitope from that recognized by the anti-LAG-3 sdAb.

[0175] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991); and creating polypeptides comprising tandem single-domain antibodies (see, e.g., U.S. Patent Application No. 20110028695; and Conrath et al. J. Biol. Chem., 2001; 276(10):7346-50). Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies," are also included herein (see, e.g., US 2006 / 0025576A1).

[0176] In some embodiments, there is provided an isolated anti-LAG-3 construct comprising an anti-LAG-3 sdAb described herein fused to a full-length antibody or antigen binding fragment thereof that comprises a V H and a V L . The full-length antibody or antigen binding fragment recognizes an antigen other than LAG-3, or an epitope from LAG-3 that is different from the epitope recognized by the anti-LAG-3 sdAb, thereby conferring a broadened targeting capability. Due to the small size of the sdAb, in some embodiments, the anti-LAG-3 MABP (e.g., anti-LAG-3 BABPs) described herein can have similar molecular weight and pharmacokinetic properties compared to those of the full-length antibody or antigen binding fragment component. For example, an anti-LAG-3 MABP can be designed by fusing one or more anti-LAG-3 sdAbs to a monoclonal antibody with proven clinical efficacy and safety to provide increased clinical benefits and desirable pharmacokinetic properties without impeding the expressibility of the multispecific construct. In some embodiments, the anti- LAG-3 sdAb is fused to the full-length antibody or antigen binding fragment by a peptide linker.

[0177] The anti-LAG-3 MABPs (e.g., anti-LAG-3 BABPs) described herein can be adopted to target a variety of disease-related epitope or antigen combinations besides LAG-3, such as LAG-3 with the combination of immune checkpoint molecules, cell surface antigens (such as tumor antigens), or pro-inflammatory molecules, thereby providing agents that are useful for treating a variety of diseases and conditions, such as cancer. The anti-LAG-3 MABP (e.g., anti-LAG-3 BABPs) can be of any format, such as those disclosed in PCT / CN2017 / 093644.

[0178] Described and not claimed per se is an isolated anti-LAG-3 construct (e.g., MABP or BABP) comprising: (a) a first antigen binding portion comprising an anti-LAG-3 sdAb comprising a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and (b) a second antigen binding portion comprising a V H and a V L , wherein the V H and V L together form an antigen-binding site that specifically binds a second epitope (e.g., immune checkpoint molecule, such as PD-1), wherein the first antigen binding portion and the second antigen binding portion are fused to each other. Described and not claimed per se is an isolated anti-LAG-3 construct (e.g., MABP or BABP) comprising: (a) a first antigen binding portion comprising an anti-LAG-3 sdAb comprising: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221; or (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223, and (b) a second antigen binding portion comprising a V H and a V L , wherein the V H and V L together form an antigen-binding site that specifically binds a second epitope (e.g., immune checkpoint molecule, such as PD-1), wherein the first antigen binding portion and the second antigen binding portion are fused to each other. The second epitope may be from an immune checkpoint molecule, such as PD-1, 4-1BB, PD-L1, TIM-3, TIGIT, CTLA-4, VISTA, B7-1, B7-H3, CD47, OX40 or GITR. The second antigen binding portion may comprise a heavy chain comprising the V H and a light chain comprising the V L . The first antigen binding portion may be fused to the second antigen binding portion at the N-terminus of the heavy chain, the N-terminus of the light chain, the N-terminus of the Fc region, the C-terminus of the heavy chain, or the C-terminus of the light chain. The second antigen binding portion may comprise a Fab or an scFv. The first antigen binding portion may be fused to the second antigen binding portion at the C-terminus of the Fab or scFv. The second antigen binding portion may comprise a full-length 4-chain antibody. The second antigen binding portion may comprise an anti-PD-1 full-length antibody or antigen binding fragment thereof. The anti-PD-1 full-length antibody or antigen binding fragment thereof (e.g., Fab, scFv) may comprise: (i) a V H comprising HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 356, and a V L comprising LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 357; or (ii) a V H comprising HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 373, and a V L comprising LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 374. The anti-PD-1 full-length antibody may comprise: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 356, and a light chain comprising the amino acid sequence of SEQ ID NO: 357; or (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 373, and a light chain comprising the amino acid sequence of SEQ ID NO: 374. The anti-PD-1 full-length antibody may be pembrolizumab or PD1-BM-min. The first antigen binding portion may be fused to the second antigen binding portion chemically. The first antigen binding portion may be fused to the second antigen binding portion via a peptide linker. The peptide linker may be no more than about 30 (such as no more than about any one of 25, 20, or 15) amino acids long. The peptide linker may comprise the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. The N-terminus or the C-terminus of the anti-LAG-3 sdAb may be fused to a peptide sequence. The peptide sequence may comprise the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. The second antigen binding fragment may comprise an Fc region, such as an IgG1 Fc, effectorless IgG1 Fc, IgG2 Fc, IgG4 Fc, or IgG4 Fc (S228P).

[0179] In some embodiments, the isolated anti-LAG-3 MABP (e.g., BABP) comprises at least two antigen binding portions that can specifically bind at least two different epitopes. Some of the at least two antigen binding portions may be identical, so long as the MABP has binding sites for two different epitopes. The anti-LAG-3 MABPs (e.g., BABPs) can be symmetric or asymmetric. For example, the anti-LAG-3 MABP (e.g., BABP) may comprise one to eight copies of the first antigen binding portion comprising the anti-LAG-3 sdAb described herein, and one or two copies of the second antigen binding portion comprising a V H and a V L . In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises two different antigen binding portions that each comprise a V H domain and a V L domain that together form a different antigen binding site. For example, the second antigen binding portion can be a bispecific antibody. In some embodiments, the second antigen binding portion is a monospecific full-length antibody or antigen binding fragment thereof, such as Fab or scFv.

[0180] In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises any one of 1, 2, 3, 4, 5, 6, 7, 8, or more different antigen binding portions that each comprises an anti-LAG-3 sdAb described herein. In some embodiments, two identical anti-LAG-3 sdAbs are fused to each other, which are further fused to the second antigen binding portion. In some embodiments, two different anti-LAG-3 sdAbs are fused to each other, which are further fused to the second antigen binding portion.

[0181] The isolated anti-LAG-3 MABP (e.g., BABP) may have any suitable number of valencies for LAG-3 and / or the second epitope (e.g., PD-1), and any suitable number of specificity. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) is bivalent, trivalent, tetravalent, pentavalent, hexavalent, or of higher valencies for LAG-3. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) is bivalent, trivalent, tetravalent, pentavalent, hexavalent, or of higher valencies for the second epitope (e.g., PD-1). In some embodiments, the anti-LAG-3 MABP is bispecific (e.g., PD-1×LAG-3 BABP). Exemplary BABPs are depicted in FIGs. 7-16. In some embodiments, the MABP is trispecific. In some embodiments, the MABP is tetraspecific. In some embodiments, the MABP has more than four specificities.

