Multi-specific binding proteins that bind to gpnmb and a blood brain barrier target and methods of use thereof

Abstract

The present disclosure is generally directed to multispecific proteins comprising (i) an antigen-binding domain that specifically binds to a blood brain barrier (BBB) target selected from human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc) and (ii) an antigen-binding domain that specifically binds to Glycoprotein nonmetastatic melanoma protein B (GPNMB).

Description

MULTI-SPECIFIC BINDING PROTEINS THAT BIND TO GPNMB AND A BLOOD BRAIN BARRIER TARGET AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application Nos. 63 / 627,526, filed January 31, 2024; 63 / 660,914, filed June 17, 2024; 63 / 744,039, filed January 10, 2025; and 63 / 745,442, filed January 15, 2025, each of which is herein incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing (Name: 4503_027PC03_SequenceListing_ST26.xml; Size: 505,079 bytes; and Date of Creation: January 27, 2025) is here in incorporated by reference in its entirety.FIELD OF THE PRESENT DISCLOSURE

[0003] The present disclosure relates to multi-specific binding proteins comprising a blood brain barrier (BBB) antigen-binding domain that specifically binds to a BBB target selected from human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc) and antigen-binding domain that specifically binds to GPNMB and uses (e.g., therapeutic uses) thereof.BACKGROUND

[0004] Glycoprotein nonmetastatic melanoma protein B (GPNMB), also known as osteoactivin (rat ortholog), dendritic cell-heparin integrin ligand (DC-HIL, mouse ortholog), or hematopoietic growth factor inducible neurokinin-1 type (HGFIN), is a type 1 transmembrane protein. As a result of alternative splicing, human GPNMB occurs as two polypeptide isoforms, one of 572 amino acids and a shorter one of 560 amino acids. GPNMB has 12 glycosylation sites, a polycystic kidney disease (PKD) domain, an integrin-recognition (RGD) motif, an immunoreceptor tyrosine-based activation-like motif (IT AM-like), and a lysosomal targeting (dileucine) motif (Abdelmagid et al, 2008, Exp Cell Res, 314:2334-2351). GPNMB can be cleaved by the metalloproteinase ADAMI 0, releasing a soluble fragment that can bind to various receptors and trigger a cellular response (Rose et al, 2010, PLoS One, 5(8):el2093).

[0005] Increased expression of GPNMB is associated with genome wide significant risk for Parkinson’s disease (Murthy et al, 2017, Neurogenetics, 18: 121-133). The rsl99347 SNP is associated with increased expression of GPNMB in brain. GPNMB protein expression has beenlinked to ALS and to certain lysosomal storage disorders, including Gaucher’s disease and Neimann-Pick type C disease (Tanaka et al, 2012, Sci Rep, 2:537; Kramer et al, 2016, FEBS Open Bio, 6:902-913; Marques et al, 2016, PLoS One l l :e0147208).

[0006] Anti-GPNMB antibodies have been previously described. See, e.g., W02006 / 071441, W02007 / 053718, US2007 / 0190575, US2013 / 0156784, USPN 7,115,265, WO2008 / 133641, WO2010 / 135547, WO2016 / 145022, WO2017 / 046061, WO2018 / 217945, and WO2019 / 137138.

[0007] However, such anti-GPNMB antibody therapies intended for central nervous system (CNS) disorders may be limited in their effectiveness due to their limited ability to cross the blood brain barrier (BBB). Therefore, there is a need for novel anti-GPNMB therapies having improved delivery across the BBB.SUMMARY OF THE PRESENT DISCLOSURE

[0008] Provided herein are multispecific proteins comprising (i) an antigen-binding domain that specifically binds to a blood brain barrier (BBB) target selected from human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc) and (ii) an antigen-binding domain that specifically binds to Glycoprotein nonmetastatic melanoma protein B (GPNMB).

[0009] In some aspects, the anti-GPNMB antigen-binding domain is an antibody. In some aspects, the antibody is a full-length antibody. In some aspects, the antibody is an IgG antibody. In some aspects, the IgG antibody is an IgGl antibody or an IgG4 antibody. In some aspects, the anti-GPNMB antibody comprises a heavy chain with a Fc hole mutation. In some aspects, the anti-GPNMB antibody comprises a heavy chain with a Fc knob mutation.

[0010] In some aspects, the anti-BBB antigen-binding domain is a scFv. In some aspects, the anti-BBB scFv is linked to the C-terminus of the anti-GPNMB antibody heavy chain with a Fc hole mutation. In some aspects, the anti-BBB scFv is linked to the C-terminus of the anti- GPNMB antibody heavy chain with a Fc knob mutation.

[0011] In some aspects, the multispecific protein comprises two antigen-binding domains that specifically bind to a BBB target selected from human TfR and human CD98hc. In some aspects, both of the anti-BBB antigen-binding domains are scFvs. In some aspects, the first anti-BBB scFv is linked to the C-terminus of the anti-GPNMB antibody heavy chain with a Fc hole mutation. In some aspects, the second anti-BBB scFv is linked to the C-terminus of the anti- GPNMB antibody heavy chain with a Fc knob mutation.

[0012] In some aspects, the anti-BBB antigen-binding domain is an antibody. In some aspects, the antibody is a full-length antibody. In some aspects, the antibody is an IgG antibody. In some aspects, the IgG antibody is an IgGl antibody or an IgG4 antibody. In some aspects, the anti- BBB antibody comprises a heavy chain with a Fc hole mutation. In some aspects, the anti-BBB antibody comprises a heavy chain with a Fc knob mutation.

[0013] In some aspects, the anti-GPNMB antigen-binding domain is a scFv. In some aspects, the anti-GPNMB scFv is linked to the C-terminus of the anti-BBB antibody heavy chain with a Fc hole mutation. In some aspects, the anti-GPNMB scFv is linked to the C-terminus of the anti- BBB antibody heavy chain with a Fc knob mutation.

[0014] In some aspects, the multispecific protein comprises two antigen-binding domains that specifically bind to a GPNMB, wherein both of the anti-GPNMB antigen-binding domains are scFvs.

[0015] In some aspects, the first anti-GPNMB scFv is linked to the C-terminus of the anti-BBB antibody heavy chain with a Fc hole mutation. In some aspects, the second anti-GPNMB scFv is linked to the C-terminus of the anti-BBB antibody heavy chain with a Fc knob mutation.

[0016] In some aspects, the antibody is a monoclonal antibody.

[0017] In some aspects, the antibody is a humanized antibody.

[0018] In some aspects, the anti-GPNMB antigen-binding domain is selected from the group consisting of a VHH, a Fab, a Fab’, a Fab’-SH, a F(ab’)2, a Fv, or a scFv.

[0019] In some aspects, the anti-BBB antigen-binding domain is a VHH, a Fab, a Fab’, a Fab’- SH, a F(ab’)2, a Fv, or a scFv.

[0020] In some aspects, the multispecific protein further comprises iii) an Fc region.

[0021] In some aspects, the Fc region is fully active.

[0022] In some aspects, the Fc region is partially active.

[0023] In some aspects, the Fc region is not silent.

[0024] In some aspects, the anti-GPNMB antigen-binding domain is a VHH.

[0025] In some aspects, the anti-BBB antigen-binding domain is a VHH.

[0026] In some aspects, the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a VHH that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigen-binding domain is a VHH that is N-terminal to the other protein fragment of the Fc region.

[0027] In some aspects, the anti-GPNMB antigen-binding domain is a Fab.

[0028] In some aspects, the anti-BBB antigen-binding domain is a Fab.

[0029] In some aspects, the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a Fab that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigen-binding domain is a Fab that is N-terminal to the other protein fragment of the Fc region.

[0030] In some aspects, the anti-BBB antigen-binding domain is a scFv.

[0031] In some aspects, the anti-GPNMB antigen-binding domain is a scFv.

[0032] In some aspects, the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a scFv that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigen-binding domain is a scFv that is N-terminal to the other protein fragment of the Fc region.

[0033] In some aspects, the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a scFv that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigen-binding domain is a Fab that is N-terminal to the other protein fragment of the Fc region.

[0034] In some aspects, the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a Fab that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigen-binding domain is a Fab that is N-terminal to the other protein fragment of the Fc region

[0035] In some aspects, the IgG Fc region is an IgGl Fc region or an IgG4 Fc region.

[0036] In some aspects, the IgG Fc region comprises knob and hole mutations.

[0037] In some aspects, the multispecific protein is bivalent.

[0038] In some aspects, the multispecific protein is trivalent.

[0039] In some aspects, the trivalent protein comprises one antigen-binding domain that binds to the BBB target and two antigen-binding domains that bind to GPNMB.

[0040] In some aspects, the trivalent protein comprises two antigen-binding domains that bind to the BBB target and one antigen-binding domain that binds to GPNMB.

[0041] In some aspects, the multispecific protein is tetravalent. In some aspects, the tetravalent protein comprises two of antigen-binding domains that bind to the BBB target and two antigenbinding domains that bind to GPNMB.

[0042] In some aspects, the multispecific protein is a bispecific T-cell engager (BiTE), dualaffinity re-targeting protein (DARTs), or Tandem diabody (TandAb).

[0043] In some aspects, the anti-BBB antigen-binding domain and the anti-GPNMB antigenbinding domain are contained within a single polypeptide chain. In some aspects, the single polypeptide chain further comprises an Fc domain.

[0044] In some aspects, the anti-GPNMB antigen-binding domain is N-terminal to the anti- BBB antigen-binding domain.

[0045] In some aspects, the anti-GPNMB antigen-binding domain thereof is C-terminal to the anti-BBB antigen-binding domain.

[0046] In some aspects, the anti-GPNMB antigen-binding domain and the anti-BBB antigenbinding domain are directly connected via a peptide bond.

[0047] In some aspects, the anti-GPNMB antigen-binding domain and the anti-BBB antigenbinding domain are connected via a linker.

[0048] In some aspects, the linker is a peptide linker.

[0049] In some aspects, the anti-BBB antigen-binding domain and the anti-GPNMB antigenbinding domain are N-terminal to the Fc domain.

[0050] In some aspects, the anti-BBB antigen-binding domain and the anti-GPNMB antigenbinding domain are C-terminal to the Fc domain.

[0051] In some aspects, the anti-BBB antigen-binding domain is N-terminal to the Fc domain and the anti-GPNMB antigen-binding domain is C-terminal to the Fc domain.

[0052] In some aspects, the anti-GPNMB antigen-binding domain is N-terminal to the Fc domain and the anti-BBB antigen-binding domain is C-terminal to the Fc domain.

[0053] In some aspects, the multispecific protein comprises two copies of the anti-GPNMB antigen-binding domain and / or two copies of the anti-BBB antigen-binding domain.

[0054] In some aspects, the anti-GPNMB antigen-binding domain antagonizes GPNMB activity.

[0055] In some aspects, the anti-GPNMB antigen-binding domain modulates expression of activation surface markers on myeloid cells.

[0056] In some aspects, the anti-GPNMB antigen-binding domain increases cell surface expression of PD-L1 in human macrophages.

[0057] In some aspects, the anti-GPNMB antigen-binding domain increases cell surface expression of CD40 in human macrophages.

[0058] In some aspects, the anti-GPNMB antigen-binding domain increases cell surface expression of CD80 in human macrophages.

[0059] In some aspects, the anti-GPNMB antigen-binding domain modulates lysosome function in myeloid cells.

[0060] In some aspects, the anti-GPNMB antigen-binding domain increases glucocerebrosidase activity in human macrophages.

[0061] In some aspects, the anti-GPNMB antigen-binding domain decreases cell surface expression of GPNMB in human macrophages.

[0062] In some aspects, the anti-GPNMB antigen-binding domain changes interferon pathway gene expression patterns in human macrophages.

[0063] In some aspects, the anti-GPNMB antigen-binding domain reduces tumor volume in a murine tumor model, such as in an MC38 model.

[0064] In some aspects, the anti-GPNMB antigen-binding domain reduces tumor growth rate in a murine tumor model, such as in an MC38 model.

[0065] In some aspects, wherein the anti-GPNMB antigen-binding domain increases levels of IL-12p40 in serum.

[0066] In some aspects, the anti-GPNMB antigen-binding domain increases levels of CCL5 in serum.

[0067] In some aspects, the anti-GPNMB antigen-binding domain is a GPNMB ligand blocking antigen-binding domain.

[0068] In some aspects, the anti-GPNMB antigen-binding domain is a non-blocking antigenbinding domain.

[0069] In some aspects, the anti-GPNMB antigen-binding domain antagonizes GPNMB activity independent of ligand blocking activity.

[0070] In some aspects, the anti-GPNMB antigen-binding domain antagonizes GPNMB activity in vivo.

[0071] In some aspects, the anti-GPNMB antigen-binding domain binds to GPNMB expressed on a cell surface.

[0072] In some aspects, the anti-GPNMB antigen-binding domain promotes macrophage activation.

[0073] In some aspects, the anti-GPNMB antigen-binding domain overcomes a decrease in glucocerebrosidase activity associated with reduced progranulin levels.

[0074] In some aspects, the anti-GPNMB antigen-binding domain decreases GPNMB expression levels in cells. In some aspects, the anti-GPNMB antigen-binding domain decreases GPNMB expression levels in macrophages.

[0075] In some aspects, the anti-GPNMB antigen-binding domain decreases LAMP2 expression levels in cells. In some aspects, the anti-GPNMB antigen-binding domain decreases LAMP2 expression levels in macrophages, monocytes, or neutrophils.

[0076] In some aspects, the anti-GPNMB antigen-binding domain inhibits or reduces inflammasome activation.

[0077] In some aspects, the anti-GPNMB antigen-binding domain inhibits IL-ip expression or release.

[0078] In some aspects, the anti-GPNMB antigen-binding domain reduces neural inflammation.

[0079] In some aspects, the anti-GPNMB antigen-binding domain reduces expression of Clq, GFAP, IB Al, and CTSD associated with neural inflammation.

[0080] In some aspects, the anti-GPNMB antigen-binding domain reduces lysosomal stress.

[0081] In some aspects, the anti-GPNMB antigen-binding domain competes with one or more antibodies selected from GPN-81, GPN-82, GPN-83, GPN-84, GPN-85, GPN-86, GPN-87, GPN-88, GPN-89, GPN-90, GPN-91, GPN-92, GPN-93, GPN-94, GPN-95, GPN-96, GPN-97, GPN-98, and any combination thereof for binding to GPNMB.

[0082] In some aspects, the anti-GPNMB antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, a VH CDR2, a VH CDR3 and a light chain variable region (VL) CDR1, a CDR2, and a CDR3, wherein: i) the VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 308 or 9; ii) the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 310, 311, 312, 313, 314, or 315; iii) the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 316, 317, 318, or 319; iv) the VL CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 320, 321, 322, 323, 324, or 325; v) the VL CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 326, 327, 328, 329, 330, 331, 332, 333, 334, or 335; and / orvi) the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 336, 337, 338, 339, 340, or 341.

[0083] In some aspects, the anti-GPNMB antigen-binding domain comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of: i) SEQ ID NOs: 308, 310, and 316; and 320, 326, and 336; respectively; ii) SEQ ID NOs: 308, 311, and 316; and 321, 327, and 337; respectively iii) SEQ ID NOs: 308, 312, and 316; and 322, 328, and 337; respectively; iv) SEQ ID NOs: 308, 312, and 317; and 322, 328, and 337; respectively; v) SEQ ID NOs: 308, 313, and 316; and 323, 329, and 337; respectively; vi) SEQ ID NOs: 308, 312, and 316; and 321, 330, and 337; respectively; vii) SEQ ID NOs: 308, 312, and 317; and 321, 330, and 337; respectively; viii) SEQ ID NOs: 308, 312, and 316; and 321, 329, and 338; respectively; ix) SEQ ID NOs: 308, 312, and 317; and 321, 329, and 337; respectively; x) SEQ ID NOs: 308, 313, and 316; and 321, 329, and 337; respectively; xi) SEQ ID NOs: 308, 312, and 316; and 321, 329, and 339; respectively; xii) SEQ ID NOs: 9, 314, and 318; and 324, 331, and 340; respectively; xiii) SEQ ID NOs: 9, 315, and 319; and 325, 332, and 341; respectively; xiv) SEQ ID NOs: 9, 315, and 319; and 325, 332, and 340; respectively; xv) SEQ ID NOs: 9, 315, and 319; and 325, 333, and 341; respectively; xvi) SEQ ID NOs: 9, 315, and 319; and 324, 334, and 340; respectively; xvii) SEQ ID NOs: 9, 315, and 319; and 325, 335, and 340; respectively; or xviii)SEQ ID NOs: 9, 315, and 319; and 324, 331, and 340; respectively.

[0084] In some aspects, the anti-GPNMB antigen-binding domain comprises a VH that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 242, 243, 244, 245, 246, 247, and 248.

[0085] In some aspects, the anti-GPNMB antigen-binding domain comprises a VL that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, and 262.

[0086] In some aspects, the anti-GPNMB antigen-binding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of:(i) SEQ ID NOs: 242 and 249, respectively;(ii) SEQ ID NOs: 243 and 250, respectively;(iii) SEQ ID NOs: 244 and 251, respectively;(iv) SEQ ID NOs: 245 and 251, respectively;(v) SEQ ID NOs: 246 and 252, respectively;(vi) SEQ ID NOs: 244 and 253, respectively;(vii) SEQ ID NOs: 245 and 253, respectively;(viii) SEQ ID NOs: 244 and 254, respectively;(ix) SEQ ID NOs: 245 and 255, respectively;(x) SEQ ID NOs: 246 and 255, respectively;(xi) SEQ ID NOs: 244 and 256, respectively;(xii) SEQ ID NOs: 247 and 257, respectively;(xiii) SEQ ID NOs: 248 and 258, respectively;(xiv) SEQ ID NOs: 248 and 259, respectively;(xv) SEQ ID NOs: 248 and 260, respectively;(xvi) SEQ ID NOs: 248 and 261, respectively;(xvii)SEQ ID NOs: 248 and 262, respectively; or(xviii) SEQ ID NOs: 248 and 257, respectively.

[0087] In some aspects, the anti-GPNMB antigen-binding domain binds mouse GPNMB, binds cynomolgus GPNMB, or binds mouse and cynomolgus GPNMB.

[0088] In some aspects, the anti-GPNMB antigen-binding domain binds human GPNMB with an affinity of about 0.4 nM to about 120 nM, of about 0.3 nM to about 5 nM, of about 0.4 nM to about 1.04 nM, of about 0.14 to about 0.65 nM and bind mouse GPNMB with an affinity of about 0.18 nM to about 0.44 nM.

[0089] In some aspects, the BBB target is human TfR.

[0090] In some aspects, the multispecific protein is a bispecific protein.

[0091] In some aspects, the anti-TfR antigen-binding domain comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and lightchain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of:SEQ ID NOs: 8, 11, 24; and 40, 55, and 61; respectively;SEQ ID NOs: 8, 11, 25; and 41, 55, and 61; respectively;SEQ ID NOs: 8, 12, 26; and 42, 55, and 61; respectively;SEQ ID NOs: 8, 12, 27; and 42, 55, and 61; respectively;SEQ ID NOs: 8, 13, 25; and 42, 55, and 61; respectively;SEQ ID NOs: 8, 14, 25; and 42, 55, and 61; respectively;SEQ ID NOs: 8, 15, 25; and 43, 55, and 61; respectively;SEQ ID NOs: 8, 16, 25; and 42, 55, and 61; respectively;SEQ ID NOs: 8, 17, 25; and 44, 55, and 61; respectively;SEQ ID NOs: 9, 18, 28; and 45, 56, and 62; respectively;SEQ ID NOs: 9, 19, 28; and 45, 56, and 62; respectively;SEQ ID NOs: 9, 20, 28; and 46, 57, and 62; respectively;SEQ ID NOs: 9, 20, 28; and 46, 58, and 62; respectively;SEQ ID NOs: 9, 20, 28; and 47, 59, and 62; respectively;SEQ ID NOs: 9, 21, 28; and 46, 57, and 62; respectively;SEQ ID NOs: 9, 21, 28; and 47, 59, and 62; respectively;SEQ ID NOs: 9, 22, 28; and 46, 57, and 62; respectively;SEQ ID NOs: 9, 22, 28; and 46, 58, and 62; respectively;SEQ ID NOs: 9, 22, 28; and 47, 59, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 30; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 31; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 32; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 33; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 34; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 35; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 36; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 37; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 38; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 39; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 49, 58, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 50, 58, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 51, 58, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 52, 58, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 53, 58, and 62; respectively; or SEQ ID NOs: 10, 22, 28; and 54, 58, and 62; respectively.

[0092] In some aspects, the anti-TfR antigen-binding domain comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of:SEQ ID NOs: 10, 22, 30; and 50, 58, and 62; respectively;SEQ ID NOs: 10, 22, 419; and 50, 58, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 420, 58, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 421, 58, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 422, 58, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 423, 58, and 62; respectively;SEQ ID NOs: 10, 22, 437; and 50, 58, and 62; respectively;SEQ ID NOs: 8, 14, 25; and 41, 55, and 61; respectively;SEQ ID NOs: 8, 15, 25; and 424, 55, and 61; respectively;SEQ ID NOs: 8, 15, 25; and 425, 55, and 61; respectively; orSEQ ID NOs: 8, 14, 25; and 426, 55, and 61; respectively.

[0093] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of:SEQ ID NOs: 64 and 129, respectively;SEQ ID NOs: 65 and 130, respectively;SEQ ID NOs: 66 and 131, respectively;SEQ ID NOs: 67 and 130, respectively;SEQ ID NOs: 68 and 131, respectively;SEQ ID NOs: 69 and 130, respectively;SEQ ID NOs: 70 and 131, respectively;SEQ ID NOs: 71 and 130, respectively;SEQ ID NOs: 72 and 131, respectively;SEQ ID NOs: 73 and 130, respectively;SEQ ID NOs: 74 and 131, respectively;SEQ ID NOs: 75 and 132, respectively;SEQ ID NOs: 76 and 131, respectively;SEQ ID NOs: 77 and 132, respectively;SEQ ID NOs: 77 and 133, respectively;SEQ ID NOs: 78 and 134, respectively;SEQ ID NOs: 77 and 135, respectively;SEQ ID NOs: 75 and 136, respectively;SEQ ID NOs: 77 and 137, respectively;SEQ ID NOs: 77 and 138, respectively;SEQ ID NOs: 79 and 131, respectively;SEQ ID NOs: 77 and 139, respectively;SEQ ID NOs: 77 and 131, respectively;SEQ ID NOs: 80 and 140, respectively;SEQ ID NOs: 81 and 141, respectively;SEQ ID NOs: 82 and 131, respectively;SEQ ID NOs: 83 and 142, respectively;SEQ ID NOs: 77 and 143, respectively;SEQ ID NOs: 75 and 131, respectively;SEQ ID NOs: 75 and 144, respectively;SEQ ID NOs: 77 and 145, respectively;SEQ ID NOs: 84 and 131, respectively;SEQ ID NOs: 75 and 146, respectively;SEQ ID NOs: 85 and 131, respectively;SEQ ID NOs: 86 and 138, respectively;SEQ ID NOs: 79 and 139, respectively;SEQ ID NOs: 77 and 147, respectively;SEQ ID NOs: 75 and 148, respectively;SEQ ID NOs: 87 and 131, respectively;SEQ ID NOs: 88 and 131, respectively;SEQ ID NOs. 75 and 149, respectively;SEQ ID NOs: 89 and 150, respectively;SEQ ID NOs: 90 and 151, respectively;SEQ ID NOs: 77 and 152, respectively;SEQ ID NOs: 79 and 153, respectively;SEQ ID NOs: 77 and 139, respectively;SEQ ID NOs: 91 and 131, respectively;SEQ ID NOs: 92 and 131, respectively;SEQ ID NOs: 79 and 154, respectively;SEQ ID NOs: 93 and 155, respectively;SEQ ID NOs: 80 and 131, respectively;SEQ ID NOs: 94 and 131, respectively;SEQ ID NOs: 95 and 131, respectively;SEQ ID NOs: 66 and 156, respectively;SEQ ID NOs: 97 and 138, respectively;SEQ ID NOs: 95 and 156, respectively;SEQ ID NOs: 98 and 157, respectively;SEQ ID NOs: 99 and 157, respectively;SEQ ID NOs: 100 and 157, respectively;SEQ ID NOs: 101 and 157, respectively;SEQ ID NOs: 102 and 158, respectively;SEQ ID NOs: 103 and 157, respectively;SEQ ID NOs: 104 and 159, respectively;SEQ ID NOs: 105 and 160, respectively;SEQ ID NOs: 106 and 161, respectively;SEQ ID NOs: 107 and 162, respectively;SEQ ID NOs: 106 and 163, respectively;SEQ ID NOs: 108 and 164, respectively;SEQ ID NOs: 106 and 165, respectively;SEQ ID NOs: 108 and 166, respectively;SEQ ID NOs: 109 and 165, respectively;SEQ ID NOs: 110 and 167, respectively;SEQ ID NOs: 111 and 168, respectively; SEQIDNOs: 112 and 160, respectively; SEQ ID NOs: 113 and 169, respectively; SEQIDNOs: 113 and 170, respectively; SEQIDNOs: 113 and 171, respectively; SEQIDNOs: 114 and 169, respectively; SEQIDNOs: 114 and 171, respectively; SEQIDNOs: 115 and 169, respectively; SEQIDNOs: 115 and 170, respectively; SEQIDNOs: 115 and 171, respectively; SEQIDNOs: 116 and 169, respectively; SEQIDNOs: 116 and 170, respectively; SEQIDNOs: 116 and 171, respectively; SEQIDNOs: 117 and 170, respectively; SEQIDNOs: 118 and 170, respectively; SEQIDNOs: 119 and 170, respectively; SEQ ID NOs: 120 and 170, respectively; SEQIDNOs: 121 and 170, respectively; SEQ ID NOs: 122 and 170, respectively; SEQ ID NOs: 123 and 170, respectively; SEQ ID NOs: 124 and 170, respectively; SEQ ID NOs: 125 and 170, respectively; SEQIDNOs: 126 and 170, respectively; SEQ ID NOs: 127 and 170, respectively; SEQIDNOs: 117 and 173, respectively; SEQIDNOs: 117 and 174, respectively; SEQIDNOs: 117 and 175, respectively; SEQIDNOs: 117 and 176, respectively; SEQIDNOs: 117 and 177, respectively; or SEQIDNOs: 117 and 178, respectively.

[0094] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of:SEQ ID NOs: 118 and 174, respectively;SEQ ID NOs: 427 and 174, respectively;SEQ ID NOs: 117 and 428, respectively;SEQ ID NOs: 117 and 429, respectively;SEQ ID NOs: 117 and 430, respectively;SEQ ID NOs: 117 and 431, respectively;SEQ ID NOs: 432 and 174, respectively;SEQ ID NOs: 101 and 154, respectively;SEQ ID NOs: 102 and 433, respectively;SEQ ID NOs: 102 and 434, respectively; orSEQ ID NOs: 101 and 435, respectively.

[0095] In some aspects, wherein the BBB target is human CD98hc.

[0096] In some aspects, the anti-CD98hc antigen-binding domain comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of:SEQ ID NOs: 181, 185, 191, 194, 198, and 201; respectively;SEQ ID NOs: 182, 186, 191, 195, 199, and 202; respectively;SEQ ID NOs: 183, 187, 192, 196, 200, and 203; respectively;SEQ ID NOs: 9, 188, 192, 196, 200, and 203; respectively;SEQ ID NOs: 184, 189, 193, 197, 198, and 204; respectively;SEQ ID NOs: 184, 190, 193, 197, 198, and 204; respectively;SEQ ID NOs: 184, 232, 193, 197, 198, and 204; respectively;SEQ ID NOs: 184, 233, 193, 197, 198, and 204; respectively; orSEQ ID NOs: 184, 234, 193, 197, 198, and 204; respectively.

[0097] In some aspects, the anti-CD98hc antigen-binding domain comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of:SEQ ID NOs: 184, 342, 193, 197, 198, and 204, respectively; SEQ ID NOs: 184, 343, 193, 197, 198, and 204, respectively; SEQ ID NOs. 184, 344, 193, 197, 198, and 204, respectively; SEQ ID NOs: 184, 345, 193, 197, 198, and 204, respectively; SEQ ID NOs: 184, 346, 193, 197, 198, and 204, respectively; SEQ ID NOs: 184, 347, 193, 197, 198, and 204, respectively; SEQ ID NOs: 184, 348, 193, 197, 198, and 204, respectively; SEQ ID NOs: 184, 190, 193, 360, 198, and 204, respectively; SEQ ID NOs: 184, 190, 193, 361, 198, and 204, respectively; SEQ ID NOs: 184, 190, 193, 362, 198, and 204, respectively; SEQ ID NOs: 184, 190, 193, 363, 198, and 204, respectively; SEQ ID NOs: 184, 190, 193, 364, 198, and 204, respectively; SEQ ID NOs: 184, 190, 193, 365, 198, and 204, respectively; SEQ ID NOs: 184, 190, 193, 366, 198, and 204, respectively; SEQ ID NOs: 184, 344, 193, 362, 198, and 204, respectively; SEQ ID NOs: 184, 349, 193, 197, 198, and 204, respectively; SEQ ID NOs: 184, 190, 351, 197, 198, and 204, respectively; SEQ ID NOs: 184, 190, 352, 197, 198, and 204, respectively; SEQ ID NOs: 184, 190, 353, 197, 198, and 204, respectively; SEQ ID NOs: 184, 190, 354, 197, 198, and 204, respectively; SEQ ID NOs: 184, 190, 355, 197, 198, and 204, respectively; SEQ ID NOs: 184, 190, 356, 197, 198, and 204, respectively; SEQ ID NOs: 184, 190, 357, 197, 198, and 204, respectively; SEQ ID NOs: 184, 190, 358, 197, 198, and 204, respectively; SEQ ID NOs: 184, 190, 359, 197, 198, and 204, respectively; SEQ ID NOs: 184, 190, 193, 367, 198, and 204, respectively; SEQ ID NOs: 184, 190, 193, 438, 198, and 204, respectively; SEQ ID NOs: 184, 190, 193, 439, 198, and 204, respectively; SEQ ID NOs: 184, 190, 193, 440, 198, and 204, respectively; SEQ ID NOs: 184, 190, 193, 441, 198, and 204, respectively; SEQ ID NOs: 184, 190, 193, 442, 198, and 204, respectively; SEQ ID NOs: 184, 190, 193, 197, 198, and 368, respectively;SEQ ID NOs: 184, 190, 193, 197, 198, and 369, respectively;SEQ ID NOs: 184, 190, 193, 197, 198, and 370, respectively;SEQ ID NOs: 184, 190, 193, 197, 198, and 371, respectively;SEQ ID NOs: 184, 190, 193, 197, 198, and 372, respectively;SEQ ID NOs: 184, 190, 193, 197, 198, and 373, respectively;SEQ ID NOs: 184, 190, 193, 197, 198, and 374, respectively;SEQ ID NOs: 184, 190, 193, 197, 198, and 375, respectively;SEQ ID NOs: 184, 190, 193, 197, 198, and 376, respectively;SEQ ID NOs: 184, 190, 193, 197, 198, and 377, respectively;SEQ ID NOs: 350, 190, 193, 197, 198, and 204, respectively; orSEQ ID NOs: 350, 190, 193, 197, 198, and 377, respectively.

[0098] In some aspects, the anti-CD98hc antigen-binding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of:SEQ ID NOs: 205 and 211, respectively;SEQ ID NOs: 206 and 212, respectively;SEQ ID NOs: 207 and 213, respectively;SEQ ID NOs: 208 and 214, respectively;SEQ ID NOs: 209 and 215, respectively;SEQ ID NOs: 210 and 216, respectively;SEQ ID NOs: 235 and 216, respectively;SEQ ID NOs: 236 and 216, respectively; orSEQ ID NOs: 237 and 216, respectively.

[0099] In some aspects, the anti-CD98hc antigen-binding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of:SEQ ID NOs: 378 and 216, respectively;SEQ ID NOs: 379 and 216, respectively;SEQ ID NOs: 380 and 216, respectively;SEQ ID NOs: 381 and 216, respectively;SEQ ID NOs: 382 and 216, respectively;SEQ ID NOs: 383 and 216, respectively;SEQ ID NOs: 384 and 216, respectively; SEQ ID NOs: 210 and 385, respectively; SEQ ID NOs: 210 and 386, respectively; SEQ ID NOs: 210 and 387, respectively; SEQ ID NOs: 210 and 388, respectively; SEQ ID NOs: 210 and 389, respectively; SEQ ID NOs: 210 and 390, respectively; SEQ ID NOs: 210 and 391, respectively; SEQ ID NOs: 210 and 391, respectively; SEQ ID NOs: 380 and 387, respectively; SEQ ID NOs: 392 and 216, respectively; SEQ ID NOs: 393 and 216, respectively; SEQ ID NOs: 394 and 216, respectively; SEQ ID NOs: 395 and 216, respectively; SEQ ID NOs: 396 and 216, respectively; SEQ ID NOs: 397 and 216, respectively; SEQ ID NOs: 398 and 216, respectively; SEQ ID NOs: 399 and 216, respectively; SEQ ID NOs: 400 and 216, respectively; SEQ ID NOs: 401 and 216, respectively; SEQ ID NOs: 210 and 402, respectively; SEQ ID NOs: 210 and 403, respectively; SEQ ID NOs: 210 and 404, respectively; SEQ ID NOs: 210 and 405, respectively; SEQ ID NOs: 210 and 406, respectively; SEQ ID NOs: 210 and 407, respectively; SEQ ID NOs: 210 and 408, respectively; SEQ ID NOs: 210 and 409, respectively; SEQ ID NOs: 210 and 410, respectively; SEQ ID NOs: 210 and 411, respectively; SEQ ID NOs: 210 and 412, respectively; SEQ ID NOs: 210 and 413, respectively;SEQ ID NOs: 210 and 414, respectively;SEQ ID NOs: 210 and 415, respectively;SEQ ID NOs: 210 and 416, respectively;SEQ ID NOs: 210 and 417, respectively;SEQ ID NOs: 418 and 215, respectively; orSEQ ID NOs: 418 and 417, respectively

[0100] In some aspects, the multispecific protein comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of SEQ ID NOs: 263-287, 291-307, or combinations thereof.

[0101] In some aspects, the multispecific protein comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of SEQ ID NOs: 263-287, 291-307, 436, or combinations thereof.

[0102] In some aspects, the multispecific protein comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of:SEQ ID NOs: 263, 275, and 283;SEQ ID NOs: 264, 276, and 283;SEQ ID NOs: 264, 275, and 283;SEQ ID NOs: 265, 275, and 283;SEQ ID NOs: 266, 277, and 284;SEQ ID NOs: 267, 278, and 284;SEQ ID NOs: 267, 277, and 284;SEQ ID NOs: 268, 277, and 284;SEQ ID NOs: 269, 279, and 284;SEQ ID NOs: 270, 280, and 284;SEQ ID NOs: 270, 279, and 284;SEQ ID NOs: 271, 279, and 284;SEQ ID NOs: 272, 281, and 285;SEQ ID NOs: 273, 282, and 285;SEQ ID NOs: 273, 281, and 285;SEQ ID NOs: 274, 281, and 285;SEQ ID NOs: 272, 281, and 286;SEQ ID NOs: 273, 282, and 286;SEQ ID NOs: 273, 281, and 286;SEQ ID NOs: 274, 281, and 286;SEQ ID NOs: 272, 281, and 287;SEQ ID NOs: 273, 282, and 287;SEQ ID NOs: 273, 281, and 287; orSEQ ID NOs: 274, 281, and 287.

[0103] In some aspects, the multispecific protein comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of SEQ ID NOs: 436, 279, and 284.

[0104] In some aspects, provided herein is a composition comprising any of the multispecific proteins disclosed herein.

[0105] In some aspects, provided herein is a host cell comprising any of the multispecific proteins disclosed herein.

[0106] In some aspects, provided herein is a pharmaceutical composition comprising i) any of the multispecific proteins disclosed herein, and iii) a pharmaceutically acceptable carrier, excipient or stabilizer.

[0107] In some aspects, provided herein is a nucleotide sequence encoding any of the multispecific proteins disclosed herein.

[0108] In some aspects, provided herein is a method of administering or transporting compositions comprising administering to the subject any of the multispecific proteins disclosed herein, any of the compositions disclosed herein, any of the pharmaceutical compositions disclosed herein, or any of the nucleotide sequences disclosed herein.

[0109] In some aspects, provided herein is a method of treating a lysosomal storage disease or disorder in a subject comprising administering to the subject any of the multispecific proteins disclosed herein, any of the compositions disclosed herein, any of the pharmaceutical compositions disclosed herein, or any of the nucleotide sequences disclosed herein.

[0110] In some aspects, the lysosomal storage disease or disorder is Gaucher’s disease or Neimann-Pick type C disease.[OHl] In some aspects, provided herein is a method of treating a CNS disease or disorder in a subject comprising administering to the subject any of the multispecific proteins disclosed herein,any of the compositions disclosed herein, any of the pharmaceutical compositions disclosed herein, or any of the nucleotide sequences disclosed herein.

[0112] In some aspects, the CNS disease or disorder is selected from Parkinson’s disease, Alzheimer’s disease, and ALS.

[0113] In some aspects, provided herein the multispecific proteins disclosed herein, any of the compositions disclosed herein, any of the the pharmaceutical compositions disclosed herein, or any of the nucleotide sequences disclosed herein for use in any of the methods disclosed herein.

[0114] In some aspects, provided herein is a use of the multi specific proteins disclosed herein, any of the the compositions disclosed herein, any of the pharmaceutical compositions disclosed herein, or any of the nucleotide sequences disclosed herein in any of the methods disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1 (FIG. 1) shows an exemplary 2+1 bispecific protein containing an scFv that binds to a BBB target (e.g., TfR or CD98hc) linked to a C-terminus of heavy chain of a full- length antibody that binds to GPNMB.

[0116] Figure 2 (FIG. 2) shows an exemplary 2+2 bispecific protein with 2 copies of an scFv that binds to a BBB target (e.g., TfR or CD98hc), wherein 1 copy is linked to the C-terminus of each heavy chain of a full-length antibody that binds to GPNMB.

[0117] Figure 3 (FIG. 3) shows exemplary bispecific proteins with heterodimeric Fc regions (e.g., wherein one polypeptide of the Fc region contains a knob mutation and the other polypeptide of the Fc region contains a hole mutation). FIG. 3 includes (i) an exemplary 2+1 bispecific protein with an scFv that binds to a BBB target (e.g., TfR or CD98hc) linked to the C- terminus of a heavy chain of an antibody that binds to GPNMB; (ii) an exemplary 1+1 bispecific protein wherein a Fab of a full-length antibody that binds to GPNMB is replaced with an scFv that binds to a BBB target (e.g., TfR or CD98hc); and (iii) an exemplary 1+1 bispecific protein where one VHH that binds to a BBB target (e.g., TfR or CD98hc) is linked to the N-terminus of one polypeptide of an Fc region and one VHH that binds to GPNMB is linked to the N-terminus of a second polypeptide of the Fc region.

[0118] Figures 4A-4B (FIGs. 4A-4B) show nonspecific binding to baculovirus particles (BVP) of anti-TfR antibodies (H6-4, L7-2, L10-1, L10-8, H3-7, LIO-16, L-35, 42Q, 24A, and 24A 42Q), isotype control, negative control antibody, and positive control antibody at differentconcentrations of antibody (FIG. 4A) and nonspecific binding (BVP) of anti-TfR antibodies (42Q, 24A, L-21, L-6, 39.38, and L-19), negative control antibody, and positive control antibody at different concentrations of antibody (FIG. 4B).

[0119] Figures 5A-5B (FIGs. 5A-5B) show nonspecific binding to double stranded DNA (dsDNA) of anti-TfR antibodies (H6-4, L7-2, L10-1, L10-8, H3-7, LIO-16, L-35, 42Q, 24A, and 24A 42Q), Isotype control, negative control antibody, and positive control antibody at at 10 pg / mL (FIG. 5 A) and nonspecific binding (dsDNA) of anti-TfR antibodies (42Q, 24A, L-21, L- 6, 39.38, and L-19), negative control antibody, and positive control antibody at 10 pg / mL (FIG. 5B).

[0120] Figure 6 (FIG. 6) shows cell microscopy of anti-TfR antibodies (H6-4, H3-7, 24A, L7- 2, LIO-16, 24A.42Q, L10-1, L-35, L10-8, and 42Q) and isotype control assessing cell uptake in hCMEC / D3 cells.

[0121] Figures 7A-7B (FIGs. 7A-7B) show brain uptake in vessel depleted brains for anti-TfR antibodies (42Q, H6-4, L7-2, L10-1, L10-8, L-35, 24A, LIO-16, H3-7, and 24A_42Q) and isotype control) after 24 hours of 5 mg / kg of antibody is administered in mice with data shown as ng / mg tissue (FIG.7A) and fold-change over isotype control (FIG. 7B).

[0122] Figure 8 (FIG. 8) shows the ratio of antibody concentration in vessel depleted brain to whole brain for anti-TfR antibodies (42Q, H6-4, L7-2, L10-1, L10-8, L-35, 24A, LIO-16, H3-7, 24A_42Q) and isotype control after 24 hours of 5 mg / kg of antibody is administered in mice.

[0123] Figure 9 (FIG. 9) shows absolute reticulocyte (K / pL) and % of reticulocytes in whole blood for anti-TfR antibodies (42Q, H6-4, L7-2, L10-1, L10-8, L-35, 24A, LIO-16, H3-7, 24A_42Q) and isotype control after 24 hours of 5 mg / kg of antibody is administered in mice.

[0124] Figure 10 (FIG. 10) shows TfR levels normalized to GAPDH in mice treated with 5 mg / kg of anti-TfR antibodies (42Q, 24A, L-21, L-6, L-35, and 39.38) and isotype control).

[0125] Figure 11 (FIG. 11) shows the effect of various Fc regions of anti-GPNMB / anti-TfR multi-specific binding proteins of the present disclosure at increasing CD40 cell surface expression in human macrophages.

[0126] Figure 12 (FIG. 12) shows the effect of various Fc regions of anti-GPNMB / anti-TfR multi-specific binding proteins of the present disclosure at increasing PD-L1 cell surface expression in human macrophages.

[0127] Figure 13 (FIG. 13) shows the effect of various Fc regions of anti-GPNMB / anti-TfR multi-specific binding proteins of the present disclosure at enhancing glucocerebrosidase (GCase) activity in human macrophages.

[0128] Figure 14 (FIG. 14) shows the effect of various Fc regions of anti-GPNMB / anti-TfR Multi-specific binding proteins of the present disclosure on ADCC activation in vitro.

[0129] Figures 15A-15B (FIGs. 15A-15B) shows exemplary anti-GPNMB / anti-TfR multispecific binding proteins of the present disclosure display enhanced brain penetration in vivo.

[0130] Figures 16A-16B (FIGs. 16A-16B) shows increased brain uptake of CD98hc.04.048.WHl in mice (FIG. 16A) and fold-change compared to isotype control antibody (FIG. 16B).

[0131] Figure 17 (FIG. 17) shows immunohistochemistry analysis of brain tissues of mice administered CD98hc.04.048.WHl compared to isotype control antibody.

[0132] Figure 18 (FIG. 18) shows no significant changes in red blood cell count in mice following administration of CD98hc.04.048.WHl compared to isotype control antibody.

[0133] Figure 19A (FIG. 19A) shows increased brain uptake in mice of CD98hc.04.048.WHl antibody variants of the present disclosure.

[0134] Figure 19B (FIG. 19B) shows increased brain uptake in mice of CD98hc.04.048.WHl antibody variants of the present disclosure, shown as fold-change compared to isotype control antibody.

[0135] Figure 20 (FIG. 20) shows no decrease in amino acid uptake in cells treated with CD98hc.04.048.WHl antibody variants of the present disclosure.

[0136] Figures 21A-21B (FIGs. 21A and 21B) show an increase in CD98hc.04.048.WHl antibody (Figure 21A) and anti-GPNMB / CD98hc.04.048.WHl multi-specific antibody (Figure 2 IB) levels in mouse brain compared to that observed with the corresponding isotype control antibody.

[0137] Figures 22A-22B (FIGs. 22A and 22B) show levels of brain uptake of various anti- CD98hc.04.048.WHl monovalent Fab variants of the present disclosure presented as ng Ab domain / mg total protein and fold-change over control, respectively.

[0138] Figure 23A (FIG. 23A) shows serum clearance of antibodies containing an anti- CD98hc.04.048.WHl binding domain following 3 weekly doses in mice.

[0139] Figure 23B (FIG. 23B) shows increased levels of antibodies containing an anti- CD98hc.04.048.WHl binding domain in vessel-depleted mouse brain fractions compared to that observed with an isotype control antibody.

[0140] Figure 24A (FIG. 24A) shows serum pharmacokinetic (PK) measurements of various anti-CD98hc.04.048.WHl antibody variants in mice dosed with 20 mg / kg.

[0141] Figure 24B (FIG. 24B) shows serum pharmacokinetic (PK) measurements of various anti-CD98hc.04.048.WHl antibody variants in mice dosed with 3 mg / kg.

[0142] Figure 24C (FIG. 24C) shows antibody concentrations in vessel-depleted brain samples from mice following administration of various anti-CD98hc.04.048.WHl antibody variants at 20 mg / kg.

[0143] Figure 24D (FIG. 24D) shows antibody concentrations in vessel-depleted brain samples from mice following administration of various anti-CD98hc.04.048.WHl antibody variants at 3 mg / kg.

[0144] Figure 25 (FIG. 25) shows the effect on GCase activity in microglia isolated from nonhuman primates administered 50 mg / kg huIgGl isotype control (column A); 50 mg / kg anti- GPNMB antibody GPN-97 (column B); 216 mg / kg anti-GPNMB antibody GPN-97 (column C); 50 mg / kg isotype control anti-TfR antibody TfR9.lb.39.38 (column D); or 50 mg / kg anti- GPNMB-TfR antibody GPT-23 (GPN-97 + TfR scFv (cis-huIgGl LALAPS + WT huIgGl) (column E).

[0145] Figures 26A-26B (FIGs. 26A and 26B) show bulk RNA-seq analysis of isolated microglia obtained from non-human primates administered an anti-GPNMB-TfR antibody of the present disclosure.DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE

[0146] The present disclosure relates to multi-specific binding proteins comprising a blood brain barrier antigen-binding domain that specifically binds to a target selected from human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc) and an antigen-binding domain that specifically binds to GPNMB and uses (e.g., therapeutic uses) of such multi-specific binding proteins.

[0147] Passive transfer of substances from blood to brain is restricted by the blood brain barrier (BBB). The BBB provides precise control of central nervous system (CNS) homeostasis allowing for proper neuronal function and also protecting neural tissue from toxins and pathogens.However, the BBB poses a problem with regard to delivering therapeutics to the CNS. While recombinant proteins and antibody therapeutics have shown much success outside the CNS, such biologies do not cross the BBB efficiently. As a result, delivery of certain therapeutics to the CNS has relied on injection of the therapeutic directly into the CNS; an invasive procedure with limited efficacy due to rapid export of cerebral spinal fluid (CSF) containing the therapeutic from the brain to the blood. Alternatively, a therapeutic intended for the CNS may be administered systemically at doses high enough to allow for sufficient penetration of the BBB by the therapeutic. However, this approach may result in unintended effects due to the high dose in the periphery or increased manufacturing and formulation burdens to achieve the high dose.

[0148] Multi-specific binding proteins provided herein are capable of crossing the blood brain barrier and capable of transporting an anti-GPNMB antigen binding domain, antibody or antigenbinding fragment thereof across the blood brain barrier, thus enhancing or increasing exposure of the anti-GPNMB antigen binding domain, antibody or antigen-binding fragment thereof to the CNS.

[0149] The present disclosure provides various multi-specific binding protein formats comprising an anti-blood brain barrier antigen-binding domain linked to an anti-GPNMB antigen-binding domain (e.g., in an antibody antigen binding domain or antibody or antigenbinding fragment thereof that binds specifically to GPNMB).

[0150] In some aspects, a multi-specific binding protein of the present disclosure with a “1 +1 multi-specific binding protein format” comprises a TfR antigen-binding domain in a bivalent, bispecific format comprising (i) one antigen-binding domain that binds to human TfR; and (ii) one antigen binding domain that binds to GPNMB. Such a “1+1” format can also comprise an Fc domain or Fc region. In some aspects, a multi-specific binding protein of the present disclosure with a “1 +1 multi-specific binding protein format” comprises a CD98hc antigen-binding domain in a bivalent, bi-specific format comprising (i) one antigen-binding domain that binds to human CD98hc; and (ii) one antigen binding domain that binds to GPNMB. Such a “1+1” format can also comprise an Fc domain or Fc region.

[0151] In some aspects, a multi-specific binding protein of the present disclosure with a “2+1 multi-specific binding protein format” comprises a TfR antigen-binding domain in a trivalent, bispecific format comprising (i) an antigen-binding domain that binds to human TfR and (ii) an antibody comprising two anti-GPNMB antigen-binding domains, wherein the antibody comprises two heavy chains and two light chains; wherein the antigen-binding domain that binds to humanTfR is linked to the C-terminus of one of the two antibody heavy chains. In some aspects, a multi-specific binding protein of the present disclosure with a “2+1 multi-specific binding protein format” comprises a CD98hc antigen-binding domain in a trivalent, bi-specific format comprising (i) an antigen-binding domain that binds to human CD98hc, and (ii) an antibody comprising two anti-GPNMB antigen-binding domains, wherein the antibody comprises two heavy chains and two light chains; wherein the antigen-binding domain that binds to human CD98hc is linked to the C-terminus of one of the two antibody heavy chains.

[0152] In some embodiments, the multi-specific protein comprises a “1+2” multi-specific protein format, wherein the multi-specific protein is trivalent and bi-specific. In some embodiments, the multi-specific protein comprises: (i) two antigen-binding domains that specifically binds to human TfR and (ii) an antibody or antigen-binding fragment thereof that specifically binds to GPNMB, wherein the antibody or antigen-binding fragment thereof is a scFv, VHH, or Fab, optionally comprising an Fc domain. In some embodiments, the multispecific protein comprises a “1+2” multi-specific protein format, wherein the multi-specific protein is trivalent and bi-specific. In some embodiments, the multi-specific protein comprises: (i) two antigen-binding domains that specifically binds to human CD98hc and (ii) an antibody or antigen-binding fragment thereof that specifically binds to GPNMB, wherein the antibody or antigen-binding fragment thereof is a scFv, VHH, or Fab, optionally comprising an Fc domain.

[0153] In some aspects, a multi-specific binding protein of the present disclosure with a “2+2 multi-specific binding protein format” comprises a TfR antigen-binding domain in a tetravalent, bi-specific format comprising (i) two antigen-binding domains that bind to human TfR and (ii) an antibody comprising two anti-GPNMB antigen-binding domains, wherein the antibody comprises two heavy chains and two light chains; wherein one antigen-binding domain that binds to human TfR is linked to the C-terminus of one of the two antibody heavy chains, and the other antigenbinding domain that binds to human TfR is linked to the C-terminus of the other of the two antibody heavy chains. The two antigen-binding domains that bind to human TfR can comprise the same amino acid sequence. The two antigen-binding domains that bind to human TfR can comprise different amino acid sequences. In some aspects, a multi-specific binding protein of the present disclosure with a “2+2 multi-specific binding protein format” comprises a CD98hc antigen-binding domain in a tetravalent, bi-specific format comprising (i) two antigen-binding domains that bind to human CD98hc and (ii) an antibody comprising two anti-GPNMB antigenbinding domains, wherein the antibody comprises two heavy chains and two light chains;wherein one antigen-binding domain that binds to human CD98hc is linked to the C-terminus of one of the two antibody heavy chains, and the other antigen-binding domain that binds to human CD98hc is linked to the C-terminus of the other of the two antibody heavy chains. In some aspects, a multi-specific binding protein of the present disclosure with a “2+2 multi-specific binding protein format” comprises a TfR antigen-binding domain in a tetravalent, bi-specific format comprising (i) two antigen-binding domains that bind to human GPNMB and (ii) an antibody comprising two anti-TfR antigen-binding domains, wherein the antibody comprises two heavy chains and two light chains; wherein one antigen-binding domain that binds to human GPNMB is linked to the C-terminus of one of the two antibody heavy chains, and the other antigen-binding domain that binds to human GPNMB is linked to the C-terminus of the other of the two antibody heavy chains. The two antigen-binding domains that bind to human GPNMB can comprise the same amino acid sequence. The two antigen-binding domains that bind to human GPNMB can comprise different amino acid sequences. In some aspects, a multi-specific binding protein of the present disclosure with a “2+2 multi-specific binding protein format” comprises a CD98hc antigen-binding domain in a tetravalent, bi-specific format comprising (i) two antigen-binding domains that bind to human GPNMB and (ii) an antibody comprising two anti-CD98hc antigen-binding domains, wherein the antibody comprises two heavy chains and two light chains; wherein one antigen-binding domain that binds to human GPNMB is linked to the C-terminus of one of the two antibody heavy chains, and the other antigen-binding domain that binds to human GPNMB is linked to the C-terminus of the other of the two antibody heavy chains. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000).Definitions

[0154] The terms “GPNMB” or “GPNMB polypeptide” or “GPNMB protein” are used interchangeably herein refer herein to any native GPNMB from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (cynos)) and rodents (e.g., mice and rats), unless otherwise indicated. GPNMB is also referred to as osteoactivin (ratortholog), dendritic cell-heparin integrin ligand (DC-HIL, mouse ortholog), or hematopoietic growth factor inducible neurokinin-1 type (HGFIN). In some aspects, the term encompasses both wild-type sequences and naturally occurring variant sequences, e.g, splice variants or allelic variants. In some aspects, the term encompasses “full-length,” unprocessed GPNMB as well as any form of GPNMB that results from processing in the cell. In some aspects, the GPNMB is human GPNMB. As used herein, the term “human GPNMB” refers to a polypeptide with the amino acid sequence of SEQ ID NO:241.

[0155] The terms “anti-GPNMB antibody,” an “antibody that binds to GPNMB,” and “antibody that specifically binds GPNMB” refer to an antibody that is capable of binding GPNMB with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting GPNMB. In some aspects, the extent of binding of an anti-GPNMB antibody to an unrelated, non-GPNMB polypeptide is less than about 10% of the binding of the antibody to GPNMB as measured, e.g, by a radioimmunoassay (RIA). In some aspects, an antibody that binds to GPNMB has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10'8M or less, e.g., from 10'8M to 10'13M, e.g., from 10'9M to 10'13M). In some aspects, an anti-GPNMB antibody binds to an epitope of GPNMB that is conserved among GPNMB from different species.

[0156] The terms “central nervous system” or “CNS” refer to the complex of nerve tissues that control bodily function and includes the brain and spinal cord.

[0157] The terms “blood brain barrier” or “BBB” refer to a network of brain capillary endothelial cells that are closely sealed by tight junctions.

[0158] A “central nervous system antigen” or “CNS antigen” is an antigen expressed in the CNS, including the brain.

[0159] A “brain antigen” is a CNS antigen expressed in the brain.

[0160] The terms “BBB target,” “BBB protein,” “BBB receptor,” and “BBB antigen” refer to a target / protein / receptor / antigen expressed on blood brain barrier cells (e.g., TfR or CD98hc). In some aspects, an antigen-binding domain (e.g., in an antibody, scFv, or Fab) that binds to the BBB target / protein / receptor / antigen allows transportation of a molecule or compound associated with said antigen-binding domain across the BBB.

[0161] A “neurological disorder” as used herein refers to a disease or disorder which affects the CNS and / or which has an etiology in the CNS. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, an ocular disease or disorder, viral ormicrobial infection, inflammation, ischemia, neurodegenerative disease, seizure, behavioral disorders, and a lysosomal storage disease.

[0162] A “lysosomal storage disorder” or “LSD” as used herein refers to a metabolic disease (e.g., inherited metabolic disease) characterized by the accumulation of substrates, such as undigested or partially digested macromolecules, in excess in various cells of organs, which ultimately results in cellular dysfunction and clinical abnormalities. LSDs have been defined as deficiencies in lysosomal function generally classified by the accumulated substrate and include sphingolipidoses, oligosaccharidoses, mucolipidoses, mucopolysaccharidoses, lipoprotein storage disorders, neuronal ceroid lipofuscinoses, and others. LSDs may also include other deficiencies or defects in proteins that result in accumulation of macromolecules, such as proteins necessary for normal post-translational modification of lysosomal enzymes, or proteins important for proper lysosomal trafficking. LSDs are diseases caused by defects in single genes. Enzyme defects cause nearly seventy percent of the LSDs, and the rest are defects in enzyme activator or associated proteins. Lysosomal storage disorders include, but are not limited to, Gaucher’s disease and Neimann-Pick type C disease.

[0163] The terms “Transferrin receptor,” “TfR,” “TfR polypeptide,” and “TfR protein” are used interchangeably herein to refer to any native TfR from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (cynos)) and rodents (e.g., mice and rats), unless otherwise indicated. TfR is also referred to as transferrin receptor protein 1, TR, tfRl, Trfr, T9, and p90. In some aspects, the term encompasses both wild-type sequences and naturally occurring variant sequences, e.g., splice variants or allelic variants. In some aspects, the term encompasses “full-length,” unprocessed TfR, as well as any form of TfR that results from processing in the cell. Full-length transferrin receptor protein includes a short N- terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain. In some aspects, the TfR is human TfR. As used herein, the term “human TfR” refers to a polypeptide with the following amino acid sequence:MMDQARS AF SNLFGGEPLS YTRF SLARQ VDGDNSHVEMKL AVDEEENADNNTKANVT KPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPA ARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFK LSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHA NFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSD WKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPG AAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSL HLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVE KLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEV AGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFR ATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLP ALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO: 1).

[0164] As used herein, the terms “CD98hc,” “CD98hc polypeptide,” and “CD98hc protein” are used interchangeably herein to refer to any native CD98hc from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (cynos)) and rodents (e.g., mice and rats), unless otherwise indicated. CD98hc is also referred to as 4F2 cell-surface antigen heavy chain, 4F2hc, 4F2 heavy chain antigen, lymphocyte activation antigen 4F2 large subunit, solute carrier family 3 member 2, and CD98. CD98hc protein is encoded by the SLC3A2 gene and is part of the large amino acid transporter (LAT) complex. In some aspects, the term encompasses both wild-type sequences and naturally occurring variant sequences, e.g., splice variants or allelic variants. In some aspects, the term encompasses “full-length,” unprocessed CD98hc, as well as any form of CD98hc that results from processing in the cell. In some aspects, the CD98hc is human CD98hc. As used herein, the term “human CD98hc” refers to a polypeptide with the following amino acid sequence:MELQPPEASIAVVSIPRQLPGSHSEAGVQGLSAGDDSELGSHCVAQTGLELLASGDPLPS ASQNAEMIETGSDCVTQAGLQLLASSDPPALASKNAEVTGTMSQDTEVDMKEVELNEL EPEKQPMNAASGAAMSLAGAEKNGLVKIKVAEDEAEAAAAAKFTGLSKEELLKVAGSP GWVRTRWALLLLFWLGWLGMLAGAVVIIVRAPRCRELPAQKWWHTGALYRIGDLQAF QGHGAGNLAGLKGRLDYLSSLKVKGLVLGPIHKNQKDDVAQTDLLQIDPNFGSKEDFD SLLQSAKKKSIRVILDLTPNYRGENSWFSTQVDTVATKVKDALEFWLQAGVDGFQVRDI ENLKDASSFLAEWQNITKGFSEDRLLIAGTNSSDLQQILSLLESNKDLLLTSSYLSDSGST GEHTKSL VTQ YLNATGNRWC SWSLSQ ARLLTSFLP AQLLRL YQLMLFTLPGTPVF S YGD EIGLDAAALPGQPMEAPVMLWDESSFPDIPGAVSANMTVKGQSEDPGSLLSLFRRLSDQ RSKERSLLHGDFHAFSAGPGLFSYIRHWDQNERFLVVLNFGDVGLSAGLQASDLPASAS LPAKADLLLSTQPGREEGSPLELERLKLEPHEGLLLRFPYAA (SEQ ID NO: 2).