[0182] In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises: (a) one or more copies (such as 2) of a first antigen binding portion comprising an anti-LAG-3 sdAb, and (b) a single copy of a second antigen binding portion comprising a V H and a V L , wherein the V H and V L together form an antigen-binding site that specifically binds a second epitope (e.g., PD-1), wherein each copy of the first antigen binding portion is fused to the second antigen binding portion. In some embodiments, there is provided an anti-LAG-3 MABP (e.g., BABP) comprising: (a) a plurality (such as 2, 3, 4, 5, 6, 7, 8, or more) of identical or different anti-LAG-3 sdAbs, and (b) a plurality (such as 2, 3, 4, 5, 6, or more) of a second antigen binding portion comprising a V H and a V L , wherein the V H and V L together form an antigen-binding site that specifically binds a second epitope (e.g., PD-1), and, wherein the anti- LAG-3 sdAbs are fused to each other, and / or to the second antigen binding portion. In some embodiments, there is provided an anti-LAG-3 MABP (e.g., BABP) comprising: (a) a plurality (such as 2, 3, or 4) of identical or different anti-LAG-3 sdAbs, and (b) two copies of a second antigen binding portion each comprising a V H and a V L , wherein the V H and V L together form an antigen-binding site that specifically binds a second epitope (e.g., PD-1), and wherein the anti- LAG-3 sdAbs are fused to each other, and / or to the second antigen binding portion. In some embodiments, one or more of the anti-LAG-3 sdAb is each further fused to another identical or different anti-LAG-3 sdAb.

[0183] In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises: (a) a single copy of a first antigen binding portion comprising an anti-LAG-3 sdAb, and (b) two copies of a second antigen binding portion each comprising a V H and a V L , wherein the V H and V L together form an antigen-binding site that specifically binds a second epitope, wherein the first antigen binding portion is fused to one of the two copies of the second antigen binding portion. In some embodiments, the anti-PD-1 MABP (e.g., BABP) comprises: (a) two copies of a first antigen binding portion each comprising an anti-LAG-3 sdAb, (b) two copies of a second antigen binding portion each comprising a V H and a V L , wherein the V H and V L together form an antigen-binding site that specifically binds a second epitope, wherein one copy of the first antigen binding portion is fused to each copy of the second antigen binding portion. In some embodiments, one or more of the anti-LAG-3 sdAbs are each further fused to another identical or different anti-LAG-3 sdAb.

[0184] In some embodiments, the anti-LAG-3 construct comprises an anti-LAG-3 sdAb described herein fused to a second antibody, wherein the second antibody is a full-length antibody consisting of two heavy chains and two light chains (such as full-length antibody specifically recognizing PD-1). In some embodiments, the anti-LAG-3 sdAb and the full-length antibody are fused to each other via a linker, such as a peptide linker. In some embodiments, the N-terminus or the C-terminus of the anti-LAG-3 sdAb is fused to a peptide sequence.

[0185] Described and not claimed per se is an isolated anti-LAG-3 construct comprising an anti-LAG-3 sdAb and a full-length antibody consisting of two heavy chains and two light chains (such as a full-length antibody specifically recognizing PD-1), wherein the anti-LAG-3 sdAb comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. Described and not claimed per se is an isolated anti-LAG-3 construct comprising an anti-LAG-3 sdAb and a full-length antibody consisting of two heavy chains and two light chains (such as a full-length antibody specifically recognizing PD-1), wherein the anti-LAG-3 sdAb comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221; or (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223. The Fc fragment of the full-length antibody may be hIgG1 Fc, effectorless hIgG1 Fc, hIgG2 Fc, hIgG4 Fc, or hIgG4 Fc (S228P). The full-length antibody may be an activator of a stimulatory immune checkpoint molecule. The full-length antibody may be an immune checkpoint inhibitor, such as an inhibitor of TIGIT, TIM-3, CTLA-4, or PD-1. The full-length antibody may be pembrolizumab, PD1-BM-min or nivolumab. The anti-LAG-3 sdAb and the full-length antibody may be fused to each other via a peptide linker. The peptide linker may comprise the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. The N-terminus or the C-terminus of the anti-LAG-3 sdAb may be fused to a peptide sequence. The peptide sequence may comprise the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372.

[0186] The multispecific anti-LAG3 construct may be of any suitable format. Exemplary bispecific anti-LAG3 constructs are shown in FIGs. 7-16.

[0187] In some embodiments, the anti-LAG-3 construct comprises two anti-LAG-3 sdAbs. In some embodiments, the N-terminus of each anti-LAG-3 sdAb is fused to the C-terminus of a heavy chain of the full-length antibody (exemplified as FIG. 8). In some embodiments, the C-terminus of each anti- LAG-3 sdAb is fused to the N-terminus of a heavy chain of the full-length antibody (exemplified as FIG. 7). In some embodiments, the N-terminus of each anti-LAG-3 sdAb is fused to the C-terminus of a light chain of the full-length antibody (exemplified as FIG. 10). In some embodiments, the C-terminus of each anti-LAG-3 sdAb is fused to the N-terminus of a light chain of the full-length antibody (exemplified as FIG. 9). In some embodiments, the C-terminus of each anti-LAG-3 sdAb is fused to the N-terminus of a C H 2 domain of the full-length antibody, and the N-terminus of each anti-LAG-3 sdAb is fused to the C-terminus of a C H 1 domain of the full-length antibody (exemplified as FIG. 13). In some embodiments, the N-terminus of each anti-LAG-3 sdAb is fused to the C-terminus of an scFv, and the C-terminus of each anti-LAG-3 sdAb is fused to the N-terminus of an C H 2 domain of a Fc region (exemplified as FIG. 14).

[0188] In some embodiments, the anti-LAG-3 construct comprises four anti-LAG-3 sdAbs. In some embodiments, the C-terminus of a first anti-LAG-3 sdAb is fused to the N-terminus of a heavy chain of the full-length antibody, and the C-terminus of a second anti-LAG-3 sdAb is fused to the N-terminus of a light chain of the full-length antibody (exemplified as FIG. 11). In some embodiments, the C-terminus of a first anti-LAG-3 sdAb is fused to the N-terminus of a heavy chain of the full-length antibody, and the C-terminus of a second anti-LAG-3 sdAb is fused to the N-terminus of the first anti-LAG-3 sdAb (exemplified as FIG. 12). In some embodiments, the N-terminus of a first anti-LAG-3 sdAb is fused to the C-terminus of a C H 1 domain of a Fab, and the C-terminus of the first anti-LAG-3 sdAb is fused to the N-terminus of a second C H 1 domain; and the N-terminus of a second anti-LAG-3 sdAb is fused to the C-terminus of a C L domain of a Fab, and the C-terminus of the second anti-LAG-3 sdAb is fused to the N-terminus of a second C L domain (exemplified as FIG. 15). In some embodiments, the N-terminus of a first anti-LAG-3 sdAb is fused to the C-terminus of an scFv, and the C-terminus of the first anti-LAG-3 sdAb is fused to the N-terminus of a C H 1 domain; and the C-terminus of a second anti-LAG-3 sdAb is fused to the N-terminus of a second C L domain (exemplified as FIG. 16).a) Fusion polypeptides

[0189] The first antigen binding portion comprising an anti-LAG-3 sdAb described herein and the second antigen binding portion comprising a V H and a V L of the anti-LAG-3 MABP (e.g., BABP) are fused (i.e., covalently linked) to each other. Thus, the anti-LAG-3 MABPs (e.g., BABPs) of the present application comprise one or more fusion polypeptides. Each fusion polypeptide may comprise the first antigen binding portion comprising an anti-LAG-3 sdAb described herein, and a polypeptide from the second antigen binding portion.

[0190] The first antigen binding portion comprising an anti-LAG-3 sdAb described herein and the second antigen binding portion comprising a V H and a V L may be linked directly by a single chemical bond (such as peptide bond) or via a peptide linker. The first antigen binding portion comprising an anti-LAG-3 sdAb may be fused at either the N-terminus or the C-terminus of any one (including each) polypeptide of the second antigen binding portion, or may be fused at an internal position of any one (including each) polypeptide of the second antigen binding portion, such as at the N-terminus of the Fc region in the heavy chain of the second antigen binding portion. The fusion polypeptides may be obtained either recombinantly or chemically. In some embodiments, the C-terminus of the first antigen binding portion comprising an anti-LAG-3 sdAb is fused to the N-terminus of any (including each) polypeptide of the second antigen binding portion via a chemical bond (such as peptide bond) or a peptide linker. In some embodiments, the N-terminus of the first antigen binding portion comprising an anti-LAG-3 sdAb is fused to the C-terminus of any (including each) polypeptide of the second antigen binding portion via a chemical bond (such as peptide bond) or a peptide linker. In some embodiments, the first antigen binding portion comprising an anti-LAG-3 sdAb is fused to the second antigen binding portion via a chemical bond that is not a peptide bond involving the main chain chemical groups of amino acids.