[0165] As used herein, the terms “antibody” and “immunoglobulin” are used interchangeably and refer to an antibody molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing (e.g., a glycoprotein), through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term “antibody” encompasses monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multi-specific (e.g., bispecific) antibodies, and any other immunoglobulin molecule so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), based on the identity of their heavy-chain constant regions referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of antibodies have different and well-known subunit structures and three-dimensional configurations. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0166] The terms “anti-TfR antibody,” “antibody that binds to TfR,” and “antibody that specifically binds TfR” refer to an antibody that is capable of binding TfR with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting TfR. In some aspects, the anti-TfR antibody is capable of transporting another diagnostic and / or therapeutic agent into the brain. In certain aspects, an anti-TfR antibody binds to an epitope of TfR that is conserved among TfR from different species.

[0167] The terms “anti-TfR antigen-binding domain,” “antigen-binding domain that binds to TfR,” “anti-TfR antigen-binding region,” “antigen-binding region that binds to TfR,” and “TfR binding domain” refer to an antigen-binding domain that binds to TfR with sufficient affinity such that the antigen-binding domain is useful as a diagnostic and / or therapeutic agent in targeting TfR. In one aspect, the extent of binding of an anti-TfR antigen-binding domain to an unrelated, non-TfR polypeptide is less than about 10% of the binding of the antigen-binding domain to TfR as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to TfR has a dissociation constant (KD) of < 20 pM, <15 pM, <12 pM, <10 pM, < 7.5 pM, < 5 pM, <2.5 pM, < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10'8M or less, e.g., from 10'8M to 10'13M, e.g., from 10'9M to 10'13M). Incertain embodiments, an anti-TfR antigen-binding domain binds to an epitope of TfR that is conserved among TfR from different species.

[0168] The terms “anti-CD98hc antibody,” “antibody that binds to CD98hc,” and “antibody that specifically binds CD98hc” refer to an antibody that is capable of binding CD98hc with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD98hc. In some aspects, the anti-CD98hc antibody is capable of transporting another diagnostic and / or therapeutic agent into the brain. In certain aspects, an anti-CD98hc antibody binds to an epitope of CD98hc that is conserved among CD98hc from different species.

[0169] The terms “anti-CD98hc antigen-binding domain,” “antigen-binding domain that binds to CD98hc,” “anti-CD98hc antigen-binding region,” “antigen-binding region that binds to CD98hc,” and “CD98hc binding domain” refer to an antigen-binding domain that binds to CD98hc with sufficient affinity such that the antigen-binding domain is useful for targeting CD98hc and / or useful as a diagnostic agent, a therapeutic agent, or for transporting a molecule or compound across the BBB. In one aspect, the extent of binding of an anti-CD98hc antigenbinding domain to an unrelated, non-CD98hc polypeptide is less than about 10% of the binding of the antigen-binding domain to CD98hc as measured, e.g., by a radioimmunoassay (RIA). In certain aspects, an antibody that binds to CD98hc has a dissociation constant (KD) of < 0.1 pM, < 1 pM, <10 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., IO’8M or less, e.g., from 10'8M to 10'13M, e.g., from 10'9M to 10'13M). In certain aspects, an anti- CD98hc antigen-binding domain binds to an epitope of CD98hc that is conserved among CD98hc from different species.

[0170] An “antigen-binding domain” or “antigen-binding region” refers to a monovalent portion of an antibody that binds to an antigen. An “antigen-binding domain” can comprise the antigenic determining regions of an antibody (e.g., the complementarity determining regions (CDRs)). An antibody or antigen-binding fragment thereof (including mono-specific and multispecific (e.g., bi-specific) antibodies or antigen-binding fragments thereof can comprise an antigen-binding domain. In some aspects, an antigen-binding domain is not present in the context of an antibody. In some aspects, provided herein is an antibody, a VHH, a Fab, a Fab’, a Fab’- SH, a F(ab’)2, a Fv, or a scFv comprising an antigen-binding domain.

[0171] 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, whole antibodies include those with heavy and light chains including anFc region. The constant regions can be native sequence constant regions (e.g., human native sequence constant regions) or amino acid sequence variants thereof. In some cases, the intact antibody can have one or more effector functions. The C-terminal lysine (residue 447 according to the EU numbering system) of an intact antibody can 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 “full-length antibodies,” “intact antibodies,” or “whole antibodies” can 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

[0172] The term “native IgG antibodies” refers to heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (“L”) chains and two identical heavy (“H”) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intra-chain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[0173] The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody.

[0174] As used herein, the term “heavy chain” when used in reference to an antibody can refer to any distinct type, e.g., alpha (a), delta (5), epsilon (a), gamma (y), and mu (p), based on the amino acid sequence of the constant region, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgGi, IgG2, IgGs, and IgG4. Heavy chain amino acid sequences are well known in the art. In some aspects, the heavy chain is a human heavy chain.

[0175] The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody.

[0176] As used herein, the term “light chain” when used in reference to an antibody can refer to any distinct type, e.g., kappa (K) or lambda (X) based on the amino acid sequence of theconstant regions. Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0177] The terms “variable region” or “variable domain” 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 “VH” and “VL”, 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. Generally, the variable region or variable domain is typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain.

[0178] The term “Fv” or “variable fragment” refers to the minimum antibody fragment which comprises a complete antigen-binding site and consists of a dimer of one heavy-chain variable region (VH) and one light-chain variable region (VL). 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.

[0179] As used herein, the term “constant region” is a region of an antibody that is not the variable region of the antibody, e.g., a carboxyl terminal portion of a light and / or heavy chain which is not directly involved in binding of an antibody to antigen, but which can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain. In certain aspects, an antibody or antigen-binding fragment comprises a constant region or portion thereof that is sufficient for antibody-dependent cell- mediated cytotoxicity (ADCC).

[0180] A “constant domain” means a domain within a constant region that is capable of forming an immunoglobulin fold. Constant domains include the CHI, CH2, CH3, and CL domains.

[0181] The term “antibody fragment” refers to a portion of an antibody. An “antigen-binding fragment” of an antibody refers to a portion of an antibody that binds to an antigen. An antigenbinding fragment of an antibody can comprise the antigenic determining regions of an antibody (e.g., the complementarity determining regions (CDRs)). Examples of antigen-binding fragments of antibodies include, but are not limited to Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, and single chain antibodies. An antigen-binding fragment of an antibody can be monovalent or multi-valent (e.g., bi-valent). An antigen-binding fragment of an antibody can bemonospecific or multi-specific (e.g., bi-specific.) An antigen-binding fragment of an antibody can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans or can be artificially produced.

[0182] The term “Fab” or “fragment antigen-binding region” refers to a region on an antibody that binds to antigens. It is composed of one constant domain, one variable domain of the heavy chain and one variable domain of the light chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.

[0183] The term “F(ab’)2 fragment” refers to antibody fragments that are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving intact some of the hinge region. F(ab’)2 fragments have two antigen-binding F(ab) portions linked together by disulfide bonds, and therefore are divalent with a molecular weight of about 110 kDa. Fab’ fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CHI 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.

[0184] As used herein, a “Fc fragment,” “fragment crystallizable region,” or “Fc region” is composed of two or more polypeptides, each being an antibody heavy chain fragment and each containing at least one (e.g., two or three) heavy chain constant domains. In some aspects, an Fc region is composed of two heavy chain fragments, each containing a CH2 domain and a CH3 domain. 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 can 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, an Fc region may not contain any K447 residues, may contain at least one polypeptide containing a K447 residue and at least one polypeptide that does not contain a K447 residue, or may only contain polypeptides that include a K447 residue. Suitable native-sequence Fc regions for use in the present disclosure include human IgGl, IgG2, IgG3 and IgG4. In a native antibody, an Fc region refers to the region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. However, as used herein, an Fc region can be modified to increase, decrease, or eliminate interaction with Fc receptors and / or proteins of the complimentsystem. In native IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody’s two heavy chains. However, as used herein, the two or more polypeptides in an “Fc region” do not need to have identical sequences. In some aspects, an “Fc region” comprises a first polypeptide comprising an Fc domain (e.g., IgGl Fc domain) with a knob mutation and a second polypeptide comprising an Fc domain (e.g., IgGl Fc domain) with a hole mutation. In native IgM and IgE antibody isotypes, the Fc region contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

[0185] The term “Fc domain” refers to one or more constant region domains within an Fc region, such as a CH2 or CH3 domain, in a single polypeptide. In some aspects, the Fc domain includes at least one amino acid deletion, addition, or substitution as compared to the amino acid sequence of a native Fc domain, such as by including a set of “knob-into-hole” deletions, additions, or substitutions or including amino acid deletions, additions, or substitutions to effect electrostatic steering of the Fc domain to favor attractive interactions among different polypeptide chains. In some aspects, the Fc domain is in a “knob” format. In some aspects, the Fc domain is in a “hole” format.

[0186] The term “single-chain Fv”, also abbreviated as “sFv” or “scFv”, refers to antibody fragments that comprise the VH and VL antibody domains that form a single polypeptide chain. In some aspects, the scFv polypeptide comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0187] The term “diabodies” refers to small antibody fragments prepared by constructing scFv fragments with short linkers (about 5-10 residues) between the VH and VL domains, such that inter-chain but not intra-chain pairing of the variable 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” scFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.

[0188] The term “CDR” or “complementarity determining region” refers to hypervariable regions in the variable region of an immunoglobulin that determine antibody diversity and antigen specificity.

[0189] The term “Kabat numbering” and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or an antigen-binding fragment thereof. In certain aspects, CDRs can be determinedaccording to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35 A and 35B) (CDRH1), amino acid positions 50 to 65 (CDRH2), and amino acid positions 95 to 102 (CDRH3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDRL1), amino acid positions 50 to 56 (CDRL2), and amino acid positions 89 to 97 (CDRL3).

[0190] The term “Chothia” refers to the location of the structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927- 948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontane A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226). In some aspects, the Chothia residues are numbered as shown in the table below.

[0191] The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops and are used by Oxford Molecular’s AbM antibody modeling software.

[0192] In some aspects, the CDRs can be “contact” CDRs. The “contact” CDRs are based on an analysis of the available complex crystal structures.

[0193] The residues from each of these CDRs are noted below.Loop _ Kabat _ AbM _ Chothia _ ContactLI L24-L34 L24-L34 L26-L32 L30-L36L2 L50-L56 L50-L56 L50-L52 L46-L55L3 L89-L97 L89-L97 L91-L96 L89-L96Hl H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering)Hl H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering)H2 H50-H65 H50-H58 H52-H56 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

[0194] CDRs can comprise “extended CDRs” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (Hl), 50-65 or 49-65 (H2), and 93-102, 94- 102, or 95-102 (H3) in the VH. The variable-domain residues are numbered according to Kabat et al., supra, for each of these extended-CDR definitions.

[0195] CDRs can also be identified according to the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97.

[0196] The term “monoclonal” when referring to an antibody or antigen-binding fragment thereof refers to a homogeneous antibody or antigen-binding fragment population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal” antibody or antigen-binding fragment thereof encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv) mutants, fusion proteins or complexes comprising an antibody or antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, a “monoclonal” antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0197] The term “chimeric” antibodies or antigen-binding fragments thereof refers to antibodies or antigen-binding fragments thereof wherein the amino acid sequence is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies or antigen-binding fragments thereof derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability, while the constant regions are homologous to the sequences in antibodies or antigen-binding fragments thereof derived from another (usually human) to avoid eliciting an immune response in that species.

[0198] The term “humanized” antibody or antigen-binding fragment thereof refers to forms of non-human (e.g., murine) antibodies or antigen-binding fragments that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies or antigen-binding fragments thereof are human immunoglobulins in which residues from the complementarity determining regions (CDRs) are replaced by residues from the CDRs of a molecule originating from a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity,affinity, and capability (“CDR grafted”) (Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988)). The humanized antibody or antigen-binding fragment thereof can be further modified by the substitution of additional residues either in the Fv framework region and / or within the replaced non-human residues to refine and optimize the specificity, affinity, and / or capability of the antibody or antigen-binding fragment thereof. In general, the humanized antibody or antigenbinding fragment thereof will comprise VH and VL that comprise substantially all of at least one, and typically two or three, of the CDR regions that correspond to the non-human immunoglobulin, whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody or antigen-binding fragment thereof can also comprise at least a portion of an immunoglobulin constant region or Fc region, typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some aspects, a “humanized antibody” is a resurfaced antibody.

[0199] The term “human” antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin gene locus, where such antibody or antigen-binding fragment is made using any technique known in the art. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0200] “Framework” or “FR” residues are those variable-domain residues other than the CDR residues as herein defined.

[0201] An “acceptor human framework” as used herein is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence thereof, or it can comprise pre-existing amino acid sequence changes. In some aspects, 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. Where pre-existing amino acid changes are present in a VH, in some aspects those changes occur at only three, two, or one of positions 71H, 73H and 78H; for instance, the amino acid residues at those positions can by 71 A, 73T and / or 78A. Insome aspects, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0202] A “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH 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 VL, the subgroup can be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al., supra. Additionally, for the VH, the subgroup can be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra.

[0203] An “amino-acid modification” at a specified position, e.g., of an antibody of the present disclosure, refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent the specified residue. Insertion “adjacent” to a specified residue means insertion within one to two residues thereof. The insertion can be N-terminal or C- terminal to the specified residue. In some aspects, an amino acid modification is a substitution.

[0204] 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.

[0205] A “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human IgGl Fc region (non- A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well.

[0206] A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, in some aspects two or more amino acid substitution(s). In some aspects, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and in some aspects from about one to about five amino acid substitutions compared to a native sequence Fc region or in the Fc region of the parent polypeptide. In some aspects, the variant Fc region possesses at least 80% homology with a native sequence Fc region and / or with an Fc region of a parent polypeptide, at least 90% homology therewith, or at least 95% homology therewith.

[0207] “Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. In some aspects, an FcR is a native sequence human FcR. In some aspects, a FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (“IT AM”) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (“ITIM”) in its cytoplasmic domain. Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. FcRs can also increase the serum half-life of antibodies.

[0208] “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 or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody or antigen-binding fragment thereof and antigen). The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD). Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of k0ff / k0n, whereas KA is calculated from the quotient of k0n / k0ff. konrefers to the association rate constant of, e.g., an antibody or antigen-binding fragment thereof to an antigen, and koir refers to the dissociation rate constant of, e.g., an antibody or antigen-binding fragment thereof from an antigen. The konand koir can be determined by techniques known to one of ordinary skill in the art, such as BIAcore® or KinExA. Dissociation constants may also be determined through any analytical technique, including any biochemical or biophysical technique such as ELISA, surface plasmon resonance (SPR), bio-layer interferometry see, e.g., Octet System by ForteBio), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped-flow analysis, and colorimetric or fluorescent protein melting analyses. (See, e.g., Estep et al, (2013) MAbs 5(2):270-8.)

[0209] With regard to the binding of an antibody to a target molecule, the term “specific binding” or “specifically binds” or is “specific for” a particular polypeptide or an epitope on aparticular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term “specific binding” or “specifically binds” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of about any of 10'4M or lower, 10'5M or lower, 10'6M or lower, 10'7M or lower, 10'8M or lower, 10'9M or lower, IO'10M or lower, 10'11M or lower, 10'12M or lower or a KD in the range of 10'4M to 10'6M or 10'6M to IO'10M or 10'7M to 10'9M. As will be appreciated by the skilled artisan, affinity and KD values are inversely related. A high affinity for an antigen is measured by a low KD value. In some aspects, the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0210] The term “linker” or “linked” refers to the covalent linkage between two polypeptides or two heterologous molecules. In some aspects, a linker is a chemical linker. In some aspects, the linker comprises a peptide bond, and the two polypeptides or two heterologous molecules are linked to each other either directly to or via one or more additional amino acids. A glycine linker is one that comprises one or more glycines, but no other amino acids, e.g., GGGG (SEQ ID NO: 3). A glycine-rich linker is one that comprises one or more glycines and can contain other amino acids as long as glycine is the predominant species in the linker e.g., GGGNGG (SEQ ID NO: 4), wherein N is any amino acid. A glycine-serine linker is one which contains both glycine and serine in any proportion, e.g., GGGS (SEQ ID NO: 5). Similarly, a proline linker is one that comprises one or more prolines but no other amino acids. A proline-rich linker is one that comprises one or more prolines and can contain other amino acids so long as proline is the predominant species in the linker.

[0211] As used herein, “percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence refers to 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 conservativesubstitutions as identical matches. 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 known in the art needed to achieve maximal alignment over the full-length of the sequences being compared.

[0212] A polypeptide, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some aspects, an antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

[0213] As used herein, “substantially pure” refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0214] The term “expression system” refers to one or more nucleic acid molecules comprising coding sequence and control sequence(s) in operable linkage, along with a host cell and / or other in vitro transcription and translation machinery, such that one or more proteins encoded by the nucleic acid molecule(s) are capable of being produced.

[0215] The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors,” or simply, “expression vectors.” In general, expression vectors of utility in recombinant DNA techniquesare often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.

[0216] “Polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.

[0217] A “host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this disclosure. In some aspects, the host cell is an isolated host cell.

[0218] “ Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.

[0219] As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of progression, ameliorating or palliating the pathological state, and remission or improved prognosis of a particular disease, disorder, or condition. An individual is successfully “treated”, for example, if one or more symptoms associated with a particular disease, disorder, or condition are mitigated or eliminated.

[0220] The terms “administer,” “administering,” “administration,” and the like, as used herein, refer to methods that can be used to deliver a drug, e.g., an anti -human antibody or antigenbinding fragment thereof, to the desired site of biological action.

[0221] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. An effective amount can be provided in one or more administrations. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effectof another medication such as via targeting, delaying the progression of the disease, and / or prolonging survival. An effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” can be considered in the context of administering one or more therapeutic agents, and a single agent can be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result can be or is achieved.

[0222] As used herein, the terms “subject” and “patient” are used interchangeably. The subject can be a mammal such as a non-human animal (e.g., cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.). In some aspects, the subject is a cynomolgus monkey. In some aspects, the subject is a human.

[0223] As used herein, administration “in conjunction” or “in combination” with another compound or composition includes simultaneous administration and / or administration at different times. Administration in conjunction also encompasses administration as a coformulation or administration as separate compositions, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration. In some aspects, administration in conjunction is administration as a part of the same treatment regimen.

[0224] As used herein, a “complex” refers to one or more proteins comprising connected parts. The parts can be connected e.g., via a peptide bond (e.g., in a fusion protein or complex), a linker (e.g., a peptide linker), or via noncovalent protein-protein interactions such as disulfide bonds (e.g., in an antibody). Exemplary parts that can be included in a complex include an antigenbinding domain that specifically binds to GPNMB and an antigen-binding domain than specifically binds to CD98hc or TfR, an Fc region, and / or an Fc domain. Accordingly, nonlimiting examples of a “complex” comprising an anti-GPNMB antigen-binding domain and an anti-CD98hc or anti-TfR antigen-binding domain include (a) a fusion protein or complex comprising the anti-GPNMB antigen-binding domain and the anti-CD98hc or anti-TfR antigenbinding domain in a single polypeptide chain and (b) four proteins connected via noncovalent protein-protein interactions, wherein the first protein contains a heavy chain of anti-GPNMB antibody comprising an anti-GPNMB antigen-binding domain, the second protein contains aheavy chain of the anti-GPNMB fused to an scFv containing an anti-CD98hc or anti-TfR scFv containing an anti-CD98hc or anti-TfR antigen-binding domain, and the third and fourth proteins contain light chains of the anti-GPNMB antibody (e.g., as shown in Figure 1). Formats of other exemplary complexes are provided in Figures 1-3.

[0225] As used herein, the term “composition” refers to pharmaceutical combination of components. The components can be connected to each other via covalent bonds or non-covalent bonds or can merely be contained in the same mixture or solution. Accordingly, a “composition” comprising an antigen-binding domain can refer, e.g., to an antibody, scFv, or Fab comprising the antigen-binding domain, can refer to a fusion protein or complex comprising the antigenbinding domain, or can refer to a mixture of solution comprising such an antigen-binding domain, antibody, scFv, Fab, fusion protein or complex. In some aspects, a composition is a “pharmaceutical composition.”

[0226] As used herein the term “pharmaceutical composition” refers to a combination comprising an active agent (e.g., an antigen-binding domain, an antibody, a scFv, or Fab comprising the antigen-binding domain disclosed herein) with at least one inert pharmaceutically acceptable agent (e.g., an excipient or a carrier).

[0227] As used herein, the terms “about” and “approximately,” when used to modify a numeric value or numeric range, indicate that deviations of up to 10% above and down to 10% below the value or range remain within the intended meaning of the recited value or range. It is understood that wherever aspects are described herein with the language “about” or “approximately” a numeric value or range, otherwise analogous aspects referring to the specific numeric value or range are also provided.

[0228] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly indicates otherwise. For example, reference to an “antibody” is a reference to from one to many antibodies, such as molar amounts, and includes equivalents thereof known to those skilled in the art, and so forth.

[0229] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can mean “includes,” “including,” and the like; “consisting essentially of’ or “consists essentially of’ are open-ended, allowing for the presenceof more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art aspects.

[0230] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0231] The present disclosure will be more fully understood by reference to the following Examples. They should not, however, be construed as limiting the scope of the present disclosure. All citations throughout the disclosure are hereby expressly incorporated by reference.I. Glycoprotein nonmetastatic melanoma protein B (GPNMB), glucocerebrosidase (GCase), and disorders associated therewith

[0232] Human GPNMB is a type 1 transmembrane glycoprotein that, as a result of alternative splicing, occurs as two polypeptide isoforms, one of 572 amino acids and a shorter of 560 amino acids. GPNMB has 12 glycosylation sites, a polycystic kidney disease (PKD) domain, an integrin-recognition (RGD) motif, an immunoreceptor tyrosine-based activation-like motif (IT AM-like), and a lysosomal targeting (dileucine) motif (Abdelmagid et al, 2008, Exp Cell Res, 314:2334-2351). GPNMB can be cleaved by the metalloproteinase ADAM10, releasing a soluble fragment that can bind to various receptors and trigger a cellular response (Rose et al, 2010, PLoS One, 5(8):el2093).

[0233] Increased expression of GPNMB in the brain has been detected in a diverse number of neurodegenerative disorders, including Parkinson’s disease, Alzheimer’s disease, and ALS (Huttenrauch et al, 2018, Acta Neuropathol Commun, 6: 108; Tanaka et al, 2012, Sci Rep, 2:573; Ono et al, 2016, Sci Rep, 6:23241; Moloney et al, 2018, Neurobiol Dis, 120: 1-11). GPNMB levels are elevated in the substantia nigra of sporadic Parkinson’s disease patients (Moloney et al, 2018, Neurobiol Dis, 120: 1-11). Furthermore, the single nucleotide polymorphism (SNP) rsl99347, a known top risk SNP for Parkinson’s disease, is located in the GPNMB gene and results in increased GPNMB expression (Murthy et al, 2017, Neurogenetics, 18: 121-133).

[0234] Additionally, GPNMB protein levels are elevated and correlate with disease severity and progression in various lysosomal storage disorders, such as, for example, Gaucher’s disease (Zigdon et al, 2015, PLoS One, 10:e0120194; Kramer et al, 2016, FEBS Open Bio, 6:902-913; Murugesan et al, 2018, Blood Cells, Mol Dis, 68:47-53). Glucocerebrosidase (GCase) is a lysosomal P-glucosidase-degrading glucosylceramide. Inherited deficiency of glucocerebrosidase is the cause of autosomal recessive Gaucher’s disease, the most common lysosomal storagedisease (Brady et al, 1966, J Clin Invest, 45: 1112-1115). This disease is caused by mutations in the lysosomal hydrolase P-glucosidase glycocerebrosidase, resulting in an accumulation of its substrate glycosylceramide (van der Lienden et al, 2018, Int Journal of Molecular Sciences, 20:66). Genetic deficiency of glucocerebrosidase is also a risk factor for Parkinson’s disease and Lewy body dementia (Sidransky et al, 2009, N Eng J Med, 361 :1651-1661; Tsuang et al, 2012, Neurology, 79: 1944-1950). Conduritol P-epoxide (CBE) is an irreversible inhibitor of glucocerebrosidase and is used to generate in vitro and in vivo models for investigating Gaucher diseases and Parkinson’s disease. Systemic inhibition of lysosomal glucocerebrosidase using conduritol-P-epoxide (CBE) is associated with specific Parkinson’s disease-relevant pathologies, including accumulation of a-synuclein aggregates, elevations of glycosphingolipids, and widespread neuroinflammation (Rocha et al, 2015, Antioxid Redox Signal, 23:550-564).

[0235] Recent studies have shown a link between mutations in the glucocerebrosidase gene and increased risk of Parkinson’s disease, with more severe mutations imparting higher levels of risk. Reduced glucocerebrosidase activity has been reported in the substantia nigra, cerebellum, and caudate of Parkinson’s disease patients (Alcalay et al., 2015, Brain 138:2648).Glucocerebrosidase mutations result in a gain of toxic function and / or altered cellular function due to a diversion of cellular resources (Gregg et al., 2012, Ann. Neurol. 72:455-46; Schondorf et al., 2014, Nat. Commun. 5:4028; Kilpatrick et al., 2016, Cell Calcium. 59: 12-20; Cullen et al., 2011, Ann. Neurol.69:940-953). Studies in rodent models of Parkinson’s disease have also suggested a link between glucocerebrosidase activity and a-synuclein accumulation (Rocha et al., 2015, Antioxidants & Redox Signaling 23: 550; Rocha et al., 2015, Neurobiology of Disease 82:495).

[0236] Pharmacological inhibition of glucocerebrosidase with conduritol -beta-epoxide (CBE) resulted in increased GPNMB levels in brain (Zigdon et al, 2015, PLoS One, 10:e0120194; Vardi et al, 2016, J Pathol, 239:496-509; Moloney et al, 2018, Neurobiol Dis, 120: 1-11).

[0237] Parkinson’s disease is a progressive disorder that affects movement, and it is recognized as the second most common neurodegenerative disease after Alzheimer’s disease. Common symptoms of Parkinson’s disease include resting tremor, rigidity, and bradykinesia, and nonmotor symptoms, such as depression, constipation, pain, sleep disorders, genitourinary problems, cognitive decline, and olfactory dysfunction, are also increasingly being associated with this disorder.

[0238] Provided herein are multi-specific binding proteins comprising a BBB antigen-binding domain linked to an antigen binding domain (e.g., in an antibody or antigen-binding fragment thereof that binds specifically to GPNMB (e.g., human GPNMB). Multi-specific binding proteins provided herein are useful, e.g., for the treatment of GPNMB associated disorders, including neurodegenerative disorders (e.g., Parkinson’s disease), and lysosomal storage disorders. In some aspects, the BBB antigen-binding domain of a multi-specific binding protein provided herein binds to TfR (e.g., human TfR). In some aspects, the BBB antigen-binding domain of a multispecific binding protein provided herein binds to CD98hc (e.g., human CD98hc).

[0239] In some aspects, multi-specific binding proteins of the present disclosure bind human GPNMB, bind mouse GPNMB, bind cynomolgus monkey GPNMB, bind both human and mouse GPNMB, or bind human, mouse, and cynomolgus monkey GPNMB.

[0240] In some aspects, multi-specific binding proteins of the present disclosure bind human GPNMB with an affinity of about 0.4 nM to about 120 nM. In some aspects, multi-specific binding proteins of the present disclosure bind human GPNMB with an affinity of about 4 nm to about 15 nm. In some aspects, multi-specific binding proteins of the present disclosure bind mouse GPNMB with an affinity of about 0.3 nM to about 5 nM. In some aspects, multi-specific binding proteins of the present disclosure bind mouse GPNMB with an affinity of about 1 nM to about 3 nM. In some aspects, multi-specific binding proteins of the present disclosure bind cynomolgus GPNMB with an affinity of about 0.4 nM to about 1.04 nM. In some aspects, multispecific binding proteins of the present disclosure bind mouse GPNMB with an affinity of about 5 nM to about 15 nM. In other aspects, multi-specific binding proteins of the present disclosure bind human GPNMB with an affinity of about 0.14 to about 0.65 nM and bind mouse GPNMB with an affinity of about 0.18 nM to about 0.44 nM.

[0241] In some aspects, multi-specific binding proteins of the present disclosure are antagonistic to GPNMB expression and / or activity. In some aspects, multi-specific binding proteins of the present disclosure increase expression levels (e.g., increase cell surface expression) of PDL1 (e.g., in macrophages, including human macrophages); increase expression levels (e.g., increase cell surface expression) of CD40 (e.g., in macrophages, including human macrophages); increase expression levels (e.g., increase cell surface expression) of CD80 (e.g., in macrophages, including human macrophages); increase serum cytokine expression levels of IL- 12p40 and CCL5. In some aspects, multi-specific binding proteins of the present disclosure promote macrophage activation.

[0242] In some aspects, multi-specific binding proteins of the present disclosure increase GCase activity in cells (e.g., in macrophages, including human macrophages). In some aspects, multi-specific binding proteins of the present disclosure are effective at overcoming a decrease in GCase activity associated with reduced progranulin levels.

[0243] In some aspects, multi-specific binding proteins of the present disclosure decrease GPNMB expression levels (e.g., decrease cell surface expression) in cells (e.g., in macrophages, including human macrophages). In some aspects, multi-specific binding proteins of the present disclosure decrease LAMP2 expression levels in cells (e.g., in macrophages, monocytes, neutrophils).

[0244] In some aspects, multi-specific binding proteins of the present disclosure inhibit or reduce inflammasome activation. In some aspects, multi-specific binding proteins of the present disclosure inhibit IL-1 [3 expression or release.

[0245] In some aspects, multi-specific binding proteins of the present disclosure reduce neural inflammation. In some aspects, multi-specific binding proteins of the present disclosure reduce expression of Clq, GFAP, IB Al, and CTSD associated with neural inflammation.

[0246] In some aspects, multi-specific binding proteins of the present disclosure reduce lysosomal stress.II. Antigen-Binding Domains and Compositions Comprising Antigen-Binding Domains

[0247] In some aspects, compositions provided herein may comprise an antigen-binding domain.

[0248] In some aspects, compositions provided herein may comprise a fusion protein or complex. In some aspects, the fusion protein or complex comprises an antigen-binding domain.

[0249] In some aspects, compositions provided herein may comprise a multi-specific protein. In some aspects, the multi-specific protein comprises an antigen-binding domain.

[0250] In some aspects, compositions provided herein may comprise an antibody or an antibody fragment thereof.

[0251] In some aspects, compositions provided herein may comprise an antigen-binding fragment. Antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. Fordiscussion of Fab and F(ab’)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0252] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent and / or bispecific. See, for example, EP404097; WO 1993 / 01161; Hudson et al. Nat. Med. 9: 129- 134 (2003). Triabodies and tetrabodies are also described in Hudson et al. Nat. Med. 9:129-134 (2003). Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In some aspects, a single-domain antibody is a human single-domain antibody (see, e.g., U.S. Patent No. 6,248,516).

[0253] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0254] As provided herein, a composition comprising an antigen-binding domain provided herein can be chimeric. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody.

[0255] As provided herein, a composition comprising an antigen-binding domain provided herein can be humanized. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some aspects, a humanized antibody is substantially non-immunogenic in humans. In some aspects, a humanized antibody has substantially the same affinity for a target as an antibody from another species from which the humanized antibody is derived. See, e.g., U.S. Pat. No. 5,530,101; 5,693,761; 5,693,762; and 5,585,089. In some aspects, amino acids of an antibody variable domain that can be modified without diminishing the native affinity of the antigen-binding domain while reducing its immunogenicity are identified. See, e.g., U.S. Pat. Nos. 5,766,886 and 5,869,619. Generally, a humanized antibody comprises one or more variable domains in which CDRs (or portions thereof) are derived from a non-human antibody, and framework regions (FRs) (or portions thereof) are derived from human antibody sequences. A humanized antibody can comprise at least a portion of a human constant region. In some aspects,some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0256] Humanized antibodies and methods of making them are reviewed, for example, in Almagro et al. Front. Biosci. 13: 161 9-1633 (2008), and are further described, e.g., in U.S. Patent Nos. 5,821,337; 7,527,791; 6,982,321; and 7087409. Human framework regions that can 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. Set. 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)).

[0257] As provided herein, a composition comprising an antigen-binding domain provided herein can be human. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk et al. Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg Curr. Opin. Immunol. 20:450-459 (2008).

[0258] Human antibodies can 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. One can engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci in anticipation that such mice would produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can preserve the large variable gene diversity as well as the proper regulation of antibody production and expression. By exploiting the mouse machinery for antibody diversification and selection and the lack of immunological tolerance to human proteins, the reproduced human antibody repertoire in these mouse strains can yield high affinity fully human antibodies against any antigen of interest, including human antigens. Using the hybridoma technology, antigen-specific human mAbs with the desired specificity can be produced and selected. Certain exemplary methods are described in U.S. Pat. No. 5,545,807, EP546073, and EP546073. See also, for example, 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 can be further modified, e.g., by combining with a different human constant region.

[0259] Human antibodies 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) and Boemer 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, 1 03:3557-3562 (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). Human hybridoma technology (Trioma technology) is also described in Vollmers et al. Histology and Histopathology 20(3):927-937 (2005) and Vollmers et al. Methods and Findings in Experimental and Clinical Pharmacology 27(3): 185-91 (2005). Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0260] In some aspects, an antibody is a human antibody isolated by in vitro methods and / or screening combinatorial libraries for antibodies with the desired activity or activities. Suitable examples include but are not limited to phage display (CAT, Morphosys, Dyax, Biosite / Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed) ribosome display (CAT), yeast display (Adimab), and the like. In certain phage display methods, repertoires of VH and VL 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). 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. See also 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(2): 1 19-132 (2004). Phage typically display antibody fragments, either as single-chain Fv (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 selfantigens 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 comprising random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom et al. J. Mol. Biol., 227: 381-388, 1992. Patent publications describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2007 / 0292936 and 2009 / 0002360. Antibodies isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.III. Fc Domains and Regions

[0261] A composition provided herein can comprise an Fc domain or region or fragment thereof. In some aspects, an Fc domain or region is of IgG class, the IgM class, or the IgA class. In some aspects, an Fc domain or region or fragment thereof is an IgG Fc domain or region or fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a human IgG Fc domain or region or fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a human IgGl Fc domain or region or fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a human IgG2 Fc domain or region or fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a human IgG4 Fc domain or region or fragment thereof.

[0262] In some aspects provided herein, a composition provided herein comprises a modified Fc domain or region or fragment thereof. In some aspects, the modified Fc domain or region or fragment thereof is a modified IgGl Fc comprising one or more modifications. For example, in some aspects, the IgGl modified Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc domain of the same isotype).

[0263] In some aspects, the Fc domain or region or fragment thereof is a wildtype IgG2. In some aspects, the modified Fc domain or region or fragment thereof is a modified IgG2 Fc comprising one or more modifications. For example, in some aspects, the modified IgG2 Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type IgG2 Fc).

[0264] In some aspects, the Fc domain or region or fragment thereof is a wildtype IgG4. In some aspects, the modified Fc domain or region or fragment thereof is a modified IgG4 Fc comprising one or more modifications. For example, in some aspects, the modified IgG4 Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type IgG4 Fc). In someaspects, the one or more amino acid substitutions in the modified IgG4 Fc are IgG4-S228P or IgG4-S228P / L235E, where the amino acid position is according to the EU numbering convention.

[0265] In some aspects, the one or more amino acid substitutions are selected from N297A (Bolt S et al. (\99 ) Eur J Immunol 23:403-411), D265A (Shields et al. (2001) A. J. Biol. Chem. 276, 6591-6604), L234A, L235A (Hutchins et al. (1995) Proc Natl Acad Sci USA, 92: 11980- 11984; Alegre et al., (1994) Transplantation 57: 1537-1543. 31; Xu et al., (2000) Cell Immunol, 200: 16-26), G237A (Alegre et al. (1994) Transplantation 57: 1537-1543. 31; Xu et al. (2000) Cell Immunol, 200: 16-26), C226S, C229S, E233P, L234V, L234F, L235E (McEarchern et al., (2007) Blood, 109: 1185-1192), P331S (Sazinsky et al., (2008) Proc Natl Acad Sci USA 2008, 105:20167-20172), K322A (Hezareh et al. (2001) J Virol. 75(24) 12161-12168), S267E, L328F, A330L, M252Y, S254T, E430G, and / or T256E, where the amino acid position is according to the EU numbering convention. In some aspects, the Fc comprises the amino acid substitutions L234A, L235A, and P331S (LALAPS) according to EU numbering. In some aspects, the Fc comprises N325S and L328F mutations according to EU numbering. In some aspects, the Fc comprises P329G or P329S according to EU numbering. In some aspects, the Fc comprises K322A according to EU numbering.Table 1: Exemplary Fc Domains

[0266] In some aspects provided herein, a composition provided herein comprises one or more mutations to promote heterodimerization of Fc domains. In some aspects, a Fc region of a bispecific complex provided herein is formed by Fc domains that contain amino acid mutations, substitutions, additions, or deletions to promote heterodimerization in which different polypeptides comprising different Fc domains can dimerize to yield a heterodimer configuration. In some aspects, a bispecific of the present disclosure comprises a first Fc sequence comprising a first CH3 region, and a second Fc sequence comprising a second CH3 region, wherein the sequences of the first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions.

[0267] Methods to promote heterodimerization of Fc domains include amino acid deletions, additions, or substitutions of the amino acid sequence of the Fc domain, such as by including a set of “knob-into-hole” deletions, additions, or substitutions or including amino acid deletions, additions, or substitutions to effect electrostatic steering of the Fc to favor attractive interactions among different polypeptide chains. Methods for promoting heterodimerization of complementary Fc polypeptides have been previously described in, for example, Ridgway et al, 1996, Protein Eng, 9:617-621; Merchant et al, 1998, Nature Biotechnol, 16:677-681; Moore et al,2011, MAbs, 3:546-557; Von Kreudenstein et al, 2013, 5:646-654; Gunasekaran et al, 2010, J Biol Chem, 285: 19637-19464; Leaver-Fay et al, 2016, Structure, 24:641-651; Ha et al, 2016, Frontiers in Immunology, 7: 1; Davis et al, 2010, Protein Eng Des Sei, 23: 195-202; PCT Pub. Nos. W01996 / 027011; WO 1998 / 050431; W02006 / 028936; W02009 / 089004;WO201 1 / 143545; WO2014 / 067011; WO2012 / 058768; WO2018 / 027025; US Pub. Nos. US2014 / 0363426; US2015 / 0307628; US2018 / 0016354; US2015 / 0239991; US2017 / 0058054; U.S. Pat. Nos. 5,731,168; 7,183,076; 9,701,759; 9,605,084; 9,650,446; 8,216,805; 8,765,412; and 8,258,268.

[0268] In some aspects, complementary Fc polypeptides of an Fc heterodimer include a mutation to alter charge polarity across the Fc dimer interface such that co-expression of electrostatically matched Fc domains support favorable attractive interactions, thereby promoting desired Fc heterodimer formation; whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation (Guneskaran et al, 2010, J Biol Chem, 285:19637-19646). When co-expressed in a cell, association between the polypeptide chains is possible but the chains do not substantially self-associate due to charge repulsion.

[0269] “Knob-hole” or “knob-into-hole” configurations are complementary Fc polypeptides of an Fc heterodimer that promote heterodimerization of two Fc polypeptides. “Knob-into-hole” technology is described in U.S. Pat. Nos. 5,731,168; 7,695,936; 8,216,805; 8,765,412; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan).Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis.

[0270] In some aspects, a composition provided herein comprises a Fc dimer comprising a “knob” mutation in one Fc domain and a “hole” mutation in the other Fc domain. In some aspects, the “knob” mutation comprises the amino acid substitution T366W according to EU numbering. In some aspects, the “hole” mutation comprises the amino acids substitutions T366S,L368A, and Y407V according to EU numbering. In some aspects, the “knob” mutation comprises the amino acid substitution T366W in one of the two subunits of the Fc dimer, and the “hole” mutation comprises the amino acid substitutions T366S, L368A and Y407V in the other subunit of the Fc dimer. In some aspects, the subunit of the Fc dimer comprising the “knob” mutation additionally comprises the amino acid substitution S354C, and the subunit of the Fc dimer comprising the “hole” mutation additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc dimer, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). Thus, in such configurations, a first Fc polypeptide comprises amino acid modifications to form the “knob” and a second Fc polypeptide comprises amino acid modifications to form the “hole” thus forming an Fc heterodimer comprising complementary Fc polypeptides.

[0271] Exemplary paired amino acid modifications of complementary Fc polypeptides of an Fc heterodimeric configuration are set forth below in Table 2 (EU numbering).Table 2: Exemplary paired Fc modifications for heterodimeric Fc domainsIV. Multi-specific Binding Proteins that Bind to Blood-Brain Barrier Receptors or Proteins

[0272] Provided herein are multi-specific binding proteins that specifically bind to human receptors or proteins of the blood-brain barrier. Such multi-specific binding proteins are capable of crossing the blood brain barrier (BBB) and capable of transporting other agents (e.g.,therapeutically active agents) associated with the composition across the blood brain barrier. Exemplary human receptors or proteins of the blood-brain barrier are transferrin receptor (TfR) and CD98 heavy chain (CD98hc).

[0273] In some aspects, the compositions provided herein comprise a TfR antigen-binding domain. In some aspects, the compositions provided herein comprise a CD98hc antigen-binding domain.A. Antigen-Binding Domains That Bind to TfR

[0274] Provided herein are antigen-binding domains that specifically bind to human TfR. Such anti-TfR antigen-binding domains are capable of crossing the blood brain barrier (BBB) and capable of transporting other agents (e.g., therapeutically active agents) associated with the antigen-binding domain across the blood brain barrier. Accordingly, in some aspects, provided herein are antigen-binding domains that specifically bind to human TfR and are capable of being internalized in blood brain barrier epithelial cells.

[0275] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six CDRs of an antibody listed in Tables 3, 4, 13, and 14 below (i.e. the three VH CDRs of the antibody listed in Table 3 or 13 and the three VL CDRs of the same antibody listed in Table 4 or 14). In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six CDRs of an antibody listed in Tables 3, 4, 5, 13, 14, or 15 below. In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six CDRs of an antibody listed in Tables 3, 4, 5, 13, 14, or 15 as determined by Kabat numbering. In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six Chothia CDRs of an antibody listed in Tables 3, 4, 5, 13, 14, or 15.

[0276] In some aspects, the CDRs of an antigen-binding domain that specifically binds to human TfR can be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732- 745. See also, e.g., Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In some aspects, provided herein are antigen-binding domains that specifically bind to human TfR and comprise VH and VL CDRs of an antibody listed in Tables 3, 4, 5, 13, 14, or 15 as determined by the method in MacCallum RM et al.

[0277] In some aspects, the CDRs of an antigen-binding domain that specifically binds to human TfR can be determined according to the AbM numbering scheme, which refers to AbM hypervariable regions, which represent a compromise between the Kabat CDRs and Chothiastructural loops, and are used by Oxford Molecular’s AbM antibody modeling software (Oxford Molecular Group, Inc.). In some aspects, provided herein are antigen-binding domains that specifically bind to human TfR and comprise VH and VL CDRs of an antibody listed in Tables 3, 4, 5, 13, 14, or 15 as determined by the AbM numbering scheme.

[0278] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six IMGT CDRs of an antibody listed in listed in Tables 3, 4, 5, 13, 14, or 15 according to the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL- CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97.

[0279] In some aspects, an antigen-binding domain that specifically binds to human TfR provided herein is described by its VL domain alone, or its VH domain alone, or by its 3 VL CDRs alone, or its 3 VH CDRs alone. See, for example, Rader C et al., (1998) PNAS 95: 8910- 8915, which is incorporated herein by reference in its entirety, describing the humanization of the mouse anti-avP3 antibody by identifying a complementing light chain or heavy chain, respectively, from a human light chain or heavy chain library, resulting in humanized antibody variants having affinities as high or higher than the affinity of the original antibody. See also Clackson T et al., (1991) Nature 352: 624-628, which is incorporated herein by reference in its entirety, describing methods of producing antibodies that bind a specific antigen by using a specific VL domain (or VH domain) and screening a library for the complementary variable domains. The screen produced 14 new partners for a specific VH domain and 13 new partners for a specific VL domain, which were strong binders, as determined by ELISA. See also Kim SJ & Hong HJ, (2007) J Microbiol 45: 572-577, which is incorporated herein by reference in its entirety, describing methods of producing antibodies that bind a specific antigen by using a specific VH domain and screening a library (e.g., human VL library) for complementary VL domains; the selected VL domains in turn could be used to guide selection of additional complementary (e.g., human) VH domains.

[0280] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the VH of an antibody listed in Table 5 or 15.

[0281] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the VL of antibody listed in Table 5 or 15.

[0282] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the VH and the VL of an antibody listed in Table 5 or 15 (i.e., the VH of the antibody listed in Table 5 or 15 and the VL of the same antibody listed in Table 5.

[0283] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 80% identical to a VH amino acid sequence of an antibody listed in Table 5 or 15 and (ii) a VL comprising an amino acid sequence that is at least 80% identical to the VL amino acid sequence of the same antibody in Table 5 or 15. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3, 4, 13, and 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs). In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 85% identical to a VH amino acid sequence of an antibody in Table 5 or 15 and (ii) a VL comprising an amino acid sequence that is at least 85% identical to the VL amino acid sequence of the same antibody in Table 5 or 15.

[0284] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 90% identical to a VH amino acid sequence of an antibody in Table 5 or 15, and (ii) a VL comprising an amino acid sequence that is at least 90% identical to the VL amino acid sequence of the same antibody in Table 5 or 15. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of an antibody in listed in Tables 3, 4, 13, and 14 (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs).

[0285] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 95% identical to a VH amino acid sequence of an antibody in Table 5 or 15 and (ii) a VL comprising an amino acid sequence that is at least 95% identical to the VL amino acid sequence of the same antibody in Table 5 or 15. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3, 4, 13, and 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0286] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 96% identical to a VH amino acid sequence of an antibody in Table 5 or 15 and (ii) a VL comprising an amino acid sequence that is at least 96% identical to the VL amino acid sequence of the same antibody inTable 5 or 15. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3, 4, 13, and 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0287] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 97% identical to a VH amino acid sequence of an antibody in Table 5 or 15 and (ii) a VL comprising an amino acid sequence that is at least 97% identical to the VL amino acid sequence of the same antibody in Table 5 or 15. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3, 4, 13, and 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0288] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 98% identical to a VH amino acid sequence of an antibody in Table 5 or 15 and (ii) a VL comprising an amino acid sequence that is at least 98% identical to the VL amino acid sequence of the same antibody in Table 5 or 15. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3, 4, 13, and 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0289] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 99% identical to a VH amino acid sequence of an antibody in Table 5 or 15 and (ii) a VL comprising an amino acid sequence that is at least 99% identical to the VL amino acid sequence of the same antibody in Tables 3, 4, 13, and 14. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3, 4, 13, and 14 (e.g., the non- identical amino acids in the VH and / or VL are outside of the CDRs).

[0290] In some aspects, provided herein is an antigen-binding domain that competitively inhibits binding to TfR of as an antibody comprising a VH amino acid sequence of an antibody in Table 5 or 15 and a VL amino acid sequence of the same antibody in Table 5 or 15.

[0291] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises a VH and a VL on a single polypeptide chain (e.g., a VH and VL in Table 5 or 15).

[0292] In some aspects, the antigen-binding domain comprises an scFv. In some aspects, an scFv comprises a VH and a VL of an antibody listed in Table 5 or 15. The scFv can comprise a VH that is N-terminal to a VL or a VL that is N-terminal to a VH. The scFv can comprise alinker, e.g., between a VH and a VL. Accordingly, the scFv can be in the orientation VH-linker- VL or VL-linker-VH. Such a linker can be about 5 to about 25 amino acids in length. Such a linker can be about 5 to about 20 amino acids in length. Such a linker can be about 10 to about 25 amino acids in length. Such a linker can be about 10 to about 20 amino acids in length. Such a linker can be, e.g., a glycine linker, a glycine-rich linker, or a glycine-serine linker. Such a linker can comprise the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 6). Such a linker can comprise the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 7).

[0293] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises a VH on a first polypeptide and a VL on a second polypeptide (e.g., a Fab). The Fab comprises one constant domain, one VH, and one VL. In some aspects, the antigen-binding domain comprises a Fab comprising a constant domain, a VH of an antibody listed in Table 5 or 15 and a VL of the same antibody in Table 5 or 15. In some aspects, the antigen-binding domain that comprises a Fab comprises the CDRs of the antibody in Tables 3, 4, 13, and 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0294] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the antigen-binding fragment of a heavy chain only antibody (e.g., a VHH or nanobody). In some aspects, an antigen-binding domain comprises a VHH comprising the VH of an antibody listed in Table 5 or 15. In other aspects, the antigen-binding domain comprises a VHH comprises the heavy chain CDRs of an antibody listed in Table 3 or 13.

[0295] In some aspects, an antigen-binding domain that specifically binds to human TfR is a murine antigen-binding domain. In some aspects, an antigen-binding domain that specifically binds to human TfR is a chimeric antigen-binding domain. In some aspects, an antigen-binding domain that specifically binds to human TfR is a humanized antigen-binding domain. In some aspects, an antigen-binding domain that specifically binds to human TfR is a human antigenbinding domain.

[0296] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR also binds to cynomolgus monkey TfR.

[0297] In certain aspects, an antibody that binds to TfR has a dissociation constant (KD) of about 0.01 nM to about 10,000 nM, less than about 20 pM, less than about 15 pM, less than about 12 pM, less than about 10 pM, less than about 7.5 pM, less than about 5 pM, less than about 2.5 pM, less than about 1 pM, less than about 100 nM, less than about 10 nM, less thanabout 1 nM, less than about 0.1 nM, less than about 0.01 nM, or less than about 0.001 nM (e.g., 10'8M or less, e.g., from 10'8M to 10'13M, e.g., from 10'9M to 10'13M).

[0298] In some aspects, the anti-TfR antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 8, 15, 25, 43, 55, and 61, respectively.

[0299] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 102 and 158, respectively.

[0300] In some aspects, the anti-TfR antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 10, 22, 28, 420, 58, and 62, respectively.

[0301] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 117 and 428, respectively.

[0302] In some aspects, the anti-TfR antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 10, 22, 30, 46, 58, and 62, respectively.

[0303] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 118 and 170, respectively.

[0304] In some aspects, the anti-TfR antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 10, 22, 28, 423, 58, and 62, respectively.

[0305] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 117 and 431, respectively.

[0306] In some aspects, an antigen-binding domain provided herein bind to human TfR with “low” affinity. In some aspects, the antigen-binding domain binds human TfR with an affinity between 500 nM and 10 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 2 pM and 5 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 1 pM and 5 pM. In some aspects, the antigen-binding domain bindshuman TfR with an affinity between 2 pM and 8 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 750 nM and 2 pM.

[0307] In some aspects, the antigen-binding domain binds human TfR with an affinity between about 750 nM and about 10,000 nM (or about 10 pM), such as about 751 nM to about 10,000 nM, such as about 751 nM to about 2,500 nM, about 751 nM to about 5,000 nM, about 2,500 nM to about 5,000 nM, about 2,500 nM to about 10,000 nM, about 5,000 nM to about 10,000 nM, and values and ranges there between. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 2 pM and about 5 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 1 pM and about 5 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 2 pM and about 8 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 750 nM and about 2 pM.

[0308] In some aspects, an antigen-binding domain provided herein bind to human TfR with “medium” affinity. In some aspects, an antigen-binding domain provided herein bind to human TfR with “medium” affinity. In some aspects, the antigen-binding domain binds human TfR with an affinity between 10 nM and 500 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 50 nM and 500 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 100 nM and 250 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 10 nM and 100 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 250 nM and 500 nM.

[0309] In some aspects, the antigen-binding domain binds human TfR with an affinity between 51 nM and 750 nM, such as about 51 nM to about 100 nM, about 51 nM to about 250 nM, about 51 nM to about 500 nM, about 100 nM to about 250 nM, about 100 nM to about 500 nM, about 100 nM to about 750 nM, about 250 nM to about 500 nM, about 250 nM to about 750 nM, about 500 nM to about 750 nM, and values and ranges therebetween. In some aspects, the antigenbinding domain binds human TfR with an affinity between about 50 nM and about 500 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 100 nM and about 250 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 10 nM and about 100 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 250 nM and about 500 nM.

[0310] In some aspects, an antigen-binding domain provided herein bind to human TfR with “high” affinity. In some aspects, the antigen-binding domain binds human TfR with an affinity less than 10 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 0.01 nM and 10 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 0.1 nM and 10 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 0.01 nM and 1 nM.

[0311] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR binds with an affinity of about 0.01 nM to about 50 nM (e.g., about 1 nM to about 50 nM, about 5 nM to about 50 nM, about 10 nM to about 50 nM, about 10 nM to about 25 nM, or about 15 nM to about 25 nM). In some embodiments, the antigen-binding domain binds human TfR with an affinity less than about 50 uM. In some aspects, the antigen-binding domain binds human TfR with an affinity less than about 10 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 0.01 nM about about 50 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 0.01 nM and about 10 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 0.1 nM and about 10 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 0.01 nM and about 1 nM.

[0312] In some aspects, an antigen-binding domain provided herein specifically binds to human TfR with an affinity of no more than 250 nM (e.g., 10 pM to 250 nM, 5 pM to 250 nM, 1 nM to 250 nM or 3 nM to 250 nM), an affinity of no more than 200 nM (e.g., 10 pM to 200 nM, 5 pM to 200 nM, 1 nM to 200 nM or 3 nM to 200 nM), or an affinity of no more than 150 nM (e.g., 10 pM to 150 nM, 5 pM to 150 nM, 1 nM to 150 nM or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance.

[0313] In some aspects, an antigen-binding domain provided herein specifically binds to human TfR with an affinity of less than about 250 nM (e.g., about 10 pM to about 250 nM, about 5 pM to about 250 nM, about 1 nM to about 250 nM or about 3 nM to about 250 nM), an affinity of less than about 200 nM (e.g., about 10 pM to about 200 nM, about 5 pM to about 200 nM, about 1 nM to about 200 nM or about 3 nM to about 200 nM). In some aspects, an antigenbinding domain provided herein specifically binds to human TfR with an affinity of less than about 150 nM (e.g., about 10 pM to about 150 nM, about 5 pM to about 150 nM, about 1 nM to about 150 nM or about 3 nM to about 150 nM).

[0314] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR also binds to cynomolgus TfR with an affinity of about 0.01 nM to about 50 nM (such as about 1 nM to about 50 nM, about 5 nM to about 50 nM, about 5 nM to about 30 nM, about 5 nM to about 25 nM, or about 5 nM to about 15 nM), of about 51 nM to about 1250 nM (such as about 100 nM to about 1250 nM or about 150 nM to about 1250 nM), or of about 1251 nM to about 10,000 nM (such as about 1250 nM to about 5000 nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, e.g., using the BIACORE™.

[0315] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR binds to (i) human TfR with an affinity of about 0.01 nM to about 50 nM (such as about 1 nM to about 50 nM, about 5 nM to about 50 nM, about 10 nM to about 50 nM, about 10 nM to about 25 nM, or about 15 nM to about 25 nM) and cynomolgus TfR with an affinity of about 0.01 nM to about 50 nM (such as about 1 nM to about 50 nM, about 5 nM to about 50 nM, about 5 nM to about 30 nM, about 5 nM to about 25 nM, or about 5 nM to about 15 nM); (ii) human TfR with an affinity of about 51 nM to about 750 nM (such as about 100 nM to about 750 nM or about 100 nM to about 700 nM) and cynomolgus TfR wih an affinity of about 51 nM to about 1250 nM (such as about 100 nM to about 1250 nM or about 150 nM to about 1250 nM); or (iii) human TfR with an affinity of about 751 nM to about 10,000 nM (such as about 1000 nM to about 5000 nM) and cynomolgus TfR with an affinity of about 1251 nM to about 10,000 nM (such as about 1250 nM to about 5000 nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, e.g., using BIACORE™.

[0316] In some aspects, the binding of an antigen-binding domain provided herein that specifically binds to human TfR is measured using surface plasmon resonance. Surface plasmon resonance can be measured, e.g., using the Carterra LSA platform or BIACORE®. In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR binds to human TfR as measured by, for example, radioimmunoassay (RIA), Western blot, or ELISA OD450.

[0317] In some aspects, an antigen-binding domain provided herein that binds to human TfR with a moderate to higher affinity (e.g., an affininty of 0.01 nM to 750 nM) results in more rapid brain uptake and clearance. This improved property can be particularly useful, e.g., for complexing with enzymes or proteins that tend to have a faster clearance in the periphery.

[0318] In some aspects, an antigen-binding domain provided herein specifically binds to human TfR with an affinity of about 127 nM to about 4720 nM. In some aspects, an antigenbinding domain provided herein specifically binds to cynomolgus TfR with an affinity of about 158 nM to about 2810 nM. In some aspects, the affinity is measured using surface plasmon resonance (BIACORE®). See Example 5.

[0319] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR reduces cell surface expression by more than 40%, 60%, or 80% relative to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS.

[0320] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR does not significantly increase cell surface expression of TfR on HCMED / D3 cells relative to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS.

[0321] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR does not significantly increase cell surface expression of TfR on HCMED / D3 cells relative to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS.

[0322] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR accumulates at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40 or 50-fold more than an isotype control after peripheral injection. In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR at least 5-fold more than binding to an irrelevant protein and / or specifically binds to cynomolgus TfR at least 5-fold more than binding to an irrelevant protein.

[0323] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR accumulates at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15- fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold more than an isotype control after peripheral injection. In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR at least 5-fold more than binding to an irrelevant protein and / or specifically binds to cynomolgus TfR at least 5-fold more than binding to an irrelevant protein.

[0324] Also provided herein are antigen-binding domains that bind to the same epitope of TfR as a TfR antigen-binding domain provided herein. Also provided herein are antigen-binding domains that competitively inhibit binding to TfR of TfR antigen-binding domain provided herein.

[0325] As provided herein, antigen-binding domains provided herein that bind to TfR are capable of crossing the BBB. In some aspects, such antigen-binding domains provided herein are internalized in blood-brain barrier epithelial cells greater than 5 fold, greater than 10-fold, or greater than 15-fold as compared to internalization by an isotype control. The blood-brain barrier endothelial cells can be, e.g., HCMEC / D3 cells.

[0326] As provided herein, antigen-binding domains provided herein are capable of crossing the BBB. In some aspects, antigen-binding domains provided herein are internalized in bloodbrain barrier epithelial cells greater than about 5-fold, about 10-fold, about 20-fold, or about 30- fold as compared to internalization by an isotype control. The blood-brain barrier endothelial cells can be, e.g., HCMEC / D3 cells.

[0327] In some aspects, antigen-binding domains provided herein that bind to TfR do not reduce cell-surface expression of TfR on HCMEC / D3 cells by more than 20%, 40%, or 60% relative to cell-surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS. In some aspects, compositions provided herein do not significantly increase cell-surface expression of TfR on HCMEC / D3 cells relative to cell-surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS.

[0328] In some aspects, compositions provided herein accumulate at least 4-fold, 5 -fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more than an isotype control in vessel-depleted mouse brain.

[0329] In some aspects, compositions provided herein accumulate at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold or at least about 50-fold or more than an isotype control in vessel-depleted mouse brain.

[0330] In some aspects, compositions provided herein bind human TfR with an equilibrium dissociation constant (KD) of about 6.7 nM to about 340 nM and binds cynomolgus monkey TfR with a KD of about 18 nM to about 870 nM.Table 3. Heavy Chain CDR Sequences of Anti-TfR AntibodiesTable 4: Light Chain CDR Sequences of Anti-TfR AntibodiesTable 5: VH and VL Sequences of Anti-TfR AntibodiesB. Antigen-Binding Domains That Bind to CD98hc

[0331] Provided herein are antigen-binding domains that specifically bind to human CD98hc.

[0332] Such antigen-binding domains can be capable of crossing the blood brain barrier (BBB) and capable of transporting other agents (e.g., therapeutically active agents) associated with the antigen-binding domain across the blood brain barrier. Accordingly, in some aspects, provided herein are antigen-binding domains that specifically bind to human CD98hc that are capable of being internalized in blood brain barrier epithelial cells.

[0333] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the six CDRs of an antibody listed in Tables 6A, 6B, 7A and 7B (i.e. the three VH CDRs of the antibody listed in Table 6A or 6B and the three VL CDRs of the same antibodylisted in Table 7A or 7B). In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the six CDRs of an antibody listed in Tables 6A, 6B, 7A, and 7B as determined by Kabat numbering. In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the six Chothia CDRs of an antibody listed in Tables 6A, 6B, 7A, and 7B.