[0191] In some embodiments, the second antigen binding portion comprises a single-chain antibody fragment comprising the V H and V L . In some embodiments, the second antigen binding portion comprises an scFv. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises a fusion polypeptide comprising in the N-terminus to C-terminus direction: the first antigen binding portion comprising the anti-LAG-3 sdAb described herein, an optional peptide linker, the V H domain and the V L domain. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises a fusion polypeptide comprising in the N-terminus to C-terminus direction: the first antigen binding portion comprising the anti-LAG-3 sdAb described herein, an optional peptide linker, the V L domain and the V H domain. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises a fusion polypeptide comprising in the N-terminus to C-terminus direction: the V H domain, the V L domain, an optional peptide linker, and the first antigen binding portion comprising the anti-LAG-3 sdAb described herein. In some embodiments, anti-LAG-3 MABP (e.g., BABP) comprises a fusion polypeptide comprising in the N-terminus to C-terminus direction: the V L domain, the V H domain, an optional peptide linker, and the first antigen binding portion comprising the anti-LAG-3 sdAb described herein.

[0192] In some embodiments, the second antigen binding portion comprises a heavy chain comprising the V H domain, and a light chain comprising the V L domain. In some embodiments, the heavy chain further comprises one or more heavy chain constant domains, such as C H 1, C H 2, C H 3, and C H 4, and / or an antibody hinge region (HR). In some embodiments, the light chain further comprises a light chain constant domain (C L ), such as the lambda C L domain or kappa C L domain. In some embodiments, the N-terminus of the first antigen binding portion comprising the anti-LAG-3 sdAb described herein is fused to the C-terminus of the heavy chain. In some embodiments, the C-terminus of the first antigen binding portion comprising the anti-LAG-3 sdAb is fused to the N-terminus of the heavy chain. In some embodiments, the N-terminus of the first antigen binding portion comprising the anti-LAG-3 sdAb is fused to the C-terminus of the light chain. In some embodiments, the C-terminus of the first antigen binding portion comprising the anti-LAG-3 sdAb is fused to the N-terminus of the light chain. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises a first polypeptide comprising from the N-terminus to the C-terminus: the heavy chain, an optional peptide linker, and the first antigen binding portion comprising the anti-LAG-3 sdAb and an optional peptide sequence; and a second polypeptide comprising the light chain. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises a first polypeptide comprising from the N-terminus to the C-terminus: the first antigen binding portion comprising an optional peptide sequence and the anti-LAG-3 sdAb, an optional peptide linker, and the heavy chain; and a second polypeptide comprising the light chain. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises a first polypeptide comprising from the N-terminus to the C-terminus: the light chain, an optional peptide linker, and the first antigen binding portion comprising the anti-LAG-3 sdAb and an optional peptide sequence; and a second polypeptide comprising the heavy chain. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises a first polypeptide comprising from the N-terminus to the C-terminus: the first antigen binding portion comprising an optional peptide sequence and the anti-LAG-3 sdAb, an optional peptide linker, and the light chain; and a second polypeptide comprising the heavy chain. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises two identical first polypeptides and two identical second polypeptides described herein.

[0193] In some embodiments, the second antigen binding portion comprises a full-length antibody consisting of two heavy chains and two light chains. In some embodiments, the full-length antibody is a full-length monoclonal antibody consisting of two identical heavy chains and two identical light chains. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises two identical first polypeptides each comprising from the N-terminus to the C- terminus the heavy chain, an optional peptide linker, the first antigen binding portion comprising the anti-LAG-3 sdAb and an optional peptide sequence; and two identical second polypeptides each comprising the light chain (e.g., FIG. 8). In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises two identical first polypeptides each comprising from the N-terminus to the C-terminus: the first antigen binding portion comprising an optional peptide sequence and the anti-LAG-3 sdAb, an optional peptide linker, and the heavy chain; and two identical second polypeptides each comprising the light chain (e.g., FIG. 7). In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises two identical first polypeptides each comprising from the N-terminus to the C-terminus: the light chain, an optional peptide linker, and the first antigen binding portion comprising the anti-LAG-3 sdAb and an optional peptide sequence; and two identical second polypeptides each comprising the heavy chain (e.g., FIG. 10). In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises two identical first polypeptides each comprising from the N-terminus to the C-terminus: the first antigen binding portion comprising an optional peptide sequence and the anti-LAG-3 sdAb, an optional peptide linker, and the light chain; and two identical second polypeptides comprising the heavy chain (e.g., FIG. 9).

[0194] In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises: (a) a full-length antibody consisting of a first and a second heavy chains and a first and a second light chains, wherein the full-length antibody specifically recognizes a first epitope (e.g., PD-1); (b) a first anti-LAG-3 sdAb; (c) a second anti-LAG-3 sdAb; (d) a third anti-LAG-3 sdAb; and (e) a fourth anti-LAG-3 sdAb; wherein the C-terminus of the first anti-LAG-3 sdAb is fused to the N-terminus of the first light chain, wherein the C-terminus of the second anti-LAG-3 sdAb is fused to the N-terminus of the second light chain, wherein the C-terminus of the third anti-LAG-3 sdAb is fused to the N-terminus of the first heavy chain, and wherein the C-terminus of the fourth anti-LAG-3 sdAb is fused to the N-terminus of the second heavy chain. In some embodiments, the four anti-LAG-3 sdAbs are different. In some embodiments, the four anti-LAG-3 sdAbs are identical. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises two identical first polypeptides each comprising from the N-terminus to the C-terminus: an optional peptide sequence, the third or the fourth anti-LAG-3 sdAb, an optional peptide linker, and the heavy chain; and two identical second polypeptides each comprising an optional peptide sequence, the first or the second anti-LAG-3 sdAb, an optional peptide linker, and the light chain. See, for example, FIG. 11.

[0195] In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises: (a) a full-length antibody consisting of two heavy chains and two light chains, wherein the full-length antibody specifically recognizes a first epitope (e.g., PD-1); (b) a first anti-LAG-3 sdAb; (c) a second anti-LAG-3 sdAb; (d) a third anti-LAG-3 sdAb; and (e) a fourth anti-LAG-3 sdAb; wherein the C-terminus of the first anti-LAG-3 sdAb is fused to the N-terminus of the second anti-LAG-3 sdAb, and the C-terminus of the second anti-LAG-3 sdAb is fused to the N-terminus of one heavy chain, and wherein the C-terminus of the third anti-LAG-3 sdAb is fused to the N-terminus of the fourth anti-LAG-3 sdAb, and the C-terminus of the fourth anti-LAG-3 sdAb is fused to the N-terminus of the other heavy chain. In some embodiments, the four anti-LAG-3 sdAbs are different. In some embodiments, the four anti-LAG-3 sdAbs are identical. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises two identical first polypeptides each comprising from the N-terminus to the C-terminus: an optional peptide sequence, the first or the third anti-LAG-3 sdAb, an optional peptide linker, the second or the fourth anti-LAG-3 sdAb, an optional peptide linker, and the heavy chain; and two identical second polypeptides each comprising the light chain. See, for example, FIG. 12.

[0196] In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises: (a) a full-length antibody consisting of two heavy chains and two light chains, wherein the full-length antibody specifically recognizes a first epitope (e.g., PD-1); (b) a first anti-LAG-3 sdAb; and (c) a second anti-LAG-3 sdAb, wherein the N-terminus of the first or the second anti-LAG-3 sdAb is fused to the C-terminus of the C H 1 region of the heavy chain, and the C-terminus of the first or the second anti-LAG-3 sdAb is fused to the N-terminus of the C H 2 region of the heavy chain. In some embodiments, the two anti-LAG-3 sdAbs are identical. In some embodiments, the two anti-LAG-3 sdAbs are different. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises two identical first polypeptides each comprising from the N-terminus to the C-terminus: V H -C H 1-an optional peptide linker-anti-LAG-3 sdAb-C H 2-C H 3; and two identical second polypeptides each comprising the light chain. See, for example, FIG. 13.

[0197] In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises: (a) a first scFv that specifically recognizes a first epitope (e.g., PD-1); (b) a second scFv that specifically recognizes a second epitope (e.g., PD-1); (c) an Fc region; (d) a first anti-LAG-3 sdAb; and (e) a second anti-LAG-3 sdAb, wherein the N-terminus of each anti-LAG-3 sdAb is fused to the C-terminus of an scFv and the C-terminus of the anti-LAG-3 sdAb is fused to the N-terminus of the Fc region. In some embodiments, the two anti-LAG-3 sdAbs are identical. In some embodiments, the two anti-LAG-3 sdAbs are different. In some embodiments, the two scFvs are identical. In some embodiments, the two scFvs are different. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises two identical polypeptides each comprising from the N-terminus to the C-terminus: scFv-an optional peptide linker-anti-LAG-3 sdAb-CH 2 -CH 3 , such as V H -V L -an optional peptide linker-anti-LAG-3 sdAb-CH 2 -CH 3 , or V L -V H -an optional peptide linker-anti-LAG-3 sdAb-CH 2 -CH 3 . See, for example, FIG. 14.

[0198] In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises: (a) a first Fab that specifically recognizes a first epitope (e.g., PD-1); (b) a second Fab that specifically recognizes a second epitope (e.g., PD-1); (c) an Fc region; (d) a first Fab-like domain comprising a first anti-LAG-3 sdAb and a second anti-LAG-3 sdAb; (e) a second Fab-like domain comprising a third anti-LAG-3 sdAb and a fourth anti-LAG-3 sdAb, wherein the N-termini of each Fab-like domain are fused to the C-termini of a Fab and one of the two C-termini of the Fab-like domain is fused to the N-terminus of the Fc region. In some embodiments, the four anti-LAG-3 sdAbs are identical. In some embodiments, the four anti-LAG-3 sdAbs are different. In some embodiments, the two Fabs are identical. In some embodiments, the two Fabs are different. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises two identical first polypeptides each comprising from the N-terminus to the C-terminus: V H -C H 1-an optional peptide linker-anti-LAG-3 sdAb-C H 1-C H 2-C H 3; and two identical second polypeptides each comprising from the N-terminus to the C-terminus: V L -C L -an optional peptide linker-anti-LAG-3 sdAb-C L . See, for example, FIG. 15.

[0199] In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises: (a) a first scFv that specifically recognizes a first epitope (e.g., PD-1); (b) a second scFv that specifically recognizes a second epitope (e.g., PD-1); (c) an Fc region; (d) a first Fab-like domain comprising a first anti-LAG-3 sdAb and a second anti-LAG-3 sdAb; (e) a second Fab-like domain comprising a third anti-LAG-3 sdAb and a fourth anti-LAG-3 sdAb, wherein one of the two N-termini of each Fab-like domain is fused to the C-terminus of an scFv and one of the two C-termini of the Fab-like domain is fused to the N-terminus of the Fc region. In some embodiments, the four anti-LAG-3 sdAbs are identical. In some embodiments, the four anti-LAG-3 sdAbs are different. In some embodiments, the two scFvs are identical. In some embodiments, the two scFvs are different. In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises two identical first polypeptides each comprising from the N-terminus to the C-terminus: scFv-an optional peptide linker-anti-LAG-3 sdAb-C H 1-C H 2-C H 3; and two identical second polypeptides each comprising from the N-terminus to the C-terminus: anti-LAG-3 sdAb-C L . In some embodiments, the scFv comprises from the N-terminus to the C-terminus: V H -V L or V L -V H . See, for example, FIG. 16.

[0200] The anti-LAG-3 MABPs (e.g., BABPs) described herein may comprise one or more peptide linkers situated between the first antigen binding portion and the second antigen binding portion. In some embodiments, the peptide linker between the heavy chain polypeptide of the second antigen binding portion and the first antigen binding portion is the same as the peptide linker between the light chain polypeptide of the second antigen binding portion and the first antigen binding portion. In some embodiments, the peptide linker between the heavy chain polypeptide of the second antigen binding portion and the first antigen binding portion is different from the peptide linker between the light chain polypeptide of the second antigen binding portion and the first antigen binding portion. In some embodiments, the first antigen binding portion and the second antigen binding portion are directly fused to each other without a peptide linker disposed therebetween. The peptide linker between the two or more anti-LAG-3 sdAbs may be the same as or different from that between the anti-LAG-3 sdAb and the second antigen binding portion. Any of the peptide linkers described below in the "Peptide linkers" section above can be employed in any of the anti-LAG-3 MABPs (e.g., BABPs) described herein.b) Second antigen binding portion comprising V H and V L

[0201] The anti-LAG-3 MABPs (e.g., BABPs) comprise at least one second antigen binding portion comprising a V H and a V L . Such antigen binding portion can be a full-length conventional antibody consisting of two heavy chains and two light chains, or an antigen binding fragment derived therefrom, e.g., Fab, scFv.

[0202] In some embodiments, the second antigen binding portion is an antigen binding fragment comprising a heavy chain comprising the V H domain and a light chain comprising the V L domain. Exemplary antigen binding fragments contemplated herein include, but are not limited to, Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (such as scFv); and multispecific antibodies formed from antibody fragments.

[0203] In some embodiments, the second antigen binding portion comprises an Fc region, such as a human Fc region. In some embodiments, the Fc region is derived from an IgG molecule, such as any one of the IgG1, IgG2, IgG3, or IgG4 subclass. In some embodiments, the Fc region is capable of mediating an antibody effector function, such as ADCC and / or CDC. For example, antibodies of subclass IgG1, IgG2, and IgG3 with wildtype Fc sequences usually show complement activation including CIq and C3 binding, whereas IgG4 does not activate the complement system and does not bind CIq and / or C3. In some embodiments, the Fc region comprises a modification that reduces binding affinity of the Fc region to an Fc receptor. In some embodiments, the Fc region is an IgG1 Fc. In some embodiments, the IgG1 Fc comprises one or mutations in positions 233-236, such as L234A and / or L235A. In some embodiments, the Fc region is an effectorless IgG1 Fc. In some embodiments, the Fc region is an IgG4 Fc. In some embodiments, the IgG4 Fc comprises a mutation in positions 327, 330 and / or 331. See, for example, Armour KL et al., Eur J. Immunol. 1999; 29: 2613; and Shields RL et al., J. Biol. Chem. 2001; 276: 6591. In some embodiments, the Fc region comprises a P329G mutation. In some embodiments, the Fc region is an IgG4 Fc (S228P). In some embodiments, the Fc region comprises an amino acid sequence of any one of SEQ ID NOs: 363-365.