[0334] In some aspects, the CDRs of an antigen-binding domain that specifically binds to human CD98hc can be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732-745. See also, e.g., Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422- 439, Springer-Verlag, Berlin (2001). In some aspects, provided herein are antigen-binding domains that specifically bind to human CD98hc and comprise VH and VL CDRs of an antibody listed in Table 6 A, 6B, 7 A, and 7B as determined by the method in MacCallum RM et al.

[0335] In some aspects, the CDRs of an antigen-binding domain that specifically binds to human CD98hc can be determined according to the AbM numbering scheme (as described above). In some aspects, provided herein are antigen-binding domains that specifically bind to human CD98hc and comprise VH and VL CDRs of an antibody listed in Tables 6A, 6B, 7A, and 7B as determined by the AbM numbering scheme.

[0336] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the six IMGT CDRs of an antibody listed in Tables 6A, 6B, 7A, and 7B according to the IMGT numbering system as described above.

[0337] In some aspects, an antigen-binding domain that specifically binds to human CD98hc provided herein is described by its VL domain alone, or its VH domain alone, or by its 3 VL CDRs alone, or its 3 VH CDRs alone.

[0338] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the VH of an antibody listed in Table 8A or 8B.

[0339] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the VL of antibody listed in Table 8A or 8B.

[0340] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the VH and the VL of an antibody listed in Table 8A or 8B (i.e., the VH of the antibody listed in Table 8A or 8B and the VL of the same antibody listed in Table 8A or 8B.

[0341] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 80% identical to a VHamino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 80% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Tables 6A, 6B, 7A, and 7B (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs). In some aspects, an antigenbinding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 85% identical to a VH amino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 85% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B.

[0342] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 90% identical to a VH amino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 90% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Tables 6A, 6B, 7A, and 7B (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs).

[0343] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 95% identical to a VH amino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 95% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Tables 6A, 6B, 7A, and 7B (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs).

[0344] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 96% identical to a VH amino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 96% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Tables 6A, 6B, 7A, and 7B (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs).

[0345] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 97% identical to a VHamino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 97% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Tables 6A, 6B, 7A, and 7B (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs).

[0346] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 98% identical to a VH amino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 98% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Tables 6A, 6B, 7A, and 7B (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs).

[0347] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 99% identical to a VH amino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 99% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Tables 6A, 6B, 7A, and 7B (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs).

[0348] In some aspects, provided herein is an antigen-binding domain that competitively inhibits binding to CD98hc of as an antibody comprising a VH amino acid sequence of an antibody in Table 8A or 8B and a VL amino acid sequence of the same antibody in Table 8A or 8B

[0349] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises a VH and a VL on a single polypeptide chain (e.g., a VH and VL in Table 8A or 8B). In some aspects, the antigen-binding domain comprises an scFv. The scFv can comprise a VH that is N-terminal to a VL or a VL that is N-terminal to a VH. The scFv can comprise a linker, e.g., between a VH and a VL. Accordingly, the scFv can be in the orientation VH-linker-VL or VL-linker-VH. Such a linker can be about 5 to about 25 amino acids in length. Such a linker can be about 5 to about 20 amino acids in length. Such a linker can be about 10 to about 25 amino acids in length. Such a linker can be about 10 to about 20 amino acids in length. Such a linker can be, e.g., a glycine linker, a glycine-rich linker, or a glycine-serine linker. Such a linker cancomprise the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 6). Such a linker can comprise the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 7).

[0350] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises a VH on a first polypeptide and a VL on a second polypeptide (e.g., a Fab).

[0351] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the antigen-binding fragment of a heavy chain only antibody (e.g., a VHH or nanobody).

[0352] In some aspects, an antigen-binding domain that specifically binds to human CD98hc is a murine antigen-binding domain. In some aspects, an antigen-binding domain that specifically binds to human CD98hc is a chimeric antigen-binding domain. In some aspects, an antigenbinding domain that specifically binds to human CD98hc is a humanized antigen-binding domain. In some aspects, an antigen-binding domain that specifically binds to human CD98hc is a human antigen-binding domain.

[0353] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc also binds to cynomolgus monkey CD98hc.

[0354] In certain aspects, an antibody that binds to CD98hc has a dissociation constant (KD) of about 10 nM to about 1500 nM, about 100 nM to about 500 nM, about 500 nM to about 10 pM, or less than about 20 pM, less than about 15 pM, less than about 12 pM, less than about 10 pM, less than about 7.5 pM, less than about 5 pM, less than about 2.5 pM, less than about 1 pM, less than about 10 pM, less than about 100 nM, less than about 10 nM, less than about 1 nM, less than about 0.1 nM, less than about 0.01 nM, or less than about 0.001 nM (e.g., 10'8M or less, e.g., from 10'8M to 10'13M, e.g., from 10'9M to 10'13M).

[0355] In some aspects, the anti-CD98hc antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 184, 190, 193, 197, 198, and 204, respectively.

[0356] In some aspects, the anti-CD98hc antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 210 and 216, respectively.

[0357] In some aspects, the anti-CD98hc antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and alight chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 184, 190, 193, 362, 198, and 204, respectively.

[0358] In some aspects, the anti-CD98hc antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 210 and 387, respectively.

[0359] In some aspects, the anti-CD98hc antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 184, 343, 193, 197, 198, and 204, respectively.

[0360] In some aspects, the anti-CD98hc antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 379 and 216, respectively.

[0361] In some aspects, the anti-CD98hc antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 184, 348, 193, 197, 198, and 204, respectively.

[0362] In some aspects, the anti-CD98hc antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 384 and 216, respectively.

[0363] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of less than about 250 nM (e.g., 10 pM to 250 nM, 5 pM to 250 nM, 1 pM to 250 nM, 1 nM to 250 nM or 3 nM to 250 nM), an affinity of less than about 200 nM (e.g., 10 pM to 200 nM, 5 pM to 200 nM, 1 pM to 200 nM, 1 nM to 200 nM or 3 nM to 200 nM), or an affinity of less than about 150 nM (e.g., 10 pM to 150 nM, 5 pM to 150 nM, 1 pM to 150 nM, 1 nM to 150 nM or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, e.g., using the Carterra LSA platform. In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc with an affinity of 0.1 pM to 10 pM, 0.1 pM to 100 pM, 0.1 pM to 100 pM, 0.1 pM to InM, 1 pM to 10 pM, 1 pM to 100 pM, 1 pM to 1 nM, 1 pM to 10 nM, 1 pM to 100 nM, 1 pM to 150 nM, or 1 pM to 250 nM.

[0364] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of less than about 250 nM (e.g., 10 pM to 250 nM, 5 pM to 250 nM, 1 pM to 250 nM, 1 nM to 250 nM or 3 nM to 250 nM), an affinity of less than about 200 nM (e.g., 10 pM to 200 nM, 5 pM to 200 nM, 1 pM to 200 nM, 1 nM to 200 nM or 3 nM to 200 nM), or an affinity of less than about 150 nM (e.g., 10 pM to 150 nM, 5 pM to 150 nM, 1 pM to150 nM, 1 nM to 150 nM or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance.

[0365] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 18 nM to about 87 nm.

[0366] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of less than about 100 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 100 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 200 nM to about 1500 nM. In some aspects, an antigenbinding domain provided herein specifically binds to human CD98hc with an affinity of about 300 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 400 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 500 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 600 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 700 nM to about 1500 nM. In some aspects, an antigenbinding domain provided herein specifically binds to human CD98hc with an affinity of about 800 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 900 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1000 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1100 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1200 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1300 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1400 nM to about 1500 nM.

[0367] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 100 nM to about 200 nM. In some aspects, an antigenbinding domain provided herein specifically binds to human CD98hc with an affinity of about 200 nM to about 300 nM. In some aspects, an antigen-binding domain provided hereinspecifically binds to human CD98hc with an affinity of about 300 nM to about 400 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 400 nM to about 500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 500 nM to about 600 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 600 nM to about 700 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 700 nM to about 800 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 800 nM to about 900 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 900 nM to about 1000 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1000 nM to about 1100 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1100 nM to about 1200 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1200 nM to about 1300 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1300 nM to about 1400 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 1400 nM to about 1500 nM.

[0368] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 24 nM to about 1330 nM. In some aspects, an antigenbinding domain provided herein specifically binds to human CD98hc with an affinity of about 21 nM to about 826 nM.

[0369] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 58 nM to about 447 nM. In some aspects, an antigenbinding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 51 nM to about 709 nM. In some aspects, the affinity is measured using surface plasmon resonance (BIACORE®). See Example 24.

[0370] In some aspects, an antigen-binding domain provided herein with a lower affinity (e.g., an affinity of up to 1 pM) can have improved transport across the blood brain barrier and into the brain parenchyma, e.g., as a result of the fact that they would not remain associated with blood vessels for as long a time (e.g., as compared to an antigen-binding domain with a higher affinitysuch as the parental WH1). In some aspects, an antigen-binding domain provided herein with a lower affinity (e.g., an affinity of up to 1 pM) can have an improved safety profile (e.g., as compared to an antigen-binding domain with a higher affinity such as the parental WH1).

[0371] In some aspects, an antigen-binding domain provided herein binds to human CD98hc with “low” affinity. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 500 nM and 10 pM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 2 pM and 5 pM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 1 pM and 5 pM. In some aspects, the antigenbinding domain binds human CD98hc with an affinity between 2 pM and 8 pM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 750 nM and 2 pM.

[0372] In some aspects, an antigen-binding domain provided herein binds to human CD98hc with “medium” affinity. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 10 nM and 500 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 50 nM and 500 nM. In some aspects, the antigenbinding domain binds human CD98hc with an affinity between 100 nM and 250 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 10 nM and 100 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 250 nM and 500 nM.

[0373] In some aspects, an antigen-binding domain provided herein binds to human CD98hc with “high” affinity. In some aspects, the antigen-binding domain binds human CD98hc with an affinity less than 10 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 0.01 nM and 10 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 0.1 nM and 10 nM. In some aspects, the antigenbinding domain binds human CD98hc with an affinity between 0.01 nM and 1 nM.

[0374] In some aspects, an antigen-binding domain provided herein that binds to human CD98hc with a lower affinity (e.g., an affinity of up to 1 pM) can have improved transport into the brain parenchyma, e.g., as a result of the fact that they would not remain associated with blood vessels for as long a time (e.g., as compared to an antigen-binding domain with a higher affinity such as the parental WH1). In some aspects, an antigen-binding domain provided herein that binds to human CD98hc with a lower affinity (e.g., an affinity of up to 1 pM) can have an improved safety profile (e.g., as compared to an antigen-binding domain with a higher affinity such as the parental WH1).

[0375] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to human CD98hc with an ELISA OD450 of at least 0.45. In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to cynomolgus CD98hc with an ELISA OD450 of at least 0.45. In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to human CD98hc with an ELISA OD450 of at least 0.45 and binds to cynomolgus CD98hc with an ELISA OD450 of at least 0.45.

[0376] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to cynomolgus CD98hc with an affinity of less than about 250 nM (e.g., 10 pM to 250 nM, 5 pM to 250 nM, 1 pM to 250 nM, 10 pM to 250 nM, 1 nM to 250 nM or 3 nM to 250 nM), an affinity of less than about 200 nM (e.g., 10 pM to 200 nM, 5 pM to 200 nM, 1 pM to 200 nM, 10 pM to 250 nM, 1 nM to 200 nM or 3 nM to 200 nM), or an affinity of less than about 150 nM (e.g., 10 pM to 150 nM, 5 pM to 150 nM, 1 pM to 150 nM, 10 pM to 250 nM, 1 nM to 150 nM or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance.

[0377] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to cynomolgus CD98hc with an affinity of no more than 250 nM (e.g., 10 pM to 250 nM, 5 pM to 250 nM, 1 pM to 250 nM, 10 pM to 250 nM, 1 nM to 250 nM or 3 nM to 250 nM), an affinity of no more than 200 nM (e.g., 10 pM to 200 nM, 5 pM to 200 nM, 1 pM to 200 nM, 10 pM to 250 nM, 1 nM to 200 nM or 3 nM to 200 nM), or an affinity of no more than 150 nM (e.g., 10 pM to 150 nM, 5 pM to 150 nM, 1 pM to 150 nM, 10 pM to 250 nM, 1 nM to 150 nM or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, e.g., using the Carterra LSA platform. In some aspects, an antigen-binding domain provided herein that specifically binds to cyno CD98hc binds to cyno CD98hc with an affinity of 1 pM to 100 pM, 1 pM to 1 nM, 1 pM to 10 nM, 1 pM to 100 nM, 1 pM to 150 nM, or 1 pM to 250 nM.

[0378] In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 46 nM to about 539 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 100 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 200 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds tocynomolgus CD98hc with an affinity of about 300 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 400 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 500 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 600 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 700 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 800 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 900 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1000 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1100 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1200 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1300 nM to about 1500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1400 nM to about 1500 nM.

[0379] In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 100 nM to about 200 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 200 nM to about 300 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 300 nM to about 400 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 400 nM to about 500 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 500 nM to about 600 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 600 nM to about 700 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 700 nM to about 800 nM. In some aspects, anantigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 800 nM to about 900 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 900 nM to about 1000 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1000 nM to about 1100 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1100 nM to about 1200 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1200 nM to about 1300 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1300 nM to about 1400 nM. In some aspects, an antigen-binding domain provided herein specifically binds to cynomolgus CD98hc with an affinity of about 1400 nM to about 1500 nM.

[0380] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to each of human CD98hc and cynomolgus CD98hc with an affinity of less than about250 nM (e.g., 1 pM to 250 nM, 1 nM to 250 nM or 3 nM to 250 nM), an affinity of less than about 200 nM (e.g., 1 pM to 200 nM, 1 nM to 200 nM or 3 nM to 200 nM), or an affinity of less than about 150 nM (e.g., 1 pM to 150 nM, 1 nM to 150 nM or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance.

[0381] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to each of human CD98hc and cynomolgus CD98hc with an affinity of no more than 250 nM (e.g., 1 pM to 250 nM, 1 nM to 250 nM or 3 nM to 250 nM), an affinity of no more than 200 nM (e.g., 1 pM to 200 nM, 1 nM to 200 nM or 3 nM to 200 nM), or an affinity of no more than 150 nM (e.g., 1 pM to 150 nM, 1 nM to 150 nM or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, e.g., using the Carterra LSA platform. In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to each of human CD98hc and cynomolgus CD98hc with an affinity of 1 pM to 100 pM, 1 pM to 1 nM, 1 pM to 10 nM, 1 pM to 100 nM, 1 pM to 150 nM, or 1 pM to 250 nM.

[0382] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 18 nM to about 87 nM and binds to cynomolgus CD98hc with an affinity of about 46 nM to about 539 nM.

[0383] In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 18 nM to about 87 nM and binds to cynomolgus CD98hc with an affinity of about 46 nM to about 539 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 24 nM to about 1330 nM and binds to cynomolgus CD98hc with an affinity of about 27 nM to about 1430 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 21 nM to about 826 nM and binds to cynomolgus CD98hc with an affinity of about 28 nM to about 651 nM. In some aspects, an antigen-binding domain provided herein specifically binds to human CD98hc with an affinity of about 57 nM to about 533 nM and binds to cynomolgus CD98hc with an affinity of about 52 nM to about 859 nM.In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to human CD98hc is measured using surface plasmon resonance. Surface plasmon resonance can be measured, e.g., using the Carterra LSA platform. In some aspects, an antigenbinding domain provided herein that specifically binds to human CD98hc binds to human CD98hc as measured by, for example, radioimmunoassay (RIA), Western blot, or ELISA OD450.

[0384] Also provided herein are antigen-binding domains that bind to the same epitope of CD98hc as a CD98hc antigen-binding domain provided herein. Also provided herein are antigenbinding domains that competitively inhibit binding to CD98hc of CD98hc antigen-binding domain provided herein.Table 6A: Heavy Chain CDR Sequences of Anti-CD98hc AntibodiesTable 6B: Heavy Chain CDR Sequences of Anti-CD98hc AntibodiesTable 7A: Light Chain CDR Sequences of Anti-CD98hc AntibodiesTable 7B: Light Chain CDR Sequences of Anti-CD98hc AntibodiesTable 8A: VH and VL Sequences of Anti-CD98hc AntibodiesTable 8B: VH and VL Sequences of Anti-CD98hc AntibodiesV. Multi-specific Binding Proteins that Comprise an Anti-GPNMB Antigen-Binding Domain and an Antigen-Binding Domain that Binds to Blood-Brain Barrier Receptors or Proteins

[0385] Provided herein are multi-specific binding proteins that specifically bind to human receptors or proteins of the blood-brain barrier.Multi-specific Binding Proteins Comprising Anti-TfR Antigen-Binding Domains

[0386] Provided herein are antigen-binding domains that specifically bind to human TfR.

[0387] In some aspects, an antibody or antigen-binding fragment thereof provided herein comprises an antigen-binding domain that specifically binds to human TfR. In some aspects, an antibody or antigen-binding fragment thereof comprises an antigen-binding domain that specifically binds to human TfR linked to a second antigen-binding domain that specifically binds to GPNMB. Also provided herein are antibodies or antigen-binding fragments thereof that bind to the same epitope of TfR as a TfR antigen-binding domain provided herein. Also provided herein are antibodies or antigen-binding fragments thereof that competitively inhibit binding to TfR of a TfR antigen-binding domain provided herein.

[0388] In some aspects, a multi-specific binding protein provided herein comprises a first antigen-binding domain that specifically binds to human TfR linked to a second antigen-binding domain that specifically binds to human GPNMB. The first antigen-binding domain thatspecifically binds to human TfR can be any antigen-binding domain that binds to human TfR provided herein. The second antigen-binding domain can be an antigen-binding domain that specifically binds to human GPNMB.

[0389] In some aspects, a multi-specific binding protein provided herein comprises an antigenbinding domain that specifically binds to human TfR that is linked to a second antibody or antigen-binding fragment thereof. The second antibody or antigen-binding fragment thereof specifically binds to human GPNMB. In some aspects, the second antibody or antigen-binding fragment does not bind to TfR.

[0390] In some aspects, a multi-specific binding protein provided herein is bi-specific. In some aspects, a multi-specific binding protein provided herein is bivalent, trivalent, or tetravalent.

[0391] In some aspects, a multi-specific binding protein provided herein comprises a 2+1 multi-specific protein format (as shown in Figure 1) or a 2+2 multispecific-protein format (as shown in Figure 2).

[0392] In some aspects, a multi-specific binding protein provided herein comprises a TfR antigen-binding domain that is an scFv linked to an antibody that binds to human GPNMB, wherein the antibody comprises two heavy chains and two light chains. In some aspects, the scFv is linked to the C-terminus of one of the two antibody heavy chains, e.g., via a protein linker.

[0393] In some aspects, the multi-specific binding protein comprises 1) an antigen-binding domain that specifically binds TfR, 2) a second antigen-binding domain that specifically binds to human GPNMB, and 3) an Fc region, wherein the TfR antigen-binding domain and the second antigen-binding domain are connected or linked to the Fc region of the multi-specific binding protein. In other aspects, the multi-specific binding protein comprises 1) an antigen-binding domain that comprises a heavy chain variable region and specifically binds TfR, 2) a second antigen binding domain that comprises a heavy-chain variable region and specifically binds to human GPNMB, and 3) an Fc region, wherein the TfR antigen-binding domain and the second antigen-binding domain are connected or linked to the Fc region of the multi-specific binding protein.

[0394] In some aspects, the multi-specific binding protein comprises 1) an antigen-binding domain that specifically binds TfR, 2) a second antigen-binding domain that binds to human GPNMB, and 3) an Fc region, wherein the antigen-binding domain that specifically binds TfR and the second antigen-binding domain are both connected or linked to the N-terminus of the Fc portion of the multi-specific binding protein. In other aspects, the antigen-binding domain thatspecifically binds TfR is connected or linked to the N-terminus of an Fc portion of the multispecific protein and the second antigen-binding domain is linked to the C-terminus of the Fc portion of the multi-specific binding protein. In other aspects, the antigen-binding domain that specifically binds TfR is connected or linked to the C-terminus of an Fc portion of the multispecific binding protein and the second antigen-binding domain is linked to the N-terminus of the Fc portion of the multi-specific binding protein.

[0395] In some aspects, a multi-specific binding protein provided herein comprises two scFv molecules that comprise antigen-binding domains that specifically bind TfR and an antibody that binds to human GPNMB, wherein the antibody comprises two heavy chains and two light chains, and wherein one of the two scFv molecules is linked to the C-terminus of one of the antibody heavy chains, and wherein the other scFv molecule is linked to the C-terminus of the other antibody heavy chain. In some aspects, the scFvs are linked to the heavy chains of the antibody via a protein linker.

[0396] In some aspects, disclosed herein is a trivalent, bi-specific protein comprising (i) a single scFv, Fab or VHH antigen-binding domain that specifically binds to human TfR and (ii) an antibody that specifically binds to human GPNMB, wherein the antibody comprises two heavy chains and two light chains, and wherein the single scFv, Fab or VHH antigen-binding domain that specifically binds to human TfR is linked to the C-terminus or N-terminus of one of the two antibody heavy chains. The Fc may be a heterodimeric Fc, such as knob and hole Fc. An example of this 2+1 format with a knob-hole Fc is shown as (i) in Fig. 3.

[0397] In some aspects, disclosed herein is a tetravalent, bi-specific protein comprising (i) two scFv, Fab or VHH antigen-binding domains that specifically bind to human TfR and (ii) an antibody that binds to human GPNMB, wherein the antibody comprises two heavy chains and two light chains, and wherein one of the scFv, Fab, or VHH antigen-binding domains that specifically binds to human TfR is linked to the C-terminus of one of the two antibody heavy chains, and the other scFv, Fab, or VHH antigen-binding domain that specifically binds to human TfR is linked to the C-terminus or N-terminus of the other of the two antibody heavy chains. In some aspects, the two scFv, Fab or VHH antigen-binding domains that bind to human TfR can comprise the same amino acid sequence. In some aspects, the Fc is a heterodimeric Fc, such as an Fc region with knob and hole mutations.

[0398] In some aspects, disclosed herein is a bivalent, bispecific protein comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR linked to the N-terminus of an Fc domain of an Fc dimer, and (ii) a second antigen binding domain that binds to human GPNMB, wherein the second antigen binding domain is linked to the N-terminus of the second Fc domain of an Fc dimer. An example of this format is shown as (iii) in Fig. 3.

[0399] In some aspects, disclosed herein is a bivalent, bi-specific protein comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR linked to the N-terminus or C-terminus of an Fc domain of an Fc dimer and (ii) a second antigen binding domain, wherein the second antigen binding domain binds to human GPNMB and comprises a single scFv, VHH or Fab linked to N-terminus of the Fc. In some aspects, disclosed herein is a bivalent, bi-specific protein comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR linked to the N-terminus or C-terminus of an Fc domain of an Fc dimer and (ii) a second antigen binding domain, wherein the second antigen binding domain binds to human GPNMB and comprises a single scFv, VHH or Fab linked to the C- terminus of the second Fc domain of the Fc dimer. In some aspects, the Fc is a heterodimeric Fc, such as an Fc domain with knob and hole mutations. An example of this format is shown as (ii) in Fig. 3, wherein a single scFv is linked to the N terminus of a first Fc domain of the Fc region and the second antigen binding domain is linked to the N terminus of the second Fc domain of the Fc region.

[0400] In some aspects, disclosed herein is a bivalent, bispecific protein comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR and is linked to the N-terminus of an Fc domain of an Fc dimer, and (ii) a second antigen binding domain that binds to human GPNMB and is linked to the N-terminus of the second Fc domain of an Fc dimer. An example of this format is shown as (iii) in Fig. 3.

[0401] In some aspects, disclosed herein is a bivalent, bi-specific protein comprising (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human TfR and is linked to the N-terminus or C-terminus of an Fc domain of an Fc dimer and (ii) a second antigen binding domain, wherein the second antigen binding domain binds to human GPNMB and comprises a single scFv, VHH or Fab linked to N-terminus of the Fc or C-terminus of a second Fc domain of the Fc dimer. In some aspects, the Fc is a heterodimeric Fc, such as an Fc domain with knob and hole mutations. An example of this 1+1 format is shown as (ii) in Fig. 3, where a single scFv is linked to the N terminus of an Fc domain of an Fc region and a second antigen binding domain is linked to the N terminus of a second Fc domain of the Fc region.

[0402] As provided herein, an antibody or antigen-binding fragment thereof, or multi-specific binding protein provided herein can be multi-specific, e.g., bi-specific. Many different formats and uses of bi-specific binding molecules are known in the art (reviewed in, e.g., Kontermann; Drug Discov Today, 2015 July; 20(7):838-47; MAbs, 2012 March-April; 4(2): 182-97). A bispecific protein according to the present disclosure is not limited to any particular bispecific format or method of producing it. Accordingly, a bispecific protein of the present disclosure can include various configurations having a first antigen-binding domain that binds to human TfR and a second antigen-binding domain or antibody or antigen-binding fragment thereof that binds to human GPNMB.

[0403] Bispecific molecules include, e.g., a kappa-lambda body, a dual-affinity re-targeting molecule (DART), a knob-in-hole antibody, a strand-exchange engineered domain body (SEEDbody), and a DuoBody. In some aspects, a bispecific antibody or antigen-binding fragment thereof, or multi-specific protein provided herein comprises a knob mutation and a hole mutation. In some aspects, the knob mutation comprises the amino acid substitution T366W according to EU numbering. In some aspects, the hole mutation comprises the amino acids substitutions T366S, L368A, and Y407V according to EU numbering. Examples of bispecific molecules that can be used in the present disclosure include (i) a single antibody that has two arms comprising different antigen-binding domains; (ii) a single chain antibody that has specificity to two different epitopes, e.g., via two scFvs linked in tandem by an extra peptide linker; (iii) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig. TM.) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) a chemically-linked bispecific (Fab’)2 fragment; (v) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vi) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (vii) a so-called “dock and lock” molecule, based on the “dimerization and docking domain” in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (viii) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fab-arm; and (ix) a diabody. Other examples of antibody structures are described in WO2019 / 246288, which is incorporated by reference.

[0404] In some aspects, a bispecific protein provided herein is selected from one of the following formats: CrossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, Charge pair, Fab-arm exchange, Triomab, LUZ-Y, Fcab, kappalambda-body, Orthogonal Fab, DVD- IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG (four- in-one), Nanobody, Nanbody-HAS, BiTE, TandAb, scDiabody-CH3, Diabody-CH3, Triple Body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFV-CH-CL-scFV, F(ab’)2, F(ab’)2-scFv2, scFV-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Intrabody, ImmTAC, HSAbody, scDiabody-HAS, Tandem scFv-toxin, IgG- IgG, Cov-X-body, and scFvl-PEG-scFv2, as described in Spiess, C., et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol. Immunol. (2015), which is incorporated by reference herein.

[0405] In some aspects, a multi-specific binding protein provided herein is bivalent. In some aspects, a multi-specific binding protein provided herein is multivalent (e.g., bivalent). In some aspects, an antibody or antigen-binding fragment thereof, or multi-specific binding protein provided herein is trivalent (e.g., in a 2+1 format). In some aspects, a trivalent format comprises a single TfR antigen-binding domain provided herein and two antigen-binding domains that bind to GPNMB. The two antigen-binding domains that bind to a CNS antigen or a brain antigen can comprise the same amino acid sequence or can comprise different amino acid sequences. In some aspects, the TfR antigen-binding domain is an scFv. In some aspects, the TfR antigen-binding domain is a VHH. In some aspects, the TfR antigen-binding domain is a Fab.

[0406] In some aspects, a multi-specific binding protein provided herein is tetravalent (e.g., in a 2+2 format). In some aspects, a tetravalent format comprises two TfR antigen-binding domains provided herein and two antigen-binding domains that bind to a CNS antigen or a brain antigen. The two TfR antigen-binding domains can comprise the same amino acid sequence or can comprise different amino acid sequences. In some aspects, the two TfR antigen-binding domains comprise the same amino acid sequence. In some aspects, one or both of the TfR antigen-binding domains is an scFv. The two antigen-binding domains that bind to a GPNMB antigen can comprise the same amino acid sequence or can comprise different amino acid sequences

[0407] An antibody or antigen-binding fragment thereof, or multi-specific binding protein provided herein can comprise a linker. The linker can be e.g., a glycine linker, a glycine-rich linker, or a glycine-serine linker. The linker can comprise the amino acid sequence (GGGGS)x3(SEQ ID NO: 179). The linker can comprise the amino acid sequence (GGSGG)x3 (SEQ ID NO: 217).

[0408] An antibody or antigen-binding fragment thereof, or multi-specific binding protein provided herein can comprise a constant region. In some aspects, a TfR antigen-binding domain provided herein is linked to the constant region, e.g., the C-terminus of the constant region. In some aspects, a constant domain is a human constant domain. In some aspects, a constant domain is a murine, rat, rabbit, or monkey (e.g., cynomolgus) constant domain. The constant region can be a heavy chain constant region. The constant region can be a human constant region. The constant region can be a human heavy chain constant region. The constant region can be an IgG constant region. The constant region can be an IgGl constant region. The constant region can be an IgG2 constant region. The constant region can be an IgG4 constant region. The constant region can be a human IgG constant region. The constant region can be a human IgGl constant region. The constant region can be a human IgG2 constant region. The constant region can be a human IgG4 constant region. The linker can comprise the amino acid sequence GGSGG (no repeats) (SEQ ID NO: 180). The linker may be 1 to 20 amino acids in length.

[0409] In some aspects, an antibody or antigen-binding fragment thereof, or multi-specific binding protein provided herein comprises a heavy chain and a light chain. With respect to the heavy chain, in some aspects, the heavy chain of an antigen-binding protein described herein can be an alpha (a), delta (5), epsilon (a), gamma (y) or mu (p) heavy chain. In some aspects, the heavy chain can comprise a human alpha (a), delta (5), epsilon (a), gamma (y) or mu (p) heavy chain. In some aspects, the heavy chain comprises a human gamma (y) heavy chain constant region. In some aspects, the heavy chain of comprises the amino acid sequence of an IgGl heavy chain constant region. In some aspects, the heavy chain comprises the amino acid sequence of an IgG2 (e.g., IgG2a or IgG2b) heavy chain constant region. In some aspects, the heavy chain comprises the amino acid sequence of an IgG4 heavy chain constant region. With respect to the light chain, in some aspects, the light chain is a kappa light chain. In some aspects, the light chain is a lambda light chain. In some aspects, the light chain is a human kappa light chain or a human lambda light chain.