[0204] In some embodiments, the Fc region comprises a modification that promotes heterodimerization of two non-identical heavy chains. Such modified Fc regions may be of particular interest for anti-LAG-3 MABPs (e.g., BABPs) described herein having an asymmetric design. In some embodiments, said modification is a knob-into-hole modification, comprising a knob modification in one of the heavy chains or heavy chain fusion polypeptides and a hole modification in the other one of the two heavy chains or heavy chain fusion polypeptides. In one embodiment, the Fc region comprises a modification within the interface between the two heavy chains in the CH3 domain, wherein i) in the CH3 domain of one heavy chain, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance ("knob") within the interface in the CH3 domain of one heavy chain which is positionable in a cavity ("hole") within the interface in the CH3 domain of the other heavy chain, and ii) in the CH3 domain of the other heavy chain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity ("hole") within the interface in the second CH3 domain within which a protuberance ("knob") within the interface in the first CH3 domain is positionable. Examples of knob-into-hole modifications have been described, for example, in US 2011 / 0287009, US2007 / 0178552, WO 96 / 027011, WO 98 / 050431, and Zhu et al., 1997, Protein Science 6:781-788. Other modifications to the Fc region that promote heterodimerization are also contemplated herein. For example, electrostatic steering effects can be engineered into the Fc region to provide Fc-heterodimeric molecules (see, e.g., US4676980, and Brennan et al., Science, 229: 81 (1985)). In some embodiments, the Fc region comprises a modification that inhibits Fab arm exchange. For example, the S228P mutation in IgG4 Fc prevents Fab arm exchange.

[0205] In some embodiments, the second antigen binding portion comprises a kappa light chain constant region. In some embodiments, the second antigen binding portion comprises a lambda light chain constant region. In some embodiments, the second antigen binding portion comprises a heavy chain constant region.

[0206] In some embodiments, the second antigen binding portion is a full-length antibody consisting of two heavy chains and two light chains. In some embodiments, the second antigen binding portion comprises a monoclonal antibody consisting of two heavy chains and two light chains (also referred herein as "4-chain antibody"). In some embodiments, the second antigen binding portion comprises a multispecific (e.g., bispecific) full-length antibody consisting of two heavy chains and two light chains. In some embodiments, the second antigen binding portion comprises a full-length antibody of human IgG1 subclass, of effectorless hIgG1 subclass, or of human IgG1 subclass with the mutations L234A and L235A. In some embodiments, the second antigen binding portion comprises a full-length antibody of human IgG2 subclass. In some embodiments, the second antigen binding portion comprises a full-length antibody of human IgG3 subclass. In some embodiments, the second antigen binding portion comprises a full-length antibody of human IgG4 subclass or, of human IgG4 subclass with the additional mutation S228P.

[0207] Any full-length 4-chain antibody known in the art or antigen binding fragments derived therefrom can be used as the second antigen binding portion of the anti-LAG-3 MABP (e.g. BABP) described herein. Antibodies or antibody fragments with proven clinical efficacy, safety, and pharmacokinetics profile are of particular interest. In some embodiments, the antibody or antibody fragment known in the art is further engineered, such as humanized or mutagenized to select for a variant with a suitable affinity, prior to fusion with the first antigen binding portion to provide the anti-LAG-3 MABP (e.g., BABP). In some embodiments, the second antigen binding portion comprises the V H and V L domains of a monoclonal antibody or antibody fragment known in the art, and modified heavy chain constant region and / or light chain constant region. In some embodiments, the second antigen binding portion comprises the monoclonal antibody known in the art and a modified Fc region, such as an IgG4 Fc with an S228P mutation, or an effectorless IgG1 Fc. In some embodiments, the second antigen binding portion comprises a human, humanized, or chimeric full-length antibody or antibody fragments.c) Exemplary anti-LAG-3 MABPs

[0208] In some embodiments, the anti-LAG-3 construct is an anti-LAG-3 MABP comprising a second antigen binding portion that specifically recognizes PD-1. In some embodiments, the second antigen binding portion comprises an anti-PD-1 sdAb. In some embodiments, the second antigen binding portion comprises a full-length anti-PD-1 antibody consisting of two heavy chains and two light chains.

[0209] PD-1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS and BTLA. PD-1 contains a membrane proximal immunoreceptor tyrosine inhibitory motif (ITIM) and a membrane distal tyrosine-based switch motif (ITSM) (Thomas, M.L. (1995) J Exp Med 181:1953-6; Vivier, E and Daeron, M (1997) Immunol Today 18:286-91). Although structurally similar to CTLA-4, PD-1 lacks the MYPPPY motif that is critical for B7-1 and B7-2 binding.

[0210] PD-1 regulates T-cell activation and tolerance, and thus antagonistic anti-PD-1 antibodies can be useful for overcoming tolerance. PD-1 has been defined as a receptor for B7-4. B7-4 can inhibit immune cell activation upon binding to an inhibitory receptor on an immune cell. Engagement of the PD-1 / PD-L1 pathway results in inhibition of T-cell effector function, cytokine secretion and proliferation (Turnis et al., OncoImmunology 1(7):1172-1174, 2012). High levels of PD-1 are associated with exhausted or chronically stimulated T cells. Moreover, increased PD-1 expression correlates with reduced survival in cancer patients. Agents for down modulating PD-1, B7-4, and the interaction between B7-4 and PD-1 inhibitory signal in an immune cell can result in enhancement of the immune response.

[0211] In some embodiments, there is provided an anti-LAG-3 construct comprising an anti-LAG-3 sdAb described herein fused to an anti-PD-1 sdAb. In some embodiments, the anti-LAG-3 sdAb is fused to the anti-PD-1 sdAb via a linker (such as a peptide linker). In some embodiments, the anti-LAG-3 construct comprises a plurality (such as 2, 3, 4, or more) of anti-LAG-3 sdAbs, which can be the same or different. In some embodiments, the anti-LAG-3 construct comprises a plurality (such as 2, 3, 4, or more) of anti-PD-1 sdAbs, which can be the same or different. The anti-PD-1 sdAb can be derived from any known anti-PD-1 sdAbs, such as those disclosed in PCT / CN2018 / 071729. The anti-LAG-3 construct can be of any suitable format, for example, from N- to C-terminus: (anti-LAG-3 sdAb)-L 1 -(anti-PD-1 sdAb) or (anti-PD-1 sdAb)-L 1 -(anti-LAG-3 sdAb), wherein L 1 is a chemical bond or a linker, such as peptide linker.

[0212] Exemplary 4-chain anti-PD-1 antibodies that can be used for making multispecific anti-LAG_3 constructs described herein include, but are not limited to, pembrolizumab (e.g., KEYTRUDA ®< ), nivolumab (e.g., OPDIVO ®< ), PD1-BM-min, including biosimilars thereof, derivatives thereof, and antibodies having the same sequences.

[0213] Described and not claimed per se is an anti-PD-1 full-length antibody or antigen binding fragment thereof (e.g., Fab, scFv). In some embodiments, the anti-PD-1 full-length antibody or antigen binding fragment thereof (e.g., Fab, scFv) comprises: (i) a V H comprising HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 356, and a V L comprising LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 357; or (ii) a V H comprising HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 373, and a V L comprising LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 374. The anti-PD-1 full-length antibody may comprise: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 356, and a light chain comprising the amino acid sequence of SEQ ID NO: 357; or (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 373, and a light chain comprising the amino acid sequence of SEQ ID NO: 374. In some embodiments, the anti-PD-1 full-length antibody is pembrolizumab (e.g., KEYTRUDA ®< ) or PD1-BM-min.