[0410] In some aspects, an antibody or antigen-binding fragment thereof, or multi-specific binding protein provided herein comprises constant regions comprising the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some aspects, an antibodyor antigen-binding fragment thereof, or multi-specific binding protein provided herein comprises constant regions comprising the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. In some aspects, the constant regions comprise the amino acid sequences of the constant regions of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule.

[0411] Non-limiting examples of human constant region sequences have been described in e.g., U.S. Patent No. 5,693,780 and Kabat EA et a / ., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242).

[0412] In some aspects, a constant region provided herein comprises a knob mutation. In some aspects, a constant region provided herein comprises a hole mutation. Accordingly, in some aspects, an antibody or antigen-binding fragment thereof, or multi-specific binding protein provided herein can comprise a constant region comprising a knob mutation and a constant region comprising a hole mutation. Description of knob and hole mutations are provided herein.Multi-specific Binding Proteins Comprising Anti-CD98hc Antigen-Binding Domains

[0413] Provided herein are agents comprising an antigen-binding domain that specifically binds to human CD98hc.

[0414] In some aspects, an antibody or antigen-binding fragment thereof provided herein comprises an antigen-binding domain that specifically binds to human CD98hc. In some aspects, an antibody or antigen-binding fragment thereof comprises an antigen-binding domain that specifically binds to human CD98hc and an antigen-binding domain that specifically binds to GPNMB. Also provided herein are antibodies or antigen-binding fragments thereof that bind to the same epitope of CD98hc as a CD98hc antigen-binding domain provided herein. Also provided herein are antibodies or antigen-binding fragments thereof that competitively inhibit binding to CD98hc of a CD98hc antigen-binding domain provided herein.

[0415] In some aspects, a multi-specific binding protein provided herein comprises a first antigen-binding domain that specifically binds to human CD98hc and a second antigen-binding domain that binds to human GPNMB. The first antigen-binding domain that specifically binds to human CD98hc can be any antigen-binding domain that binds to human CD98hc providedherein. The second antigen-binding domain can be an antigen-binding domain that specifically binds to human GPNMB.

[0416] In some aspects, a multi-specific binding protein provided herein comprises an antigenbinding domain that specifically binds to human CD98hc that is linked to a second antibody or antigen-binding fragment thereof. The second antibody or antigen-binding fragment thereof specifically binds to human GPNMB. In some aspects, the second antibody or antigen-binding fragment does not bind to CD98hc.

[0417] In some aspects, a multi-specific binding protein provided herein comprises a 2+1 multi-specific protein format (as shown in Figure 1) or a 2+2 multi-specific protein format (as shown in Figure 2).

[0418] In some aspects, a multi-specific binding protein provided herein comprises a CD98hc antigen-binding domain that is an scFv linked to an antibody that binds to GPNMB, wherein the antibody comprises two heavy chains and two light chains. In some aspects, the scFv is linked to the C-terminus of one of the two antibody heavy chains, e.g., via a protein linker.

[0419] In some aspects, the multi-specific binding protein comprises 1) an antigen-binding domain that specifically binds CD98hc, 2) a second antigen-binding domain that specifically binds to human GPNMB, and 3) an Fc region, wherein the CD98hc antigen-binding domain and the second antigen-binding domain are connected or linked to the Fc region of the multi-specific protein. In other aspects, the multi-specific protein comprises 1) an antigen-binding domain that comprises a heavy chain variable region and specifically binds CD98hc, 2) a second antigen binding domain that comprises a heavy-chain variable region and binds to human GPNMB, and 3) an Fc region, wherein the CD98hc antigen-binding domain and the second antigen-binding domain are connected or linked to the Fc region of the multi-specific protein.

[0420] In some aspects, the multi-specific binding protein comprises 1) an antigen-binding domain that specifically binds CD98hc, 2) a second antigen-binding domain that binds to human GPNMB, and 3) an Fc region, wherein the antigen-binding domain that specifically binds CD98hc and the second antigen-binding domain are both connected or linked to the N-terminus of the Fc portion of the multi-specific protein. In other aspects, the antigen-binding domain that specifically binds CD98hc is connected or linked to the N-terminus of an Fc portion of the multispecific protein and the second antigen-binding domain is linked to the C-terminus of the Fc portion of the multi-specific protein. In other aspects, the antigen-binding domain that specifically binds CD98hc is connected or linked to the C-terminus of an Fc portion of the multi-specific protein and the second antigen-binding domain is linked to the N-terminus of the Fc portion of the multi-specific protein.

[0421] In some aspects, a multi-specific binding protein provided herein comprises two scFv molecules that comprise antigen-binding domains that specifically bind CD98hc and an antibody that binds to human GPNMB, wherein the antibody comprises two heavy chains and two light chains, and wherein one of the two scFv molecules is linked to the C-terminus of one of the antibody heavy chains, and wherein the other scFv molecule is linked to the C-terminus of the other antibody heavy chain. In some aspects, the scFvs are linked to the heavy chains of the antibody via a protein linker.

[0422] In some aspects, disclosed herein is a trivalent, bi-specific protein comprising (i) a single scFv, Fab or VHH antigen-binding domain that specifically binds to human CD98hc and (ii) an antibody that specifically binds to human GPNMB, wherein the antibody comprises two heavy chains and two light chains, and wherein the single scFv, Fab or VHH antigen-binding domain that specifically binds to human CD98hc is linked to the C-terminus or N-terminus of one of the two antibody heavy chains. The Fc may be a heterodimeric Fc, such as knob and hole Fc. An example of this 2+1 format with a knob-hole Fc is shown as (i) in Fig. 3.

[0423] In some aspects, disclosed herein is a tetravalent, bi-specific protein comprising (i) two scFv, Fab or VHH antigen-binding domains that specifically bind to human CD98hc and (ii) an antibody that binds to human GPNMB, wherein the antibody comprises two heavy chains and two light chains, and wherein one of the scFv, Fab, or VHH antigen-binding domains that specifically binds to human CD98hc is linked to the C-terminus of one of the two antibody heavy chains, and the other scFv, Fab, or VHH antigen-binding domain that specifically binds to human CD98hc is linked to the C-terminus or N-terminus of the other of the two antibody heavy chains. In some aspects, the two scFv, Fab or VHH antigen-binding domains that bind to human CD98hc can comprise the same amino acid sequence. In some aspects, the Fc is a heterodimeric Fc, such as an Fc region with knob and hole mutations.

[0424] In some aspects, disclosed herein is a bivalent, bispecific protein comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc linked to the N-terminus of an Fc domain of an Fc dimer, and (ii) a second antigen binding domain that binds to human GPNMB, wherein the second antigen binding domain is linked to the N-terminus of the second Fc domain of an Fc dimer. An example of this 1+1 format is shown as (iii) in Fig. 3.

[0425] In some aspects, disclosed herein is a bivalent, bi-specific protein comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc linked to the N-terminus or C-terminus of an Fc domain of an Fc dimer and (ii) a second antigen binding domain, wherein the second antigen binding domain binds to human GPNMB and comprises a single scFv, VHH or Fab linked to N-terminus of the Fc. In some aspects, disclosed herein is a bivalent, bi-specific protein comprising (i) a single scFv, VHH, or Fab antigenbinding domain that specifically binds to human CD98hc linked to the N-terminus or C-terminus of an Fc domain of an Fc dimer and (ii) a second antigen binding domain, wherein the second antigen binding domain binds to human GPNMB and comprises a single scFv, VHH or Fab linked to the C-terminus of the second Fc domain of the Fc dimer. In some aspects, the Fc is a heterodimeric Fc, such as an Fc region with knob and hole mutations. An example of this 1+1 format is shown as (ii) in Fig. 3, wherein a single scFv is linked to the N terminus of a first Fc domain of the Fc region and the second antigen binding domain is linked to the N terminus of the second Fc domain of the Fc region.

[0426] In some aspects, disclosed herein is a bivalent, bispecific protein comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc and is linked to the N-terminus of an Fc domain of an Fc dimer, and (ii) a second antigen binding domain that binds to human GPNMB and is linked to the N-terminus of the second Fc domain of an Fc dimer. An example of this 1+1 format is shown as (iii) in Fig. 3.

[0427] In some aspects, disclosed herein is a bivalent, bi-specific protein comprising (i) a single scFv, VHH, or Fab antigen-binding domain that binds to human CD98hc and is linked to the N-terminus or C-terminus of an Fc domain of an Fc dimer and (ii) a second antigen binding domain, wherein the second antigen binding domain binds to human GPNMB and comprises a single scFv, VHH or Fab linked to N-terminus of the Fc or C-terminus of a second Fc domain of the Fc dimer. In some aspects, the Fc is a heterodimeric Fc, such as an Fc domain with knob and hole mutations. An example of this 1+1 format is shown as (ii) in Fig. 3, where a single scFv is linked to the N terminus of an Fc domain of an Fc region and a second antigen binding domain is linked to the N terminus of a second Fc domain of the Fc region.

[0428] As provided herein, an antibody or antigen-binding fragment thereof, or multi-specific protein provided herein can be multi-specific, e.g., bi-specific. As described above, a bispecific protein of the present disclosure can include various configurations having a first antigen-bindingdomain that binds to human CD98hc and a second antigen-binding domain that binds to human GPNMB.

[0429] In some aspects, an antibody or antigen-binding fragment thereof, or multi-specific binding protein provided herein comprises a CD98hc binding domain and a knob mutation and a hole mutation. Knob and hole mutations are described in detail herein and similar apply to agents comprising CD98hc binding domains. In some aspects, the knob mutation comprises the amino acid substitution T366W according to EU numbering. In some aspects, the hole mutation comprises the amino acids substitutions T366S, L368A, and Y407V according to EU numbering. Examples of bispecific molecules that can be used are described herein.

[0430] In some aspects, bispecific protein formats are described above. In some aspects, an antibody or antigen-binding fragment thereof or multi-specific binding protein comprising a CD98hc binding domain and provided herein is bivalent or tetravalent. Formats comprising two CD98hc antigen -binding domains can comprise the same amino acid sequence or can comprise different amino acid sequences. In some aspects, the two CD98hc antigen-binding domains comprise the same amino acid sequence. In some aspects, one or both of the CD98hc antigenbinding domains is an scFv. The two antigen-binding domains that bind to a GPNMB antigen can comprise the same amino acid sequence or can comprise different amino acid sequences.

[0431] An antibody or antigen-binding fragment thereof, or multi-specific binding protein comprising a CD98hc binding domain and provided herein can comprise a linker. The linker can be e.g., a glycine linker, a glycine-rich linker, or a glycine-serine linker. The linker can comprise the amino acid sequence (GGGGS)x3 (SEQ ID NO: 179). The linker can comprise the amino acid sequence (GGSGG)x3 (SEQ ID NO: 217).

[0432] An antibody or antigen-binding fragment thereof, or multi-specific binding protein comprising a CD98hc binding domain and provided herein can comprise a constant region, as described herein.

[0433] In some aspects, an antibody or antigen-binding fragment thereof, or multi-specific binding protein comprising a CD98hc binding domain as provided herein comprises a heavy chain and a light chain, as described herein.

[0434] In some aspects, an antibody or antigen-binding fragment thereof, or multi-specific protein comprising a CD98hc binding domain as provided herein comprises constant regions comprising the amino acid sequences of the constant regions of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulinmolecule, or any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. Non-limiting examples of human constant regions and human constant region sequences are described herein.VI. Multi-specific Binding Proteins that Comprise an Antigen-Binding Domains that Binds to GPNMB and an Antigen-Binding Domain that Binds to Blood-Brain Barrier Receptors or Proteins

[0435] Provided herein are compositions that specifically bind to human receptors or proteins of the blood-brain barrier.

[0436] In some aspects, the composition comprises a multispecific protein comprising (i) an antigen-binding domain that specifically binds to human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc) and (ii) an isolated anti-Glycoprotein nonmetastatic melanoma protein B (GPNMB) antibody or antigen binding fragment thereof.

[0437] In some aspects, provided herein are multispecific proteins comprising (i) an antigenbinding domain that specifically binds to a blood brain barrier (BBB) target selected from human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc) and (ii) an antigen-binding domain that specifically binds to Glycoprotein nonmetastatic melanoma protein B (GPNMB).

[0438] In some aspects, the anti-GPNMB antigen-binding domain is an antibody. In some aspects, the antibody is a full-length antibody.

[0439] In some aspects, the antibody is an IgG antibody. In some aspects, the IgG antibody is an IgGl antibody or an IgG4 antibody.

[0440] In some aspects, the anti-GPNMB antibody comprises a heavy chain with a Fc hole mutation.

[0441] In some aspects, the anti-GPNMB antibody comprises a heavy chain with a Fc knob mutation.

[0442] In some aspects, the anti-BBB antigen-binding domain is a scFv.

[0443] In some aspects, the anti-BBB scFv is linked to the C-terminus of the anti-GPNMB antibody heavy chain with a Fc hole mutation.

[0444] In some aspects, the anti-BBB scFv is linked to the C-terminus of the anti-GPNMB antibody heavy chain with a Fc knob mutation.

[0445] In some aspects, the multispecific protein comprises two antigen-binding domains that specifically bind to a BBB target selected from human TfR and human CD98hc. In some aspects, both of the anti-BBB antigen-binding domains are scFvs.

[0446] In some aspects, the first anti-BBB scFv is linked to the C-terminus of the anti-GPNMB antibody heavy chain with a Fc hole mutation. In some aspects, the second anti-BBB scFv is linked to the C-terminus of the anti-GPNMB antibody heavy chain with a Fc knob mutation.

[0447] In some aspects, the anti-BBB antigen-binding domain is an antibody. In some aspects, the antibody is a full-length antibody.

[0448] In some aspects, the antibody is an IgG antibody. In some aspects, the IgG antibody is an IgGl antibody or an IgG4 antibody.

[0449] In some aspects, the anti-BBB antibody comprises a heavy chain with a Fc hole mutation.

[0450] In some aspects, the anti-BBB antibody comprises a heavy chain with a Fc knob mutation.

[0451] In some aspects, the anti-GPNMB antigen-binding domain is a scFv.

[0452] In some aspects, the anti-GPNMB scFv is linked to the C-terminus of the anti-BBB antibody heavy chain with a Fc hole mutation.

[0453] In some aspects, the anti-GPNMB scFv is linked to the C-terminus of the anti-BBB antibody heavy chain with a Fc knob mutation.

[0454] In some aspects, the multispecific protein comprises two antigen-binding domains that specifically bind to a GPNMB, wherein both of the anti-GPNMB antigen-binding domains are scFvs.

[0455] In some aspects, the first anti-GPNMB scFv is linked to the C-terminus of the anti-BBB antibody heavy chain with a Fc hole mutation. In some aspects, the second anti-GPNMB scFv is linked to the C-terminus of the anti-BBB antibody heavy chain with a Fc knob mutation.

[0456] In some aspects, the antibody is a monoclonal antibody.

[0457] In some aspects, the antibody is a humanized antibody.

[0458] In some aspects, the anti-GPNMB antigen-binding domain is selected from the group consisting of a VHH, a Fab, a Fab’, a Fab’-SH, a F(ab’)2, a Fv, or a scFv.

[0459] In some aspects, the anti-BBB antigen-binding domain is a VHH, a Fab, a Fab’, a Fab’- SH, a F(ab’)2, a Fv, or a scFv.

[0460] In some aspects, the multispecific protein further comprises iii) an Fc region.

[0461] In some aspects, the Fc region is fully active.

[0462] In some aspects, the Fc region is partially active.

[0463] In some aspects, the Fc region is not silent.

[0464] In some aspects, the anti-GPNMB antigen-binding domain is a VHH.

[0465] In some aspects, the anti-BBB antigen-binding domain is a VHH.

[0466] In some aspects, the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a VHH that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigen-binding domain is a VHH that is N-terminal to the other protein fragment of the Fc region.

[0467] In some aspects, the anti-GPNMB antigen-binding domain is a Fab.

[0468] In some aspects, the anti-BBB antigen-binding domain is a Fab.

[0469] In some aspects, the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a Fab that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigen-binding domain is a Fab that is N-terminal to the other protein fragment of the Fc region.

[0470] In some aspects, the anti-BBB antigen-binding domain is a scFv.

[0471] In some aspects, the anti-GPNMB antigen-binding domain is a scFv.

[0472] In some aspects, the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a scFv that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigen-binding domain is a scFv that is N-terminal to the other protein fragment of the Fc region.

[0473] In some aspects, the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a scFv that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigen-binding domain is a Fab that is N-terminal to the other protein fragment of the Fc region.

[0474] In some aspects, the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a Fab that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigen-binding domain is a Fab that is N-terminal to the other protein fragment of the Fc region

[0475] In some aspects, the IgG Fc region is an IgGl Fc region or an IgG4 Fc region.

[0476] In some aspects, the IgG Fc region comprises knob and hole mutations.

[0477] In some aspects, the multispecific protein is bivalent.

[0478] In some aspects, the multispecific protein is trivalent.

[0479] In some aspects, the trivalent protein comprises one antigen-binding domain that binds to the BBB target and two antigen-binding domains that bind to GPNMB.

[0480] In some aspects, the trivalent protein comprises two antigen-binding domains that bind to the BBB target and one antigen-binding domain that binds to GPNMB.

[0481] In some aspects, the multispecific protein is tetravalent, optionally wherein the tetravalent protein comprises two of antigen-binding domains that bind to the BBB target and two antigen-binding domains that bind to GPNMB.

[0482] In some aspects, the multispecific protein is a bispecific T-cell engager (BiTE), dualaffinity re-targeting protein (DARTs), or Tandem diabody (TandAb).

[0483] In some aspects, the anti-BBB antigen-binding domain and the anti-GPNMB antigenbinding domain are contained within a single polypeptide chain. In some aspects, the single polypeptide chain further comprises an Fc domain.

[0484] In some aspects, the anti-GPNMB antigen-binding domain is N-terminal to the anti- BBB antigen-binding domain.

[0485] In some aspects, the anti-GPNMB antigen-binding domain thereof is C-terminal to the anti-BBB antigen-binding domain.

[0486] In some aspects, the anti-GPNMB antigen-binding domain and the anti-BBB antigenbinding domain are directly connected via a peptide bond.

[0487] In some aspects, the anti-GPNMB antigen-binding domain and the anti-BBB antigenbinding domain are connected via a linker.

[0488] In some aspects, the linker is a peptide linker.

[0489] In some aspects, the anti-BBB antigen-binding domain and the anti-GPNMB antigenbinding domain are N-terminal to the Fc domain.

[0490] In some aspects, the anti-BBB antigen-binding domain and the anti-GPNMB antigenbinding domain are C-terminal to the Fc domain.

[0491] In some aspects, the anti-BBB antigen-binding domain is N-terminal to the Fc domain and the anti-GPNMB antigen-binding domain is C-terminal to the Fc domain.

[0492] In some aspects, the anti-GPNMB antigen-binding domain is N-terminal to the Fc domain and the anti-BBB antigen-binding domain is C-terminal to the Fc domain.

[0493] In some aspects, the multispecific protein comprises two copies of the anti-GPNMB antigen-binding domain and / or two copies of the anti-BBB antigen-binding domain.

[0494] In some aspects, the anti-GPNMB antigen-binding domain antagonizes GPNMB activity.

[0495] In some aspects, the anti-GPNMB antigen-binding domain modulates expression of activation surface markers on myeloid cells.

[0496] In some aspects, the anti-GPNMB anti antigen-binding domain increases cell surface expression of PD-L1 in human macrophages.

[0497] In some aspects, the anti-GPNMB antigen-binding domain increases cell surface expression of CD40 in human macrophages.

[0498] In some aspects, the anti-GPNMB antigen-binding domain increases cell surface expression of CD80 in human macrophages.

[0499] In some aspects, the anti-GPNMB antigen-binding domain modulates lysosome function in myeloid cells.

[0500] In some aspects, the anti-GPNMB antigen-binding domain increases glucocerebrosidase activity in human macrophages.

[0501] In some aspects, the anti-GPNMB antigen-binding domain decreases cell surface expression of GPNMB in human macrophages.

[0502] In some aspects, the anti-GPNMB antigen-binding domain changes interferon pathway gene expression patterns in human macrophages.

[0503] In some aspects, the anti-GPNMB antigen-binding domain reduces tumor volume in a murine tumor model, such as in an MC38 model.

[0504] In some aspects, the anti-GPNMB antigen-binding domain reduces tumor growth rate in a murine tumor model, such as in an MC38 model.

[0505] In some aspects, the anti-GPNMB antigen-binding domain increases levels of IL-12p40 in serum.

[0506] In some aspects, the anti-GPNMB antibody or antigen binding fragment thereof increases levels of CCL5 in serum.

[0507] In some aspects, the anti-GPNMB antigen-binding domain is a GPNMB ligand blocking antibody.

[0508] In some aspects, the anti-GPNMB antigen-binding domain is a non-blocking antibody.

[0509] In some aspects, the anti-GPNMB antigen-binding domain antagonizes GPNMB activity independent of ligand blocking activity.

[0510] In some aspects, the anti-GPNMB antigen-binding domain antagonizes GPNMB activity in vivo.

[0511] In some aspects, the anti-GPNMB antigen-binding domain binds to GPNMB expressed on a cell surface.

[0512] In some aspects, the anti-GPNMB antigen-binding domain promotes macrophage activation.

[0513] In some aspects, the anti-GPNMB antigen-binding domain overcomes a decrease in glucocerebrosidase activity associated with reduced progranulin levels.

[0514] In some aspects, the anti-GPNMB ant antigen-binding domain decreases GPNMB expression levels in cells, optionally in macrophages.

[0515] In some aspects, the anti-GPNMB antigen-binding domain decreases LAMP2 expression levels in cells, optionally in macrophages, monocytes, or neutrophils.

[0516] In some aspects, the anti-GPNMB antigen-binding domain inhibits or reduces inflammasome activation.

[0517] In some aspects, the anti-GPNMB antigen-binding domain inhibits IL-ip expression or release.

[0518] In some aspects, the anti-GPNMB antigen-binding domain reduces neural inflammation.

[0519] In some aspects, the anti-GPNMB antigen-binding domain reduces expression of Clq, GFAP, IB Al, and CTSD associated with neural inflammation.

[0520] In some aspects, the anti-GPNMB antigen-binding domain reduces lysosomal stress.

[0521] In some aspects, the anti-GPNMB antigen-binding domain competes with one or more antibodies selected from GPN-81, GPN-82, GPN-83, GPN-84, GPN-85, GPN-86, GPN-87, GPN-88, GPN-89, GPN-90, GPN-91, GPN-92, GPN-93, GPN-94, GPN-95, GPN-96, GPN-97, GPN-98, and any combination thereof for binding to GPNMB.

[0522] In some aspects, the anti-GPNMB antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, a VH CDR2, a VH CDR3 and a light chain variable region (VL) CDR1, a CDR2, and a CDR3, wherein: i) the VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 308 or 9; ii) the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 310, 311, 312, 313, 314, or 315; iii) the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 316, 317, 318, or 319;iv) the VL CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 320, 321, 322, 323, 324, or 325; v) the VL CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 326, 327, 328, 329, 330, 331, 332, 333, 334, or 335; and / or vi) the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 336, 337, 338, 339, 340, or 341.

[0523] In some aspects, the anti-GPNMB antigen-binding domain comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of: i) SEQ ID NOs: 308, 310, and 316; and 320, 326, and 336; respectively; ii) SEQ ID NOs: 308, 311, and 316; and 321, 327, and 337; respectively iii) SEQ ID NOs: 308, 312, and 316; and 322, 328, and 337; respectively; iv) SEQ ID NOs: 308, 312, and 317; and 322, 328, and 337; respectively; v) SEQ ID NOs: 308, 313, and 316; and 323, 329, and 337; respectively; vi) SEQ ID NOs: 308, 312, and 316; and 321, 330, and 337; respectively; vii) SEQ ID NOs: 308, 312, and 317; and 321, 330, and 337; respectively; viii) SEQ ID NOs: 308, 312, and 316; and 321, 329, and 338; respectively; ix) SEQ ID NOs: 308, 312, and 317; and 321, 329, and 337; respectively; x) SEQ ID NOs: 308, 313, and 316; and 321, 329, and 337; respectively; xi) SEQ ID NOs: 308, 312, and 316; and 321, 329, and 339; respectively; xii) SEQ ID NOs: 9, 314, and 318; and 324, 331, and 340; respectively; xiii) SEQ ID NOs: 9, 315, and 319; and 325, 332, and 341; respectively; xiv) SEQ ID NOs: 9, 315, and 319; and 325, 332, and 340; respectively; xv) SEQ ID NOs: 9, 315, and 319; and 325, 333, and 341; respectively; xvi) SEQ ID NOs: 9, 315, and 319; and 324, 334, and 340; respectively; xvii) SEQ ID NOs: 9, 315, and 319; and 325, 335, and 340; respectively; or xviii) SEQ ID NOs: 9, 315, and 319; and 324, 331, and 340; respectively.

[0524] In some aspects, the anti-GPNMB antigen-binding domain comprises a VH that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 242, 243, 244, 245, 246, 247, and 248.

[0525] In some aspects, the anti-GPNMB antigen-binding domain comprises a VL that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, and 262.

[0526] In some aspects, the anti-GPNMB antigen-binding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of:(i) SEQ ID NOs: 242 and 249, respectively;(ii) SEQ ID NOs: 243 and 250, respectively;(iii) SEQ ID NOs: 244 and 251, respectively;(iv) SEQ ID NOs: 245 and 251, respectively;(v) SEQ ID NOs: 246 and 252, respectively;(vi) SEQ ID NOs: 244 and 253, respectively;(vii) SEQ ID NOs: 245 and 253, respectively;(viii) SEQ ID NOs: 244 and 254, respectively;(ix) SEQ ID NOs: 245 and 255, respectively;(x) SEQ ID NOs: 246 and 255, respectively;(xi) SEQ ID NOs: 244 and 256, respectively;(xii) SEQ ID NOs: 247 and 257, respectively;(xiii) SEQ ID NOs: 248 and 258, respectively;(xiv) SEQ ID NOs: 248 and 259, respectively;(xv) SEQ ID NOs: 248 and 260, respectively;(xvi) SEQ ID NOs: 248 and 261, respectively;(xvii) SEQ ID NOs: 248 and 262, respectively; or(xviii) SEQ ID NOs: 248 and 257, respectively.

[0527] In some aspects, the multispecific protein comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of SEQ ID NOs: 263-287, 291-307, or combinations thereof.

[0528] In some aspects, the multispecific protein comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of:SEQ ID NOs: 263, 275, and 283;SEQ ID NOs: 264, 276, and 283;SEQ ID NOs: 264, 275, and 283;SEQ ID NOs: 265, 275, and 283;SEQ ID NOs: 266, 277, and 284;SEQ ID NOs: 267, 278, and 284;SEQ ID NOs: 267, 277, and 284;SEQ ID NOs: 268, 277, and 284;SEQ ID NOs: 269, 279, and 284;SEQ ID NOs: 270, 280, and 284;SEQ ID NOs: 270, 279, and 284;SEQ ID NOs: 271, 279, and 284;SEQ ID NOs: 272, 281, and 285;SEQ ID NOs: 273, 282, and 285;SEQ ID NOs: 273, 281, and 285;SEQ ID NOs: 274, 281, and 285;SEQ ID NOs: 272, 281, and 286;SEQ ID NOs: 273, 282, and 286;SEQ ID NOs: 273, 281, and 286;SEQ ID NOs: 274, 281, and 286;SEQ ID NOs: 272, 281, and 287;SEQ ID NOs: 273, 282, and 287;SEQ ID NOs: 273, 281, and 287;SEQ ID NOs: 274, 281, and 287; orSEQ ID NOs: 436, 279, and 284.

[0529] In some aspects, the anti-GPNMB antigen-binding domain binds human GPNMB, binds mouse GPNMB, binds cynomolgus GPNMB, binds both human and mouse GPNMB, or binds human, mouse, and cynomolgus GPNMB.

[0530] In some aspects, the anti-GPNMB antigen-binding domain binds human GPNMB with an affinity of about 0.4 nM to about 120 nM, of about 0.3 nM to about 5 nM, of about 0.4 nM to about 1.04 nM, of about 0.14 to about 0.65 nM and bind mouse GPNMB with an affinity of about 0.18 nM to about 0.44 nM.

[0531] In some aspects, the antigen-binding domain is an anti-TfR antigen-binding domain.

[0532] In some aspects, the anti-GPNMB antigen-binding domain in the multispecific protein is N-terminal to the anti-TfR antigen-binding domain.

[0533] In some aspects, the anti-GPNMB antigen-binding domain is C-terminal to the anti-TfR antigen-binding domain.

[0534] In some aspects, the anti-GPNMB antigen-binding domain and the anti-TfR antigenbinding domain are directly connected via a peptide bond.

[0535] In some aspects, the anti-GPNMB antigen-binding domain and the anti-TfR antigenbinding domain are connected via a linker.

[0536] In some aspects, the linker is a peptide linker.

[0537] In some aspects, the multispecific protein further comprises iii) an Fc region.

[0538] In some aspects, the Fc region is fully active.

[0539] In some aspects, the Fc region is partially active.

[0540] In some aspects, the Fc region is not silent.

[0541] In some aspects, the anti-TfR antigen-binding domain and the anti-GPNMB antigenbinding domain are linked to the N-terminus of the Fc portion of the multispecific protein.

[0542] In some aspects, the anti-TfR antigen-binding domain and the anti-GPNMB antigenbinding domain are both linked to the C-terminus of the Fc portion of the multispecific protein.

[0543] In some aspects, the anti-TfR antigen-binding domain is linked to the N-terminus of the Fc portion and the anti-GPNMB antigen-binding domain is linked to the C-terminus of the Fc portion of the multispecific protein.

[0544] In some aspects, the anti-GPNMB antigen-binding domain is linked to the N-terminus of the Fc portion of the multispecific protein and the anti-TfR antigen-binding domain is linked to the C-terminus of the Fc portion.

[0545] In some aspects, the anti-TfR antigen-binding domain is a single scFv, or a VHH, or a Fab.