[0214] In some embodiments, the anti-LAG-3 MABP (e.g., BABP) comprises (a) a first antigen binding portion comprising an anti-LAG-3 sdAb described herein, and (b) a second antigen binding portion comprising a V H and a V L , wherein the V H and V L together form an antigen-binding site that specifically binds PD-1, wherein the first antigen binding portion and the second antigen binding portion are fused to each other (herein after referred to as "LAG-3×PD-1 MABP" or "LAG-3×PD-1 BABP"). Exemplary anti-LAG-3 / PD-1 BABPs are shown in FIGs. 7-14 and Table 7.

[0215] Described and not claimed per se is an anti-LAG-3 BABP comprising: (a) a first polypeptide comprising from N-terminus to C-terminus: anti-LAG-3 sdAb-V H -C H 1-C H 2-C H 3; and (b) a second polypeptide comprising from N-terminus to C-terminus: V L -C L , wherein V H and V L forms an antigen binding site that specifically binds a second epitope (e.g., an immune checkpoint molecule, such as PD-1), and wherein the anti-LAG-3 sdAb comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. The anti-LAG-3 sdAb may comprise: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221; or (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223. In the present invention as defined by claim 1, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NO: 304, 288, 306, 295, or 275-278. In some embodiments, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NO: 288, 295, 304 or 306. In some embodiments, V H and V L form an antigen binding site that specifically binds PD-1. In some embodiments, the V H and V L domains are derived from pembrolizumab or PD1-BM-min. Described and not claimed per se is that the V H may comprise HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 356, and V L comprises LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 357. The V H may comprise HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 373, and V L may comprise LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 374. In some embodiments, the C H 3 and anti-LAG-3 sdAb are fused to each other via a peptide linker, such as a peptide linker comprising the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. In some embodiments, the N-terminus of the anti-LAG-3 sdAb is fused to a peptide sequence. In some embodiments, the peptide sequence comprises the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. In some embodiments, the C H 2 and C H 3 domains are derived from IgG1 Fc, effectorless IgG1 Fc, IgG2 Fc, IgG4 Fc, or IgG4 Fc (S228P).

[0216] Described and not claimed per se is an anti-LAG-3 BABP comprising: (a) a first polypeptide comprising from N-terminus to C-terminus: V H -C H 1-C H 2-C H 3-anti-LAG-3 sdAb; and (b) a second polypeptide comprising from N-terminus to C-terminus: V L -C L , wherein V H and V L forms an antigen binding site that specifically binds a second epitope (e.g., an immune checkpoint molecule, such as PD-1), and wherein the anti-LAG-3 sdAb comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. The anti-LAG-3 sdAb may comprise: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221; or (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223. In the present invention defined by claim 1, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NO: 304, 288, 306, 295, or 275-278. In some embodiments, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NO: 288, 295, 304 or 306. In some embodiments, V H and V L form an antigen binding site that specifically binds PD-1. In some embodiments, the V H and V L domains are derived from pembrolizumab or PD1-BM-min. Described and not claimed per se is that the V H comprises HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 356, and V L comprises LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 357. The V H may comprise HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 373, and V L comprises LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 374. In some embodiments, the C H 3 and anti-LAG-3 sdAb are fused to each other via a peptide linker, such as a peptide linker comprising the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. In some embodiments, the C-terminus of the anti-LAG-3 sdAb is fused to a peptide sequence. In some embodiments, the peptide sequence comprises the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. In some embodiments, the C H 2 and C H 3 domains are derived from IgG1 Fc, effectorless IgG1 Fc, IgG2 Fc, IgG4 Fc, or IgG4 Fc (S228P).

[0217] Described and not claimed per se is an anti-LAG-3 BABP comprising: (a) a first polypeptide comprising from N-terminus to C-terminus: V H -C H 1-C H 2-C H 3; and (b) a second polypeptide comprising from N-terminus to C-terminus: anti-LAG-3 sdAb-V L -C L , wherein V H and V L forms an antigen binding site that specifically binds a second epitope (e.g., an immune checkpoint molecule, such as PD-1), and wherein the anti-LAG-3 sdAb comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. The anti-LAG-3 sdAb may comprise: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221; or (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223. In the present invention defined by claim 1, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NO: 304, 288, 306, 295, or 275-278. In some embodiments, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NO: 288, 295, 304 or 306. In some embodiments, V H and V L form an antigen binding site that specifically binds PD-1. In some embodiments, the V H and V L domains are derived from pembrolizumab or PD1-BM-min. Described and not claimed per se is that the V H comprises HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 356, and V L comprises LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 357. The V H may comprise HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 373, and V L comprises LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 374. In some embodiments, the C H 3 and anti-LAG-3 sdAb are fused to each other via a peptide linker, such as a peptide linker comprising the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. In some embodiments, the N-terminus of the anti-LAG-3 sdAb is fused to a peptide sequence. In some embodiments, the peptide sequence comprises the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. In some embodiments, the C H 2 and C H 3 domains are derived from IgG1 Fc, effectorless IgG1 Fc, IgG2 Fc, IgG4 Fc, or IgG4 Fc (S228P).

[0218] Described and not claimed per se is an anti-LAG-3 BABP comprising: (a) a first polypeptide comprising from N-terminus to C-terminus: V H -C H 1-C H 2-C H 3; and (b) a second polypeptide comprising from N-terminus to C-terminus: V L -C L -anti-LAG-3 sdAb, wherein V H and V L forms an antigen binding site that specifically binds a second epitope (e.g., an immune checkpoint molecule, such as PD-1), and wherein the anti-LAG-3 sdAb comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. The anti-LAG-3 sdAb may comprise: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221; or (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223. In the present invention defined by claim 1, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NO: 304, 288, 306, 295, or 275-278. In some embodiments, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NO: 288, 295, 304 or 306. In some embodiments, V H and V L form an antigen binding site that specifically binds PD-1. In some embodiments, the V H and V L domains are derived from pembrolizumab or PD1-BM-min. Described and not claimed per se is that the V H comprises HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 356, and V L comprises LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 357. The V H may comprise HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 373, and V L comprises LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 374. In some embodiments, the C H 3 and anti-LAG-3 sdAb are fused to each other via a peptide linker, such as a peptide linker comprising the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. In some embodiments, the C-terminus of the anti-LAG-3 sdAb is fused to a peptide sequence. In some embodiments, the peptide sequence comprises the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. In some embodiments, the C H 2 and C H 3 domains are derived from IgG1 Fc, effectorless IgG1 Fc, IgG2 Fc, IgG4 Fc, or IgG4 Fc (S228P).

[0219] Described and not claimed per se is an anti-LAG-3 BABP comprising: (a) a first polypeptide comprising from N-terminus to C-terminus: V H -C H 1-anti-LAG-3 sdAb-C H 2-C H 3; and (b) a second polypeptide comprising from N-terminus to C-terminus: V L -C L , wherein V H and V L forms an antigen binding site that specifically binds a second epitope (e.g., an immune checkpoint molecule, such as PD-1), and wherein the anti-LAG-3 sdAb comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. The anti-LAG-3 sdAb may comprise: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221; or (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223. In the present invention defined by claim 1, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NO: 304, 288, 306, 295, or 275-278. In some embodiments, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NO: 288, 295, 304 or 306. In some embodiments, V H and V L form an antigen binding site that specifically binds PD-1. In some embodiments, the V H and V L domains are derived from pembrolizumab or PD1-BM-min. Described and not claimed per se is that the V H comprises HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 356, and V L comprises LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 357. The V H may comprise HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 373, and V L comprises LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 374. In some embodiments, the C H 3 and anti-LAG-3 sdAb are fused to each other via a peptide linker, such as a peptide linker comprising the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. In some embodiments, the C H 2 and C H 3 domains are derived from IgG1 Fc, effectorless IgG1 Fc, IgG2 Fc, IgG4 Fc, or IgG4 Fc (S228P).

[0220] Described and not claimed per se is an anti-LAG-3 BABP comprising a polypeptide comprising from N-terminus to C-terminus: V L -V H -anti-LAG-3 sdAb-CH 2 -CH 3 , wherein the V L and V H together forms an scFv that specifically binds a second epitope (e.g., an immune checkpoint molecule, such as PD-1), and wherein the anti-LAG-3 sdAb comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 39-76, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 115-152, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 191-228, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions. The anti-LAG-3 sdAb may comprise: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53; a CDR2 comprising the amino acid sequence of SEQ ID NO: 129; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 205; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 60; a CDR2 comprising the amino acid sequence of SEQ ID NO: 136; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 212; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 145; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 221; or (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 147; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 223. In the present invention defined by claim 1, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NO: 304, 288, 306, 295, or 275-278. In some embodiments, the anti-LAG-3 sdAb comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NO: 288, 295, 304 or 306. In some embodiments, V H and V L form an antigen binding site that specifically binds PD-1. In some embodiments, the V H and V L domains are derived from pembrolizumab or PD1-BM-min. Described and not claimed per se is that the V H comprises HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 356, and V L comprises LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 357. The V H may comprise HC-CDR1, HC-CDR2, and HC-CDR3 of the amino acid sequence of SEQ ID NO: 373, and V L comprises LC-CDR1, LC-CDR2, and LC-CDR3 of the amino acid sequence of SEQ ID NO: 374. In some embodiments, the C H 3 and anti-LAG-3 sdAb are fused to each other via a peptide linker, such as a peptide linker comprising the amino acid sequence of any one of SEQ ID NOs: 352-353 and 366-372. In some embodiments, the C H 2 and C H 3 domains are derived from IgG1 Fc, effectorless IgG1 Fc, IgG2 Fc, IgG4 Fc, or IgG4 Fc (S228P).

[0221] In some embodiments, the anti-LAG-3 construct comprises two identical copies of the first polypeptide and two identical copies of the second polypeptide. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 358, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 359. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 360, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 361. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 362, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 363. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 364, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 365. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 375, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 376. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 377, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 378. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 379, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 380. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 381, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 382. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 383, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 384. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 385, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 386. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 387, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 388. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 389, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 390. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 391, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 392. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 393, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 394. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 395, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 396. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 397, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 398. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 399, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 400. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 401, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 402. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 403, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 404. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 405, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 406. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 407, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 408. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 409, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 410. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 411, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 412. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 413, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 414. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 415, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 416. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 417, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 418. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 419, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 420. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 421, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 422. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 423, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 424. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 425, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 426. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 427, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 428. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 429, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 430. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 431, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 432. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 433, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 434. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 435, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 436. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 437, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 438. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 439, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 440. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 441, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 442. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 443, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 444. In some embodiments, there is provided an anti-LAG-3 construct (e.g., BABP) comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 445, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 446. In some embodiments, there is provided a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 358-365 and 375-446.Peptide linkers

[0222] The various domains and components, such as anti-LAG-3 sdAbs, Fc fragment, the first antigen binding portion, and the second antigen binding portion in the anti-LAG3 construct may be fused to each other via a suitable linker, such as a peptide linker. The length, the degree of flexibility and / or other properties of the peptide linker(s) used in the anti-LAG-3 construct may have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more particular antigens or epitopes. For example, longer peptide linkers may be selected to ensure that two adjacent domains do not sterically interfere with one another. In some embodiment, a peptide linker comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker.

[0223] The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids long. In some embodiments, the peptide linker is no more than about any of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids long. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0224] The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO1996 / 34103. In some embodiments, the peptide linker is a human IgG1 hinge (SEQ ID NO: 352). In some embodiments, the peptide linker is a mutated human IgG1 hinge (SEQ ID NO: 353). In some embodiments, the peptide linker is a human IgG4 hinge. In some embodiments, the peptide linker is a hIgG2 hinge. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G) n (SEQ ID NO: 368), glycine-serine polymers (including, for example, (GS) n (SEQ ID NO: 369), (GSGGS) n (SEQ ID NO: 370), (GGGS) n (SEQ ID NO: 371), and (GGGGS) n (SEQ ID NO: 372), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 366 (GGGGSGGGS) or 367 (GGGGSGGGGSGGGGS).

[0225] In some embodiments, the N-terminus or the C-terminus of the anti-LAG-3 sdAb may be fused to a peptide sequence, which may have any of the sequence features described herein for peptide linkers.(III) Antibody variants

[0226] In some embodiments, amino acid sequence variants of the anti-LAG-3 construct (e.g., anti-LAG-3 sdAb, anti-LAG-3 sdAb-Fc fusion protein (e.g., HCAb), anti-LAG-3 MABP / BABP) provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.a) Substitution, insertion, deletion and variants

[0227] In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs (or CDRs) and FRs. Conservative substitutions are shown in Table 2 under the heading of "Preferred substitutions." More substantial changes are provided in Table 2 under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. Table 2. Amino acid substitutions Original Residue Exemplary Substitutions Preferred Substitutions Ala (A)Val; Leu; IleValArg (R)Lys; Gln; AsnLysAsn (N)Gln; His; Asp, Lys; ArgGlnAsp (D)Glu; AsnGluCys (C)Ser; AlaSerGln (Q)Asn; GluAsnGlu (E)Asp; GlnAspGly (G)AlaAlaHis (H)Asn; Gln; Lys; ArgArgIle (I)Leu; Val; Met; Ala; Phe; NorleucineLeuLeu (L)Norleucine; Ile; Val; Met; Ala; PheIleLys (K)Arg; Gln; AsnArgMet (M)Leu; Phe; IleLeuPhe (F)Trp; Leu; Val; Ile; Ala; TyrTyrPro (P)AlaAlaSer (S)ThrThrThr (T)Val; SerSerTrp (W)Tyr; PheTyrTyr (Y)Trp; Phe; Thr; SerPheVal (V)Ile; Leu; Met; Phe; Ala; NorleucineLeu

[0228] Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0229] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0230] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).

[0231] Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0232] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR "hotspots" or CDRs. In some embodiments of the variant V H H sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

[0233] A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0234] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.b) Glycosylation variants

[0235] In some embodiments, an isolated anti-LAG-3 construct provided herein is altered to increase or decrease the extent to which the construct is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0236] Where the anti-LAG-3 construct comprises an Fc region (e.g., anti-LAG-3 sdAb-Fc fusion protein (e.g., HCAb), LAG-3× PD-1 MABP), the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the C H 2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an anti-LAG-3 construct of the present application may be made in order to create antibody variants with certain improved properties.

[0237] In some embodiments, anti-LAG-3 construct antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0238] Anti-LAG-3 construct variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003 / 011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).c) Fc region variants

[0239] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the anti-LAG-3 constructs provided herein (e.g., anti-LAG-3 HCAbs, or anti-LAG-3 MABPs), thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

[0240] In some embodiments, the present application contemplates an anti-PD-1 construct (e.g., anti-LAG-3 HCAb, or anti-LAG-3 MABP) variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the anti-LAG-3 construct in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI ™< non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96 ®< non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0241] Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

[0242] Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0243] In some embodiments, an anti-LAG-3 construct variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0244] In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0245] In some embodiments, there is provided an anti-LAG-3 construct (e.g., anti-PD-1 sdAb-Fc fusion protein (e.g., anti-LAG-3 HCAb, or anti-LAG-3 MABP) variant comprising a variant Fc region comprising one or more amino acid substitutions which increase half-life and / or improve binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826).