[0546] In some aspects, the multispecific protein comprises two copies of the anti-GPNMB antigen-binding domain.

[0547] In some aspects, the multispecific protein comprises an Fc domain or Fc region.

[0548] In some aspects, the single scFv, Fab or VHH is linked to the C-terminus of the Fc domain or Fc region, wherein the two copies of the anti-GPNMB antigen-binding domain are linked to the N-terminus of the Fc domain or Fc region.

[0549] In some aspects, the single scFv, Fab or VHH is linked to the N-terminus of the Fc domain or Fc region, wherein the two copies of the anti-GPNMB antigen-binding domain are linked to the C-terminus of the Fc domain or Fc region.

[0550] In some aspects, the anti-TfR antigen binding domain is an anti-TfR antibody, and wherein the antibody comprises two heavy chains and two light chains.

[0551] In some aspects, two copies of an anti-GPNMB antigen-binding domain are linked to the C-terminus of the two anti-TfR antibody heavy chains.

[0552] In some aspects, the anti-TfR antigen binding domain comprises two scFv, Fab, or VHH domains linked to the C-terminus of the heavy chain.

[0553] In some aspects, two copies of the anti-GPNMB antigen-binding domain are linked to the N-terminus of the Fc domain or Fc region.

[0554] In some aspects, the anti-TfR antigen binding domain comprises two scFv, Fab, or VHH antigen-binding domains that specifically bind to human TfR linked to the N-terminus of the heavy chain.

[0555] In some aspects, two copies of the anti-GPNMB antigen-binding domain are linked to the C-terminus of the Fc domain or Fc region.

[0556] In some aspects, the anti-TfR antigen binding domain comprises a single scFv, VHH, or Fab antigen-binding domain.

[0557] In some aspects, the multispecific protein further comprises (ii) an Fc domain or Fc region, and (iii) a single copy of the anti-GPNMB antigen-binding domain.

[0558] In some aspects, the single scFv, VHH, or Fab antigen-binding domain is linked to the C-terminus of the Fc domain or Fc region and the anti-GPNMB antigen-binding domain is linked to N-terminus of the Fc domain or Fc region.

[0559] In some aspects, the single scFv, VHH, or Fab antigen-binding domain is linked to the N-terminus of the Fc domain or Fc region and the anti-GPNMB antigen-binding domain is linked to C-terminus of the Fc domain or Fc region.

[0560] In some aspects, the single scFv, VHH, or Fab antigen-binding domain and the anti- GPNMB antigen-binding domain are both linked to the N-terminus of the Fc domain or Fc region.

[0561] In some aspects, the single scFv, VHH, or Fab antigen-binding domain and the anti- GPNMB antigen-binding domain are both linked to the C-terminus of the Fc domain or Fc region.

[0562] In some aspects, the Fc is a single chain, engineered monovalent Fc domain.

[0563] In some aspects, the multispecific protein is a bispecific protein.

[0564] In some aspects, provided herein is a composition comprising any of the multispecific proteins disclosed herein.VII. Polynucleotides and Methods of Making the Same

[0565] In some aspects, provided herein is a nucleotide sequence encoding an antigen-binding domain that specifically binds to a human blood brain barrier protein or receptor and an antigenbinding thereof domain that specifically binds to a human GPNMB protein. In some aspects, provided herein are polynucleotides encoding an antigen-binding domain that specifically binds to a human blood brain barrier protein or receptor and encoding an antigen-binding thereof domain that specifically binds to a human GPNMB protein. In some aspects, the human blood brain barrier protein or receptor is TfR. In some aspects, the human blood brain barrier protein or receptor is CD98hc.

[0566] Provided herein are polynucleotides comprising a nucleotide sequence encoding the compositions described herein, or a domain thereof described herein, that are optimized, e.g., by codon / RNA optimization, replacement with heterologous signal sequences, and / or elimination of mRNA instability elements. Methods to generate optimized nucleic acids for recombinant expression by introducing codon changes (e.g., a codon change that encodes the same amino acid due to the degeneracy of the genetic code) and / or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly.

[0567] A polynucleotide comprising a nucleotide sequence encoding the compositions described herein, or a domain thereof described herein, can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising, e.g., the sequence encoding the light chain and / or heavy chain of an antigen-binding domain, antibody, or antigen-binding fragment thereof. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to generate an antigen-binding domain that specifically bind tohuman TfR, fusion protein, complex, antibody, antigen-binding fragment thereof, or multispecific protein described herein, or a domain thereof described herein.

[0568] Polynucleotides provided herein can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and DNA can be double-stranded or single-stranded. If single stranded, DNA can be the coding strand or non-coding (anti-sense) strand. In some aspects, the polynucleotide is a cDNA or a DNA lacking one more endogenous introns. In some aspects, a polynucleotide is a non-naturally occurring polynucleotide. In some aspects, a polynucleotide is recombinantly produced. In some aspects, the polynucleotides are isolated. In some aspects, the polynucleotides are substantially pure.

[0569] In some aspects, polynucleotides provided herein are in the form of RNA. In some aspects, polynucleotides provided herein are in the form of RNA encoding a fusion protein or complex provided herein. In some aspects, a polynucleotide provided herein is a synthetic messenger RNA (mRNA). In some aspects, the synthetic mRNA has at least one nucleoside modification. In some aspects, the at least one nucleoside modification is selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4- thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3 -methyluridine, 5-carboxymethyl- uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l -methylpseudouridine, 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio-l- methyl-l-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl- cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1- methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l-methyl- 1-deaza-pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5- methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, 2- aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2- aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2- methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza- 8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7- methyl-guanosine, 6-thio-7-methyl-guanosine, 7-m ethylinosine, 6-methoxy-guanosine, 1- methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl- 8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl- 6-thio-guanosine.

[0570] In certain aspects, provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding the compositions described herein, or a domain thereof described herein, for recombinant expression in a host cell, e.g., in a mammalian host cell or in an E. coli cell. A vector for the production of the compositions described herein, or a domain thereof described herein, can be produced, e.g., by recombinant DNA technology using techniques well known in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding the compositions described herein, or a domain thereof described herein, operably linked to a promoter. Such vectors can, for example, include the nucleotide sequence encoding the constant region of an antigen-binding domain, antibody or antigen-binding fragment thereof (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464), and variable domains of the antigen-binding domain, antibody or antigen-binding fragment thereof can be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains. In some aspects, the vector is gene therapy vector (e.g., an AAV or lentiviral vector).

[0571] In certain aspects, provided herein are expression systems comprising polynucleotides comprising nucleotide sequences encoding the compositions described herein, or a domain thereof described herein and an antibody to GPNMB or antigen-binding domain thereof. An expression system can be included on a vector. An expression system can also be integrated into a host cell chromosome. In some aspects, an expression system is a cell free expression system. In some aspects, an expressions system comprises a host cell comprising a polynucleotide and / or vector provided herein.

[0572] Accordingly, also provided herein are cells, e.g., host cells, comprising polynucleotides and / or vectors for recombinantly expressing the compositions described herein and an antibody to GPNMB or antigen-binding domain thereof. In some aspects, for the expression of doublechained antigen-binding proteins, vectors encoding both the heavy and light chains, individually, can be co-expressed in the host cell for expression of the entire immunoglobulin. In some aspects, a host cell contains two different vectors, a first vector comprising a polynucleotide encoding a heavy chain of an antigen-binding protein described herein, and a second vector comprising a polynucleotide encoding a light chain of the an antigen-binding protein. In some aspects, a first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain, and a second host cell comprises a second vector comprising a polynucleotide encoding a light chain. In some aspects, provided herein is a population of host cells comprising such first host cell and such second host cell.

[0573] In some aspects, provided herein are methods for producing the compositions described herein, or a domain thereof and an antibody to GPNMB or antigen-binding domain thereof described herein in a host cell.

[0574] An expression vector can be transferred to a cell (e.g., host cell) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce the compositions described herein, or a domain thereof described herein.

[0575] A variety of host-expression vector systems can be utilized to the compositions described herein, or a domain thereof described herein (see, e.g., U.S. Patent No. 5,807,715). Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express the compositions described herein, or a domain thereof described herein in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid)containing coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, Rl. l, B-W, L-M, BSC1, BSC40, YB / 20 and BMTIO cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In some aspects, cells for expressing the compositions described herein, or a domain thereof described herein are CHO cells, for example CHO cells from the CHO GS System™ (Lonza). In some aspects, cells for expressing the compositions described herein, or a domain thereof described herein as described herein are human cells, e.g., human cell lines. In some aspects, a mammalian expression vector is pOptiVEC™ or pcDNA3.3. In some aspects, bacterial cells, such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells) are used for the expression of the compositions described herein, or a domain thereof described herein. For example, mammalian cells such as Chinese hamster ovary (CHO) cells in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-105; and Cockett MI et al., (1990) Biotechnology 8: 662-667). In some aspects, the compositions described herein, or a domain thereof described herein is produced by CHO cells or NSO cells.

[0576] In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can contribute to the function of the protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, Rl.l, B-W, L-M, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells.

[0577] Once the compositions described herein, or a domain thereof described herein has been produced by recombinant expression, it can be purified by any method known in the art for purification, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the compositions described herein, or a domain thereof described herein can be fused to heterologous polypeptide sequences to facilitate purification.

[0578] In some aspects, the compositions described herein are isolated or purified. Generally, an isolated or purified composition described herein is one that is substantially free of other proteins. For example, in some aspects, a preparation of the compositions described herein is substantially free of cellular material and / or chemical precursors.A. Polynucleotides Encoding Agents Comprising an antibody to GPNMB or antigenbinding domain thereof and Antigen Binding Domains That Bind TfR and Methods of Making the Same

[0579] In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding a composition comprising an antigen binding domain that specifically binds to human TfR, as described herein and an antigen binding domain that specifically binds to GPNMB. In some aspects, provided herein are polynucleotides comprising a first nucleotide sequence encoding an antigen-binding domain that specifically bind to human TfR, as described herein and a second nucleotide sequence encoding an antibody to GPNMB or antigen-binding domain thereof. In some aspects, the polynucleotide is DNA. In some aspects, the polynucleotide is RNA or mRNA.

[0580] In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding an antigen-binding domain that specifically bind to human TfR, a fusion protein, a complex, an antibody, an antigen-binding fragment thereof, or a multi-specific binding protein described herein or a domain thereof described herein.

[0581] In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically binds to human TfR provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the light chain of an antigen-binding domain that specifically binds to human TfR provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically binds to human TfR provided herein and a nucleic acid molecule encoding the light chain of an antigenbinding domain that specifically binds to human TfR provided herein.

[0582] In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically binds to GPNMB provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the light chain of an antigen-binding domain that specifically binds to GPNMB provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically binds to GPNMB provided herein and a nucleic acid molecule encoding the light chain of an antigenbinding domain that specifically binds to GPNMB provided herein.

[0583] In some aspects, combinations or compositions of polynucleotides are provided herein. In some aspects, a combination or composition comprises a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode a multi-specific protein provided herein, e.g., wherein the first polynucleotide encodes a first heavy chain, the second polynucleotide encodes a second heavy chain and an antigenbinding domain that specifically binds to human TfR provided herein, and the third polynucleotide encodes a light chain. In some aspects, the antigen-binding domains that bind to human TfR is a scFv. In some aspects, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation. In some aspects, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0584] In some aspects, a combination or composition comprises a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode a multi-specific protein provided herein, wherein the first polynucleotide encodes a first heavy chain and a first antigen-binding domain that specifically binds to human TfR, the second polynucleotide encodes a second heavy chain and a second antigen-binding domain that specifically binds to human TfR, and the third polynucleotide encodes a light chain. In some aspects, the first and second antigen-binding domains that bind to human TfR comprise the same amino acid sequence. In some aspects, the first and second antigen-binding domains that bind to human TfR comprise different amino acid sequences. In some aspects, the first and / or second antigen-binding domains that bind to human TfR are scFvs. In some aspects, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation. In some aspects, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0585] In some aspects, a combination or composition comprises a first polynucleotide and a second polynucleotide, wherein the first and second polynucleotides encode a multi-specific protein provided herein, wherein the first polynucleotide encodes a heavy chain and an antigenbinding domain that bind to human TfR provided herein, and wherein the second polynucleotide encodes a light chain.

[0586] In certain aspects, provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding an antigen-binding domain that specifically bind to human TfR, antibody, antigen-binding fragment thereof, or multi-specific binding protein described herein, or a domain thereof described herein, for recombinant expression in a host cell, e.g., in a mammalian host cell.

[0587] In certain aspects, provided herein are expression systems comprising polynucleotides comprising nucleotide sequences encoding the compositions described herein, or a domain thereof described herein and an antibody to GPNMB or antigen-binding domain thereof.

[0588] Accordingly, also provided herein are cells, e.g., host cells, comprising polynucleotides and / or vectors for recombinantly expressing an antigen-binding domain that specifically bind to human TfR, fusion protein, complex, antibody, antigen-binding fragment thereof, or multispecific binding protein described herein, or a domain thereof described herein and an antigenbinding domain that specifically binds to GPNMB.

[0589] In some aspects, provided herein are methods for producing an antigen-binding domain that specifically binds to human TfR, fusion protein, complex, antibody, antigen-binding fragment thereof, or multi-specific protein described herein, or a domain thereof and an antigenbinding domain that specifically binds to GPNMB described herein in a host cell.

[0590] In some aspects, provided herein are methods for producing a single chain antigenbinding domain that specifically binds to human TfR, a linker, an Fc domain, and a second antigen-binding domain, as described herein.

[0591] In some aspects, a composition that binds to human TfR as described herein is isolated or purified (e.g., is one that is substantially free of other proteins, and / or is substantially free of cellular material and / or chemical precursors).B. Polynucleotides Encoding Agents Comprising Antigen-Binding Domains that Bind GPNMB and Antigen Binding Domains That Bind CD98hc and Methods of Making the Same

[0592] In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding a composition comprising an antigen binding domain that specifically binds to human CD98hc described herein or a domain thereof described herein, and an antigen-binding domain that specifically binds to GPNMB.

[0593] In some aspects, provided herein are polynucleotides comprising a first nucleotide sequence encoding an antigen-binding domain that specifically bind to human CD98hc, as described herein and a second nucleotide sequence encoding an antigen-binding domain that specifically binds to GPNMB. In some aspects, the polynucleotide is DNA. In some aspects, the polynucleotide is RNA or mRNA.

[0594] In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding compositions that specifically bind to human CD98hc described herein or a domain thereof described herein.

[0595] In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically binds to human CD98hc provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the light chain of an antigen-binding domain that specifically binds to human CD98hc provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically binds to human CD98hc provided herein and a nucleic acid molecule encoding the light chain of an antigen-binding domain that specifically binds to human CD98hc provided herein.

[0596] In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically binds to GPNMB provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the light chain of an antigen-binding domain that specifically binds to GPNMB provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically binds to GPNMB provided herein and a nucleic acid molecule encoding the light chain of an antigenbinding domain that specifically binds to GPNMB provided herein.

[0597] In some aspects, combinations or compositions of polynucleotides are provided herein. In some aspects, a combination or composition comprises a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode a multi-specific protein provided herein, e.g., wherein the first polynucleotide encodes a first heavy chain, the second polynucleotide encodes a second heavy chain and an antigenbinding domain that specifically binds to human CD98hc provided herein, and the third polynucleotide encodes a light chain. In some aspects, the antigen-binding domains that bind to human CD98hc is a scFv. In some aspects, the first heavy chain comprises a knob mutation and the second heavy chain comprises a hole mutation. In some aspects, the first heavy chain comprises a hole mutation and the second heavy chain comprises a knob mutation.

[0598] In some aspects, a combination or composition comprises a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode a multi-specific protein provided herein, wherein the first polynucleotide encodes a first heavy chain and a first antigen-binding domain that specifically binds to human CD98hc, the second polynucleotide encodes a second heavy chain and a second antigen-binding domain that specifically binds to human CD98hc, and the third polynucleotide encodes a light chain. In some aspects, the first and second antigen-binding domains that bind to human CD98hc comprise the same amino acid sequence. In some aspects, the first and second antigen-binding domains that bind to human CD98hc comprise different amino acid sequences. In some aspects, the first and / or second antigen-binding domains that bind to human CD98hc are scFvs. In some aspects, the first heavy chain comprises a “knob” mutation and the second heavy chain comprises a “hole” mutation. In some aspects, the first heavy chain comprises a “hole” mutation and the second heavy chain comprises a “knob” mutation.

[0599] In some aspects, a combination or composition comprises a first polynucleotide and a second polynucleotide, wherein the first and second polynucleotides encode a multi-specific protein provided herein, wherein the first polynucleotide encodes a heavy chain and an antigenbinding domain that bind to human CD98hc provided herein, and wherein the second polynucleotide encodes a light chain.

[0600] In certain aspects, provided herein are vectors e.g., expression vectors) comprising polynucleotides comprising the compositions that bind CD98hc described herein, or a domain thereof described herein, for recombinant expression in a host cell, e.g., in a mammalian host cell.

[0601] In certain aspects, provided herein are expression systems comprising polynucleotides comprising nucleotide sequences encoding the compositions that bind CD98hc described herein, or a domain thereof described herein and an antigen-binding domain that specifically binds to GPNMB. Accordingly, also provided herein are cells, e.g., host cells, comprising polynucleotides and / or vectors for recombinantly expressing the compositions that bind CD98hc described herein, or a domain thereof described herein and an antigen-binding domain that specifically binds to GPNMB.

[0602] In some aspects, provided herein are methods for producing a composition that binds to human CD98hc as described herein, or a domain thereof, and an antigen-binding domain that specifically binds to GPNMB described herein in a host cell.

[0603] In some aspects, provided herein are methods for producing a single chain antigenbinding domain that specifically binds to human CD98hc, a linker, an Fc domain, and an antigenbinding domain that specifically binds to GPNMB.

[0604] In some aspects, provided herein are methods for producing a single chain antigenbinding domain that specifically binds to human CD98hc, a linker, an Fc domain, and a second antigen-binding domain, as described herein.

[0605] A variety of host-expression vector systems and host cells are described herein.

[0606] In some aspects, a composition that binds to human CD98hc as described herein is isolated or purified (e.g., is one that is substantially free of other proteins, and / or is substantially free of cellular material and / or chemical precursors).VIII. Pharmaceutical Compositions

[0607] Provided herein are pharmaceutical compositions comprising an antigen-binding domain that specifically binds to GPNMB and compositions that that bind to human TfR or a human CD98hc as described herein and a pharmaceutically acceptable carrier, excipient or stabilizer (Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Also provided herein are pharmaceutical compositions an antigen-binding domain that specifically binds to GPNMB and compositions that bind to human CD98hc as described herein and a pharmaceutically acceptable carrier, excipient or stabilizer. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can comprise antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueoussterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0608] In some aspects, a pharmaceutical composition an antigen-binding domain that specifically binds to GPNMB and compositions that bind to human TfR as described herein and a pharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). In some aspects, a pharmaceutical composition comprises an antigen-binding domain that specifically binds to GPNMB and compositions that bind to human CD98hc as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions described herein are, in some aspects, for use as a medicament. The compositions to be used for in vivo administration can be sterile. This is readily accomplished by filtration through, e.g., sterile filtration membranes.

[0609] In some aspects, provided herein are pharmaceutical compositions comprising a polynucleotide encoding an antigen-binding domain that specifically binds to GPNMB and a composition that binds to human TfR as described herein. Also provided herein are pharmaceutical compositions comprising a polynucleotide encoding an antigen-binding domain that specifically binds to GPNMB and a composition that binds to human CD98hc as described herein. In some aspects, the polynucleotide is RNA. In some aspects, the polynucleotide is a synthetic mRNA. In some aspects, the polynucleotide is a modified mRNA. In some aspects, the pharmaceutical composition comprising a polynucleotide further comprises a lipid-based transfection reagent.

[0610] A pharmaceutical composition described herein can be used to exert a biological effect(s) in vivo or in vitro. For example, a pharmaceutical composition described herein can be used to cross a blood brain barrier, e.g., in a subject.

[0611] In some aspects, a pharmaceutical composition provided herein is used to treat diseases or conditions such as a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation. In some aspects, a pharmaceutical composition provided herein is used to treat diseases or conditions such as Alzheimer’s disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS),amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman’s syndrome, Liddle syndrome, Parkinson’s disease, Pick’s disease, Paget’s disease, cancer, and traumatic brain injury. In some aspects, a pharmaceutical composition provided herein is used to treat frontotemporal dementia.

[0612] In some aspects, a pharmaceutical composition provided herein is formulated for intravenous administration. In some aspects, a pharmaceutical composition provided herein is formulated for subcutaneous administration.IX. Methods of Using Agents Comprising Antigen-Binding Domains that Bind GPNMB and Antigen-Binding Domains that Bind to Blood-Brain Barrier Receptors or Proteins

[0613] Compositions provided herein comprise (i) an anti-TfR or an anti-CD98hc antigenbinding domain and (ii) an anti-GPNMB antigen-binding domain that can advantageously be transported across a blood brain barrier.

[0614] Accordingly, provided herein are methods of administering or transporting compositions comprising (i) an antigen-binding protein that specifically binds to human TfR or human CD98hc and (ii) an antigen-binding domain the specifically binds to GPNMB across the blood brain barrier of a subject comprising administering to the subject such compositions.

[0615] In view of the ability of compositions provided herein to specifically bind to human TfR or human CD98hc and be transported across a blood brain barrier, these compositions can be used to treat a neurological disease or disorder. In some aspects, a method of treating a neurological disease or disorder in a subject comprises administering to the subject a composition comprising (i) an antigen-binding protein that specifically binds to human TfR or human CD98hc and (ii) an antigen-binding domain that specifically binds to GPNMB.

[0616] The neurological disease or disorder can be, for example, a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, or CNS inflammation. The neurological disease or disorder can be, for example, a neurodegenerative disease (such as Lewy body disease, postpoliomyelitis syndrome, Shy- Draeger syndrome, olivopontocerebellar atrophy, Parkinson’s disease, Gaucher disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), a tauopathy (such as Alzheimer disease and supranuclear palsy), a prion disease (such as bovine spongiform encephalopathy, scrapie, Creutz-feldt-Jakob syndrome, kuru, Gerstmann-Straussler- Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motorneuron disease, a nervous system heterodegenerative disorders (such as Canavan disease, Huntington’s disease, neuronal ceroid-lipofuscinosis, Alexander’s disease, Tourette’s syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden- Spatz syndrome, lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (such as Pick’s disease, and spinocerebellar ataxia), cancer of the CNS and / or brain (such as glioblastoma or brain metastases resulting from cancer elsewhere in the body), Alzheimer’s disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), limbic-predominant age-related TDP-43 encephalopathy (LATE), cystic fibrosis, Angelman’s syndrome, Liddle syndrome, Parkinson’s disease, Pick’s disease, Paget’s disease, cancer, or traumatic brain injury. In some aspects, the neurological disease or disorder is dementia. In some aspects, the neurological disease or disorder is frontotemporal dementia. In some aspects, the neurological disease or disorder is Alzheimer’s disease. In some aspects, the neurological disease or disorder is Parkinson’s disease. In some aspects, the neurological disease or disorder is frontal temporal epilepsy. In some aspects, the neurological disease or disorder is autism. In some aspects, the neurological disease or disorder is lissencephaly.

[0617] In some aspects, provided herein is a method of treating a lysosomal storage disease with a fusion protein or complex disclosed herein. In some aspects, the lysosomal storage disease is selected from Gaucher disease, Ceroid lipofuscinosis (Batten disease), Mucopolysaccharidosis (MPS) Type I, MPS Type II and MPS Type III ).

[0618] In some aspects, provided herein is a method of administering or transporting compositions comprising administering to the subject any of the multispecific proteins disclosed herein, any of the compositions disclosed herein, any of the pharmaceutical compositions disclosed herein, or any of the nucleotide sequences disclosed herein.

[0619] In some aspects, provided herein is a method of treating a lysosomal storage disease or disorder in a subject comprising administering to the subject any of the multispecific proteins disclosed herein, any of the compositions disclosed herein, any of the pharmaceutical compositions disclosed herein, or any of the nucleotide sequences disclosed herein.

[0620] In some aspects, the lysosomal storage disease or disorder is Gaucher’s disease or Neimann-Pick type C disease.

[0621] In some aspects, provided herein is a method of treating a CNS disease or disorder in a subject comprising administering to the subject any of the multispecific proteins disclosed herein,any of the compositions disclosed herein, any of the pharmaceutical compositions disclosed herein, or any of the nucleotide sequences disclosed herein.

[0622] In some aspects, the CNS disease or disorder is selected from Parkinson’s disease, Alzheimer’s disease, and ALS.

[0623] Compositions provided herein can be used to detect an antigen (e.g., a CNS antigen or a brain antigen). In some aspects, the compositions provided herein can be labeled. Exemplary labels include, for example, radioisotopes (e.g.,64CU) and fluorescent labels. Accordingly, methods of detecting the compositions provided herein are provided. In some aspects, a method of detecting an antigen in the CNS (e.g., brain) of a subject comprises administering the compositions provided herein in the CNS (e.g., brain). Such methods can further comprise, e.g., performing Positron emission tomography (PET) imaging on the subject. In some aspects, disclosed herein is a method of detecting a CNS antigen in vitro, comprising contacting an in vitro sample with a composition disclosed herein and locating the imaging agent within the sample.

[0624] Compositions provided herein can be used for prognostic, diagnostic, monitoring, and / or screening applications, including in vivo applications well known and standard to the skilled artisan and based on the present description. In some aspects, provided herein is a composition for use as a diagnostic. In some aspects, the compositions provided herein comprise a detectable label.

[0625] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0626] The present disclosure will be more fully understood by reference to the following Examples. They should not, however, be construed as limiting the scope of the present disclosure. All citations throughout the disclosure are hereby expressly incorporated by reference.EXAMPLESExample 1: Engineering of Antigen-Binding Domains against Human Transferrin Receptor

[0627] Mammalian expression of human, cynomolgus macaque (cyno) and mouse variants of Transferrin Receptor antigens (SEQ ID NOs:218-224) was performed by cloning synthetic genes based on cDNA into mammalian expression vectors, followed by transient transfection and expression in Expi293 cells and purified by Ni-NTA agarose (QIAGEN 30230) using themanufacturer’s protocol. See Example 1 of International Application Serial No.PCT / US2023 / 071239, filed July 28, 2023, published as WO 2024 / 026472.Table 9: Avi-His tagged variants of TfR

[0628] Plasmid constructs each expressing full length human TfR and mouse TfR (SEQ ID NOs: 1 and 225, respectively) were used to generate CHO cells stably expressing human TfR and mouse TfR. The resulting CHO cells stably expressing human TfR and mouse TfR were analyzed for cell surface expression by flow cytometry. See Example 2 of International Application Serial No. PCT / US2023 / 071239, published as WO 2024 / 026472.Table 10. Full-length TfR sequences

[0629] A mouse line was generated at Taconic Biosciences GmbH (Germany) expressing the extracellular domains of human TfR. CRISPR was used to replace the mouse ECD with the human version, while retaining the mouse intracellular and transmembrane portions under the control of the mouse promotor. Brain slices from these mice were evaluated by IHC and / or Western Blot to confirm expression and localization of the human ECD in vivo (data not shown). See Example 3 of International Application Serial No. PCT / US2023 / 071239, published as WO 2024 / 026472.

[0630] Hybridomas to obtain antibodies against TfR were generated by immunizing BALB / c mice or Sprague Dawley rats with purified extracellular domain polypeptides of human / cyno and / or mouse TfR. Lymph nodes were harvested from the mice or rats for hybridoma cell line generation. Sera from the animals were analyzed for reactivity to TfR and lymphocytes from animals whose sera demonstrated strong binding were used to generate hybridoma libraries. See Example 4 of International Application Serial No. PCT / US2023 / 071239, published as WO 2024 / 026472.

[0631] A total of 1536 anti-TfR hybridoma supernatants were screened via FACS for their ability to differentially bind CHO cells overexpressing human or mouse TfR compared to CHO parental cells. A total of 189 mouse hybridoma and 115 rat hybridoma clones displayed greater than 3-fold difference in binding (as determined by MFI) to CHO cells stably overexpressing human TfR compared to isotype control. See Example 5 of International Application Serial No. PCT / US2023 / 071239, published as WO 2024 / 026472.

[0632] Hybridoma culture supernatants from 319 hybridomas obtained as described above were screened for their ability to bind Avi-His-tagged huTfR ECD, huTfR apical domain, and muTfR ECD ) as compared to binding to an irrelevant Avi-His-tagged control protein. From this hybridoma supernatant screen, a total of 112 anti-TfR hybridoma clones were identified that displayed greater than 5-fold difference in binding to recombinant huTfR Avi-His over background, and 86 of these anti-TfR hybridoma clones also bound to the huTfR apical domain. See Example 6 of International Application Serial No. PCT / US2023 / 071239, published as WO 2024 / 026472.

[0633] Anti-TfR hybridoma antibodies were purified and screened for their ability to internalize into a blood-brain barrier endothelial cell line (hCMEC / D3). Internalization into endothelial cells at the blood-brain barrier is the first stage of transcytosis across the BBB and into the brain. To identify anti-TfR antibodies with internalization ability, HCMEC / D3 cells were seeded and treated with anti-TfR antibodies. A human IgG isotype and an anti-TfR antibody with known internalization ability were included in the assay as negative and positive controls, respectively. Antibodies (TfRl-32) were found to have a fold change of internalization over an irrelevant mouse IgG antibodies in a range from 1-2 fold to 714.7 fold. See Example 7 of International Application Serial No. PCT / US2023 / 071239, published as WO 2024 / 026472.

[0634] Anti-TfR antibodies from hybridomas were then cloned and out of 72 apical domain positive anti-TfR hybridoma clones, a total of 32 unique sequences were identified. Out of 24 apical domain positive anti-TfR hybridoma clones from rats, an additional 21 unique sequences were identified. See Example 8 of International Application Serial No. PCT / US2023 / 071239, published as WO 2024 / 026472.

[0635] Example anti-TfR antibody sequences are shown in Tables 3, 4, and 5 above.