[0246] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 concerning other examples of Fc region variants.

[0247] Anti-LAG-3 constructs (e.g., anti-LAG-3 HCAbs or anti-LAG-3 MABPs) comprising any of the Fc variants described herein, or combinations thereof, are contemplated.d) Cysteine engineered antibody variants

[0248] In some embodiments, it may be desirable to create cysteine engineered anti-LAG-3 constructs, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In some embodiments, any one or more of the following residues may be substituted with cysteine: A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered anti-LAG-3 constructs may be generated as described, e.g., in U.S. Patent No. 7,521,541.e) Antibody derivatives

[0249] In some embodiments, an anti-LAG-3 construct provided herein may be further modified to comprise additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

[0250] In some embodiments, conjugates of an anti-LAG-3 construct and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In some embodiments, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.

[0251] In some embodiments, an anti-LAG-3 construct provided herein (e.g., anti-LAG-3 HCAb, or anti-LAG-3 MABP) may be further modified to comprise one or more biologically active protein, polypeptides or fragments thereof. "Bioactive" or "biologically active", as used herein interchangeably, means showing biological activity in the body to carry out a specific function. For example, it may mean the combination with a particular biomolecule such as protein, DNA, etc., and then promotion or inhibition of the activity of such biomolecule. In some embodiments, the bioactive protein or fragments thereof include proteins and polypeptides that are administered to patients as the active drug substance for prevention of or treatment of a disease or condition, as well as proteins and polypeptides that are used for diagnostic purposes, such as enzymes used in diagnostic tests or in vitro assays, as well as proteins and polypeptides that are administered to a patient to prevent a disease such as a vaccine. In some embodiments, the bioactive protein or fragments thereof have immune-stimulatory / immune-regulatory, membrane transport, or enzymatic activities. In some embodiments, the biologically active protein, polypeptides or fragments thereof is an enzyme, a hormone, a growth factor, a cytokine, or a mixture thereof. In some embodiments, the biologically active protein, polypeptides or fragments can specifically recognize a target peptide (such as antigen, or other proteins).

[0252] In some embodiments, the bioactive protein or fragments thereof that can be comprised within the anti-LAG-3 construct described herein is a protein-binding protein. In some embodiments, the bioactive protein or fragments thereof that can be comprised within the anti-LAG-3 construct described herein is an antibody mimetics, which are small engineered proteins comprising antigen-binding domains reminiscent of antibodies (Geering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015). These molecules are derived from existing human scaffold proteins and comprise a single polypeptide. Exemplary antibody mimetics that can be comprised within the anti-LAG-3 construct described herein can be, but are not limited to, a Designed ankyrin repeat protein (DARPin; comprising 3-5 fully synthetic ankyrin repeats flanked by N- and C-terminal Cap domains), an avidity multimer (avimer; a high-affinity protein comprising multiple A domains, each domain with low affinity for a target), or an Anticalin (based on the scaffold of lipocalins, with four accessible loops, the sequence of each can be randomized). In some embodiments, the bioactive protein or fragments thereof that can be comprised within the anti-LAG-3 construct described herein is an Armadillo repeat protein (e.g., β-catenin, α-importin, plakoglobin, adenomatous polyposis coli (APC)), which comprises armadillo repeat units (characteristic, repetitive amino acid sequence of about 40 residues in length). Each Armadillo repeat is composed of a pair of alpha helices that form a hairpin structure. Multiple copies of the repeat form what is known as an alpha solenoid structure. Armadillo repeat proteins are able to bind different types of peptides, relying on a constant way of binding of the peptide backbone without requiring specific conserved side chains or interactions with free N- or C-termini of a peptide. The possibility of recognizing a peptide residue by residue, combined with the intrinsic modularity of a repeat protein, makes the armadillo repeat proteins promising candidates for the design of a generic scaffold for peptide binding.

[0253] In some embodiments, the biologically active protein or fragments thereof that can be comprised within the anti-LAG-3 construct described herein is a ligand, such as lymphokines and cellular factors which interact with specific cellular receptor. Lymphokines are low molecular weight proteins which are secreted by T cells when antigens or lectins stimulate T cell growth.III. Pharmaceutical compositions

[0254] Further provided by the present application are pharmaceutical compositions comprising any one of the anti-LAG-3 constructs described herein (e.g., anti-LAG-3 sdAb, anti-LAG-3 HCAb or anti-LAG-3 MABP), and optionally a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by mixing an anti-LAG-3 construct described...

Claims

1. An anti-LAG-3 construct comprising a single-domain antibody (sdAb) specifically recognizing LAG-3, wherein the sdAb comprises a VHH domain comprising the amino acid sequence of any one of SEQ ID NOs: 304, 288, 306, 295, or 275-278.

2. The anti-LAG-3 construct of claim 1, wherein the anti-LAG-3 construct is a heavy chain-only antibody (HCAb) comprising the sdAb specifically recognizing LAG-3 fused to an Fc fragment, wherein preferably the HCAb is monomeric or dimeric, and wherein optionally the sdAb is fused to the Fc fragment via a peptide linker.

3. The anti-LAG-3 construct of claim 1, comprising: (a) a first antigen binding portion comprising the sdAb specifically recognizing LAG-3; and (b) a second antigen binding portion that specifically recognizes a second epitope, wherein preferably the first antigen binding portion and the second antigen binding portion are fused to each other via a peptide linker, and wherein preferably the second antigen binding portion specifically recognizes an immune checkpoint molecule selected from the group consisting of PD-1, 4-1BB, PD-L1, TIM-3, TIGIT, CTLA-4, VISTA, B7-1, B7-H3, CD47, OX40 and GITR, in particular PD-1.

4. The anti-LAG-3 construct of claim 3, wherein the second antigen binding portion comprises: (i) HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 356, and LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 357; or (ii) HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 373, and LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 374, wherein CDRs are determined according to Kabat numbering.

5. The anti-LAG-3 construct of claim 3, wherein the second antigen binding portion is a full-length antibody comprising: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 356, and a light chain comprising the amino acid sequence of SEQ ID NO: 357; or (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 373, and a light chain comprising the amino acid sequence of SEQ ID NO: 374.

6. A pharmaceutical composition comprising the anti-LAG-3 construct of any one of claims 1 to 5, and a pharmaceutically acceptable carrier.

7. An effective amount of the pharmaceutical composition of claim 6 for use in treating an individual having a LAG-3-related disease, wherein said LAG-3-related disease is cancer, in particular colon cancer, comprising administering to the individual the effective amount of the pharmaceutical composition, and wherein anti-LAG-3 construct comprises (a) a first antigen binding portion comprising the sdAb specifically recognizing LAG-3; and (b) a second antigen binding portion that specifically recognizes a second epitope, wherein the second antigen binding portion is a full-length antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 373, and a light chain comprising the amino acid sequence of SEQ ID NO: 374.

8. A nucleic acid encoding the anti-LAG-3 construct of any one of claims 1 to 5.

9. A vector comprising the nucleic acid of claim 8.

10. An isolated host cell comprising the nucleic acid of claim 8 or vector of claim 9.

11. A kit comprising the anti-LAG-3 construct of any one of claims 1 to 5, the pharmaceutical composition of claim 6, the nucleic acid of claim 8, the vector of claim 9, or the host cell of claim 10.

12. An in vitro method of producing the anti-LAG-3 construct of any one of claims 1 to 5, comprising: (a) culturing the isolated host cell of claim 10 under conditions effective to express the encoded anti-LAG-3 construct; and (b) obtaining the expressed anti-LAG-3 construct from the isolated host cell.