[0636] Anti-TfR antibodies were then reformatted into a 2 +1 bispecific antibody format. Antibodies were selected for reformatting based on various criteria: 1) the antibodies covered a broad range of affinities based on ELISA and FACS binding assays, 2) the antibodies covered abroad range of internalization activity, 3) no outstanding high-risk modification sites were identified in the CDRs, and 4) the antibodies were phylogenetically diverse from each other within the hybridoma sequences obtained. An IgG isotype antibody with no target specificity (“inert isotype control antibody”) with knob-hole mutations in the constant domains of the heavy chains was used for formatting into a 2+1 bispecific antibody. An example structure of a 2+1 bispecific antibody comprised the following components: 1) Isotype control hlgGl wildtype antibody with a knob ((T366W) mutation and a hole mutation (T366S L368A Y407V) in the constant regions, 2) a (G4S)x3 linker (SEQ ID NO: 179) between the “hole side” of the hlgGl antibody and a scFv, 3) a VH sequence of the scFv, 4) a 20 amino acid linker sequence between the VH and VL of the scFv, and 5) a VL sequence of the scFv. See Example 9 of International Application Serial No. PCT / US2023 / 071239, published as WO 2024 / 026472.

[0637] Once converted into a 2 +1 antibody format, anti-TfR hybridoma antibodies were confirmed to retain TfR binding via ELISA and FACs. In total, 28 out of 32 reformatted antibodies retained binding affinity against TfR in the 2+1 antibody format. Importantly, only 6 of the 32 antibodies (TfR6, TfR9, TfR12, TfR15, TfR19, TfR27) showed strong cyno crossreactivity. See Example 10 of International Application Serial No. PCT / US2023 / 071239, published as WO 2024 / 026472.

[0638] Certain 2 +1 anti-TfR bispecific antibodies were tested for their ability to be internalized into the hCMEC / D3 cell line. The 2+1 anti-TfR bispecific antibodies showed a wide range of internalization capability with many showing significantly higher internalization than an anti-TfR control antibody. See Example 11 of International Application Serial No. PCT / US2023 / 071239, published as WO 2024 / 026472.

[0639] The scFv portion of certain anti-TfR antibodies were then chosen for their crossreactivity, function, functional stability, and sequence diversity for humanization. Structurebased antibody modeling was applied in the process of humanizing anti-TfR mouse monoclonal antibodies (mAbs) utilizing the BioMOE module of MOE (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada). After the BioMOE module of MOE was used to visualize and classify potential residues for back mutation, the back potential back mutations were classified based on amino acid type difference, interaction potential with CDR residues, impact potential for VL / VH pairing, and potential change in hydrophobic and charged surface area in and near the CDRs. Mutations that were near the CDRs or the VL / VH interface, have a significant charge difference or contain strong H-bond interactions were individually evaluatedand the significantly disrupting mutations were reverted back to the original query residues. As a result, humanized sequences may contain up to five back mutations. See Example 12 of International Application Serial No. PCT / US2023 / 071239, published as WO 2024 / 026472. Certain humanized anti-T...

Claims

WHAT IS CLAIMED IS:

1. A multispecific protein comprising (i) an antigen-binding domain that specifically binds to a blood brain barrier (BBB) target selected from human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc) and (ii) an antigen-binding domain that specifically binds to Glycoprotein nonmetastatic melanoma protein B (GPNMB).

2. The multispecific protein of claim 1, wherein the anti-GPNMB antigen-binding domain is an antibody, optionally wherein the antibody is a full-length antibody.

3. The multispecific protein of claim 2, wherein the antibody is an IgG antibody, optionally wherein the IgG antibody is an IgGl antibody or an IgG4 antibody.

4. The multispecific protein of claim 2 or 3, wherein the anti-GPNMB antibody comprises a heavy chain with a Fc hole mutation.

5. The multispecific protein of any one of claims 2-4, wherein the anti-GPNMB antibody comprises a heavy chain with a Fc knob mutation.

6. The multispecific protein of any one of claims 2-5, wherein the anti-BBB antigen-binding domain is a scFv.

7. The multispecific protein of claim 6, wherein the anti-BBB scFv is linked to the C- terminus of the anti-GPNMB antibody heavy chain with a Fc hole mutation.

8. The multispecific protein of claim 6, wherein the anti-BBB scFv is linked to the C- terminus of the anti-GPNMB antibody heavy chain with a Fc knob mutation.

9. The multispecific protein of claim 6, wherein the multispecific protein comprises two antigen-binding domains that specifically bind to a BBB target selected from human TfR and human CD98hc, wherein both of the anti-BBB antigen-binding domains are scFvs.

10. The multispecific protein of claim 9, wherein the first anti-BBB scFv is linked to the C- terminus of the anti-GPNMB antibody heavy chain with a Fc hole mutation, and wherein the second anti-BBB scFv is linked to the C-terminus of the anti-GPNMB antibody heavy chain with a Fc knob mutation.

11. The multispecific protein of claim 1, wherein the anti-BBB antigen-binding domain is an antibody, optionally wherein the antibody is a full-length antibody.

12. The multispecific protein of claim 11, wherein the antibody is an IgG antibody, optionally wherein the IgG antibody is an IgGl antibody or an IgG4 antibody.

13. The multispecific protein of claim 11 or 12, wherein the anti-BBB antibody comprises a heavy chain with a Fc hole mutation.

14. The multispecific protein of any one of claims 11-13, wherein the anti-BBB antibody comprises a heavy chain with a Fc knob mutation.

15. The multispecific protein of any one of claims 11-14, wherein the anti-GPNMB antigenbinding domain is a scFv.

16. The multispecific protein of claim 15, wherein the anti-GPNMB scFv is linked to the C- terminus of the anti-BBB antibody heavy chain with a Fc hole mutation.

17. The multispecific protein of claim 15, wherein the anti-GPNMB scFv is linked to the C- terminus of the anti-BBB antibody heavy chain with a Fc knob mutation.

18. The multispecific protein of claim 15, wherein the multispecific protein comprises two antigen-binding domains that specifically bind to a GPNMB, wherein both of the anti- GPNMB antigen-binding domains are scFvs.

19. The multispecific protein of claim 18, wherein the first anti-GPNMB scFv is linked to the C-terminus of the anti-BBB antibody heavy chain with a Fc hole mutation, and wherein the second anti-GPNMB scFv is linked to the C-terminus of the anti-BBB antibody heavy chain with a Fc knob mutation.

20. The multispecific protein of any one of claims 2-19, wherein the antibody is a monoclonal antibody.

21. The multispecific protein of any one of claims 2-20, wherein the antibody is a humanized antibody.

22. The multispecific protein of claim 1, wherein the anti-GPNMB antigen-binding domain is selected from the group consisting of a VHH, a Fab, a Fab’, a Fab’-SH, a F(ab’)2, a Fv, or a scFv.

23. The multispecific protein of claim 1 or 22, wherein the anti-BBB antigen-binding domain is a VHH, a Fab, a Fab’, a Fab’-SH, a F(ab’)2, a Fv, or a scFv.

24. The multispecific protein of claim 1, 22, or 23, further comprising iii) an Fc region.

25. The multispecific protein of any one of claims 1 and 22-24, wherein the anti-GPNMB antigen-binding domain is a VHH.

26. The multispecific protein any one of claims 1 and 22-25, wherein, the anti-BBB antigenbinding domain is a VHH.

27. The multispecific protein of any one of claims 1 and 22-24, wherein the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a VHH that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigenbinding domain is a VHH that is N-terminal to the other protein fragment of the Fc region.

28. The multispecific protein of any of claims 1, 22-24, and 26, wherein the anti-GPNMB antigen-binding domain is a Fab.

29. The multispecific protein of any of claims 1, 22-24, and 25, wherein the anti-BBB antigen-binding domain is a Fab.

30. The multispecific protein of any one of claims 1 and 22-24, wherein the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a Fab that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigenbinding domain is a Fab that is N-terminal to the other protein fragment of the Fc region.

31. The multispecific protein of any one of claims 1, 22-25, and 28, wherein the anti-BBB antigen-binding domain is a scFv.

32. The multispecific protein of any one of claims 1, 22-24, 26, and 29, wherein the anti- GPNMB antigen-binding domain is a scFv.

33. The multispecific protein of any one of claims 1 and 22-24, wherein the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a scFv that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigenbinding domain is a scFv that is N-terminal to the other protein fragment of the Fc region.

34. The multispecific protein of any one of claims 1 and 22-24, wherein the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a scFv that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigenbinding domain is a Fab that is N-terminal to the other protein fragment of the Fc region.

35. The multispecific protein of any one of claims 1 and 22-24, wherein the multispecific protein comprises an IgG Fc region, the anti-BBB antigen-binding domain is a Fab that is N-terminal to one protein fragment of the Fc region, and the anti-GPNMB antigenbinding domain is a Fab that is N-terminal to the other protein fragment of the Fc region36. The multispecific protein of any one of claims 24, 27, 30, and 33-35, wherein the IgG Fc region is an IgGl Fc region or an IgG4 Fc region.

37. The multispecific protein of any one of claims 24, 27, 30, and 33-36, wherein the IgG Fc region comprises knob and hole mutations.

38. The multispecific protein of any one of claims 1 and 22-37, wherein the multispecific protein is bivalent.

39. The multispecific protein of any one of claims 1-8, 11-17, and 20-37, wherein the multispecific protein is trivalent.

40. The multispecific protein of claim 39, wherein the trivalent protein comprises one antigen-binding domain that binds to the BBB target and two antigen-binding domains that bind to GPNMB.

41. The multispecific protein of claim 39, wherein the trivalent protein comprises two antigen-binding domains that bind to the BBB target and one antigen-binding domain that binds to GPNMB.

42. The multispecific protein of any one of claims 1-37, wherein the multispecific protein is tetravalent, optionally wherein the tetravalent protein comprises two of antigen-binding domains that bind to the BBB target and two antigen-binding domains that bind to GPNMB.

43. The multispecific protein of claim 1, wherein the multispecific protein is a bispecific T- cell engager (BiTE), dual-affinity re-targeting protein (DARTs), or Tandem diabody (TandAb).

44. The multispecific protein of claim 1, wherein the anti -BBB antigen-binding domain and the anti-GPNMB antigen-binding domain are contained within a single polypeptide chain, optionally wherein the single polypeptide chain further comprises an Fc domain.

45. The multispecific protein of claim 44, wherein the anti-GPNMB antigen-binding domain is N-terminal to the anti-BBB antigen-binding domain.

46. The multispecific protein of claim 44, wherein the anti-GPNMB antigen-binding domain thereof is C-terminal to the anti-BBB antigen-binding domain.

47. The multispecific protein of any one of claims 44-46, wherein the anti-GPNMB antigenbinding domain and the anti-BBB antigen-binding domain are directly connected via a peptide bond.

48. The multispecific protein of any one of claims 44-46, wherein the anti-GPNMB antigenbinding domain and the anti-BBB antigen-binding domain are connected via a linker.

49. The multispecific protein of claim 48, wherein the linker is a peptide linker.

50. The multispecific protein of claim 48, wherein the anti-BBB antigen-binding domain and the anti-GPNMB antigen-binding domain are N-terminal to the Fc domain.

51. The multispecific protein of claim 48, wherein the anti-BBB antigen-binding domain and the anti-GPNMB antigen-binding domain are C-terminal to the Fc domain.

52. The multispecific protein of claim 48, wherein the anti-BBB antigen-binding domain is N-terminal to the Fc domain and the anti-GPNMB antigen-binding domain is C-terminal to the Fc domain.

53. The multispecific protein of claim 48, wherein the anti-GPNMB antigen-binding domain is N-terminal to the Fc domain and the anti-BBB antigen-binding domain is C-terminal to the Fc domain.

54. The multispecific protein of any one of claims 48-53, wherein the multispecific protein comprises two copies of the anti-GPNMB antigen-binding domain and / or two copies of the anti-BBB antigen-binding domain.

55. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain antagonizes GPNMB activity.

56. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain modulates expression of activation surface markers on myeloid cells.

57. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain increases cell surface expression of PD-L1 in human macrophages.

58. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain increases cell surface expression of CD40 in human macrophages.

59. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain increases cell surface expression of CD80 in human macrophages.

60. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain modulates lysosome function in myeloid cells.

61. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain increases glucocerebrosidase activity in human macrophages.

62. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain decreases cell surface expression of GPNMB in human macrophages.

63. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain changes interferon pathway gene expression patterns in human macrophages.

64. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain reduces tumor volume in a murine tumor model, such as in an MC38 model.

65. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain reduces tumor growth rate in a murine tumor model, such as in an MC38 model.

66. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain increases levels of IL-12p40 in serum.

67. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain increases levels of CCL5 in serum.

68. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain is a GPNMB ligand blocking antigen-binding domain.

69. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain is a non-blocking antigen-binding domain.

70. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain antagonizes GPNMB activity independent of ligand blocking activity.

71. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain antagonizes GPNMB activity in vivo.

72. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain binds to GPNMB expressed on a cell surface.

73. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain promotes macrophage activation.

74. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain overcomes a decrease in glucocerebrosidase activity associated with reduced progranulin levels.

75. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain decreases GPNMB expression levels in cells, optionally in macrophages.

76. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain decreases LAMP2 expression levels in cells, optionally in macrophages, monocytes, or neutrophils.

77. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain inhibits or reduces inflammasome activation.

78. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain inhibits IL-ip expression or release.

79. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain reduces neural inflammation.

80. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain reduces expression of Clq, GFAP, IB Al, and CTSD associated with neural inflammation.

81. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain reduces lysosomal stress.

82. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain competes with one or more antibodies selected from GPN-81, GPN-82, GPN-83, GPN-84, GPN-85, GPN-86, GPN-87, GPN-88, GPN-89, GPN-90, GPN-91, GPN-92, GPN-93, GPN-94, GPN-95, GPN-96, GPN-97, GPN-98, and any combination thereof for binding to GPNMB.

83. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain comprises a heavy chain variable region (VH) complementaritydetermining region (CDR) 1, a VH CDR2, a VH CDR3 and a light chain variable region (VL) CDR1, a CDR2, and a CDR3, wherein: i) the VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 308 or 9; ii) the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 310, 311, 312, 313, 314, or 315; iii) the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 316, 317, 318, or 319; iv) the VL CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 320, 321, 322, 323, 324, or 325; v) the VL CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 326, 327, 328, 329, 330, 331, 332, 333, 334, or 335; and / or vi) the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 336, 337, 338, 339, 340, or 341.

84. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of: i) SEQ ID NOs: 308, 310, and 316; and 320, 326, and 336; respectively; ii) SEQ ID NOs: 308, 311, and 316; and 321, 327, and 337; respectively iii) SEQ ID NOs: 308, 312, and 316; and 322, 328, and 337; respectively; iv) SEQ ID NOs: 308, 312, and 317; and 322, 328, and 337; respectively; v) SEQ ID NOs: 308, 313, and 316; and 323, 329, and 337; respectively; vi) SEQ ID NOs: 308, 312, and 316; and 321, 330, and 337; respectively; vii) SEQ ID NOs: 308, 312, and 317; and 321, 330, and 337; respectively; viii) SEQ ID NOs: 308, 312, and 316; and 321, 329, and 338; respectively; ix) SEQ ID NOs: 308, 312, and 317; and 321, 329, and 337; respectively; x) SEQ ID NOs: 308, 313, and 316; and 321, 329, and 337; respectively; xi) SEQ ID NOs: 308, 312, and 316; and 321, 329, and 339; respectively; xii) SEQ ID NOs: 9, 314, and 318; and 324, 331, and 340; respectively; xiii) SEQ ID NOs: 9, 315, and 319; and 325, 332, and 341; respectively;xiv) SEQ ID NOs: 9, 315, and 319; and 325, 332, and 340; respectively; xv) SEQ ID NOs: 9, 315, and 319; and 325, 333, and 341; respectively; xvi) SEQ ID NOs: 9, 315, and 319; and 324, 334, and 340; respectively; xvii) SEQ ID NOs: 9, 315, and 319; and 325, 335, and 340; respectively; or xviii) SEQ ID NOs: 9, 315, and 319; and 324, 331, and 340; respectively.

85. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain comprises a VH that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 242, 243, 244, 245, 246, 247, and 248.

86. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain comprises a VL that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to an amino acid sequence selected from any one of SEQ ID NOs: 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, and 262.

87. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of(i) SEQ ID NOs: 242 and 249, respectively;(ii) SEQ ID NOs: 243 and 250, respectively;(iii) SEQ ID NOs: 244 and 251, respectively;(iv) SEQ ID NOs: 245 and 251, respectively;(v) SEQ ID NOs: 246 and 252, respectively;(vi) SEQ ID NOs: 244 and 253, respectively;(vii) SEQ ID NOs: 245 and 253, respectively;(viii) SEQ ID NOs: 244 and 254, respectively;(ix) SEQ ID NOs: 245 and 255, respectively;(x) SEQ ID NOs: 246 and 255, respectively;(xi) SEQ ID NOs: 244 and 256, respectively;(xii) SEQ ID NOs: 247 and 257, respectively;(xiii) SEQ ID NOs: 248 and 258, respectively;(xiv) SEQ ID NOs: 248 and 259, respectively;(xv) SEQ ID NOs: 248 and 260, respectively;(xvi) SEQ ID NOs: 248 and 261, respectively;(xvii) SEQ ID NOs: 248 and 262, respectively; or (xviii) SEQ ID NOs: 248 and 257, respectively.

88. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain binds mouse GPNMB, binds cynomolgus GPNMB, or binds mouse and cynomolgus GPNMB.

89. The multispecific protein of any one of the previous claims, wherein the anti-GPNMB antigen-binding domain binds human GPNMB with an affinity of about 0.4 nM to about 120 nM, of about 0.3 nM to about 5 nM, of about 0.4 nM to about 1.04 nM, of about 0.14 to about 0.65 nM and bind mouse GPNMB with an affinity of about 0.18 nM to about 0.44 nM.

90. The multispecific protein of any one of the previous claims, wherein the BBB target is human TfR.

91. The multispecific protein of any one of the previous claims, wherein the multispecific protein is a bispecific protein.

92. The multispecific protein of any one of claims 1-91, wherein the anti-TfR antigenbinding domain comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of:(i) SEQ ID NOs: 8, 11, 24, and 40, 55, and 61; respectively;(ii) SEQ ID NOs: 8, 11, 25; and 41, 55, and 61; respectively;(iii) SEQ ID NOs: 8, 12, 26; and 42, 55, and 61; respectively;(iv) SEQ ID NOs: 8, 12, 27; and 42, 55, and 61; respectively;(v) SEQ ID NOs: 8, 13, 25; and 42, 55, and 61; respectively;(vi) SEQ ID NOs: 8, 14, 25; and 42, 55, and 61; respectively;(vii) SEQ ID NOs: 8, 15, 25; and 43, 55, and 61; respectively;(viii) SEQ ID NOs: 8, 16, 25; and 42, 55, and 61; respectively;(ix) SEQ ID NOs: 8, 17, 25; and 44, 55, and 61; respectively;(x) SEQ ID NOs: 9, 18, 28; and 45, 56, and 62; respectively;(xi) SEQ ID NOs: 9, 19, 28; and 45, 56, and 62; respectively;(xii) SEQ ID NOs: 9, 20, 28; and 46, 57, and 62; respectively;(xiii) SEQ ID NOs: 9, 20, 28; and 46, 58, and 62; respectively;(xiv) SEQ ID NOs: 9, 20, 28; and 47, 59, and 62; respectively;(xv) SEQ ID NOs: 9, 21, 28; and 46, 57, and 62; respectively;(xvi) SEQ ID NOs: 9, 21, 28; and 47, 59, and 62; respectively;(xvii) SEQ ID NOs: 9, 22, 28; and 46, 57, and 62; respectively;(xviii) SEQ ID NOs: 9, 22, 28; and 46, 58, and 62; respectively;(xix) SEQ ID NOs: 9, 22, 28; and 47, 59, and 62; respectively;(xx) SEQ ID NOs: 10, 22, 28; and 46, 58, and 62; respectively;(xxi) SEQ ID NOs: 10, 22, 30; and 46, 58, and 62; respectively;(xxii) SEQ ID NOs: 10, 22, 31; and 46, 58, and 62; respectively;(xxiii) SEQ ID NOs: 10, 22, 32; and 46, 58, and 62; respectively;(xxiv) SEQ ID NOs: 10, 22, 33; and 46, 58, and 62; respectively;(xxv) SEQ ID NOs: 10, 22, 34; and 46, 58, and 62; respectively;(xxvi) SEQ ID NOs: 10, 22, 35; and 46, 58, and 62; respectively;(xxvii) SEQ ID NOs: 10, 22, 36; and 46, 58, and 62; respectively;(xxviii) SEQ ID NOs: 10, 22, 37; and 46, 58, and 62; respectively;(xxix) SEQ ID NOs: 10, 22, 38; and 46, 58, and 62; respectively;(xxx) SEQ ID NOs: 10, 22, 39; and 46, 58, and 62; respectively;(xxxi) SEQ ID NOs: 10, 22, 28; and 49, 58, and 62; respectively;(xxxii) SEQ ID NOs: 10, 22, 28; and 50, 58, and 62; respectively;(xxxiii) SEQ ID NOs: 10, 22, 28; and 51, 58, and 62; respectively;(xxxiv) SEQ ID NOs: 10, 22, 28; and 52, 58, and 62; respectively;(xxxv) SEQ ID NOs: 10, 22, 28; and 53, 58, and 62; respectively;(xxxvi) SEQ ID NOs: 10, 22, 28; and 54, 58, and 62; respectively;(xxxvii) SEQ ID NOs: 10, 22, 30; and 50, 58, and 62; respectively;(xxxviii) SEQ ID NOs: 10, 22, 419; and 50, 58, and 62; respectively; (xxxix) SEQ ID NOs: 10, 22, 28; and 420, 58, and 62; respectively;(xl) SEQ ID NOs: 10, 22, 28; and 421, 58, and 62; respectively;(xli) SEQ ID NOs: 10, 22, 28; and 422, 58, and 62; respectively;(xlii) SEQ ID NOs: 10, 22, 28; and 423, 58, and 62; respectively;(xliii) SEQ ID NOs: 10, 22, 437; and 50, 58, and 62; respectively;(xliv) SEQ ID NOs: 8, 14, 25; and 41, 55, and 61; respectively;(xlv) SEQ ID NOs: 8, 15, 25; and 424, 55, and 61; respectively;(xlvi) SEQ ID NOs: 8, 15, 25; and 425, 55, and 61; respectively; or(xlvii) SEQ ID NOs: 8, 14, 25; and 426, 55, and 61; respectively.

93. The multispecific protein of any one of claims 1-92, wherein the anti-TfR antigenbinding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of:(i) SEQ ID NOs: 64 and 129, respectively;(ii) SEQ ID NOs: 65 and 130, respectively;(iii) SEQ ID NOs: 66 and 131, respectively;(iv) SEQ ID NOs: 67 and 130, respectively;(v) SEQ ID NOs: 68 and 131, respectively;(vi) SEQ ID NOs: 69 and 130, respectively;(vii) SEQ ID NOs: 70 and 131, respectively;(viii) SEQ ID NOs: 71 and 130, respectively;(ix) SEQ ID NOs: 72 and 131, respectively;(x) SEQ ID NOs: 73 and 130, respectively;(xi) SEQ ID NOs: 74 and 131, respectively;(xii) SEQ ID NOs: 75 and 132, respectively;(xiii) SEQ ID NOs: 76 and 131, respectively;(xiv) SEQ ID NOs: 77 and 132, respectively;(xv) SEQ ID NOs: 77 and 133, respectively;(xvi) SEQ ID NOs: 78 and 134, respectively;(xvii) SEQ ID NOs: 77 and 135, respectively;(xviii) SEQ ID NOs: 75 and 136, respectively;(xix) SEQ ID NOs: 77 and 137, respectively;(xx) SEQ ID NOs: 77 and 138, respectively;(xxi) SEQ ID NOs: 79 and 131, respectively;(xxii) SEQ ID NOs: 77 and 139, respectively;(xxiii) SEQ ID NOs: 77 and 131, respectively;(xxiv) SEQIDNOs: 80 and 140, respectively;(xxv) SEQIDNOs: 81 and 141, respectively;(xxvi) SEQIDNOs: 82 and 131, respectively;(xxvii) SEQIDNOs: 83 and 142, respectively;(xxviii) SEQIDNOs: 77 and 143, respectively;(xxix) SEQIDNOs: 75 and 131, respectively;(xxx) SEQIDNOs: 75 and 144, respectively;(xxx i) SEQIDNOs: 77 and 145, respectively;(xxxii) SEQIDNOs: 84 and 131, respectively;(xxxiii) SEQIDNOs: 75 and 146, respectively;(xxxiv) SEQIDNOs: 85 and 131, respectively;(xxxv) SEQIDNOs: 86 and 138, respectively;(xxxvi) SEQIDNOs: 79 and 139, respectively;(xxxvii) SEQIDNOs: 77 and 147, respectively;(xxxviii) SEQ ID NOs: 75 and 148, respectively;(xxxix) SEQIDNOs: 87 and 131, respectively;(xl) SEQIDNOs: 88 and 131, respectively;(xli) SEQIDNOs: 75 and 149, respectively;(xlii) SEQIDNOs: 89 and 150, respectively;(xliii) SEQIDNOs: 90 and 151, respectively;(xliv) SEQ ID NOs: 77 and 152, respectively;(xlv) SEQIDNOs: 79 and 153, respectively;(xlvi) SEQIDNOs: 77 and 139, respectively;(xlvii) SEQIDNOs: 91 and 131, respectively;(xlviii) SEQIDNOs: 92 and 131, respectively;(xlix) SEQIDNOs: 79 and 154, respectively;(1) SEQIDNOs: 93 and 155, respectively;(li) SEQIDNOs: 80 and 131, respectively;(lii) SEQIDNOs: 94 and 131, respectively;(liii) SEQIDNOs: 95 and 131, respectively;(liv) SEQIDNOs: 66 and 156, respectively;(Iv) SEQIDNOs: 97 and 138, respectively;(Ivi) SEQ ID NOs: 95 and 156, respectively;(Ivii) SEQ ID NOs: 98 and 157, respectively;(Iviii) SEQ ID NOs: 99 and 157, respectively;(lix) SEQ ID NOs: 100 and 157, respectively;(lx) SEQ ID NOs: 101 and 157, respectively;(Ixi) SEQ ID NOs: 102 and 158, respectively;(Ixii) SEQ ID NOs: 103 and 157, respectively;(Ixiii) SEQ ID NOs: 104 and 159, respectively;(Ixiv) SEQ ID NOs: 105 and 160, respectively;(Ixv) SEQ ID NOs: 106 and 161, respectively;(Ixvi) SEQ ID NOs: 107 and 162, respectively;(Ixvii) SEQ ID NOs: 106 and 163, respectively;(Ixviii) SEQ ID NOs: 108 and 164, respectively;(Ixix) SEQ ID NOs: 106 and 165, respectively;(Ixx) SEQ ID NOs: 108 and 166, respectively;(Ixxi) SEQ ID NOs: 109 and 165, respectively;(Ixxii) SEQ ID NOs: 110 and 167, respectively;(Ixxiii) SEQ ID NOs: 111 and 168, respectively;(Ixxiv) SEQ ID NOs: 112 and 160, respectively;(Ixxv) SEQ ID NOs: 113 and 169, respectively;(Ixxvi) SEQ ID NOs: 113 and 170, respectively;(Ixxvii) SEQ ID NOs: 113 and 171, respectively;(Ixxviii) SEQ ID NOs: 114 and 169, respectively;(Ixxix) SEQ ID NOs: 114 and 171, respectively;(Ixxx) SEQ ID NOs: 115 and 169, respectively;(Ixxxi) SEQ ID NOs: 115 and 170, respectively;(Ixxxii) SEQ ID NOs: 115 and 171, respectively;(Ixxxiii) SEQ ID NOs: 116 and 169, respectively;(Ixxxiv) SEQ ID NOs: 116 and 170, respectively;(Ixxxv) SEQ ID NOs: 116 and 171, respectively;(Ixxxvi) SEQ ID NOs: 117 and 170, respectively;(Ixxxvii) SEQ ID NOs: 118 and 170, respectively;(Ixxxviii) SEQ ID NOs: 119 and 170, respectively;(Ixxxix) SEQ ID NOs: 120 and 170, respectively;(xc) SEQ ID NOs. 121 and 170, respectively;(xci) SEQ ID NOs: 122 and 170, respectively;(xcii) SEQ ID NOs: 123 and 170, respectively;(xciii) SEQ ID NOs: 124 and 170, respectively;(xciv) SEQ ID NOs: 125 and 170, respectively;(xcv) SEQ ID NOs: 126 and 170, respectively;(xcvi) SEQ ID NOs: 127 and 170, respectively;(xcvii) SEQ ID NOs: 117 and 173, respectively;(xcviii) SEQ ID NOs: 117 and 174, respectively;(xcix) SEQ ID NOs: 117 and 175, respectively;(c) SEQ ID NOs: 117 and 176, respectively;(ci) SEQ ID NOs: 117 and 177, respectively;(cii) SEQ ID NOs: 117 and 178, respectively;(ciii) SEQ ID NOs: 118 and 174, respectively;(civ) SEQ ID NOs: 427 and 174, respectively;(cv) SEQ ID NOs: 117 and 428, respectively;(cvi) SEQ ID NOs: 117 and 429, respectively;(cvii) SEQ ID NOs: 117 and 430, respectively;(cviii) SEQ ID NOs: 117 and 431, respectively;(cix) SEQ ID NOs: 432 and 174, respectively;(ex) SEQ ID NOs: 101 and 154, respectively;(cxi) SEQ ID NOs: 102 and 433, respectively;(cxii) SEQ ID NOs: 102 and 434, respectively; or(cxiii) SEQ ID NOs. 101 and 435, respectively.

94. The multispecific protein of any one of claims 1-90, wherein the BBB target is human CD98hc.

95. The multispecific protein of any one of claims 1-90 or 94, wherein the anti-CD98hc antigen-binding domain comprises heavy chain variable region (VH) complementaritydetermining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of:(i) SEQ ID NOs: 181, 185, 191, 194, 198, and 201; respectively;(ii) SEQ ID NOs: 182, 186, 191, 195, 199, and 202; respectively;(iii) SEQ ID NOs: 183, 187, 192, 196, 200, and 203; respectively;(iv) SEQ ID NOs: 9, 188, 192, 196, 200, and 203; respectively;(v) SEQ ID NOs: 184, 189, 193, 197, 198, and 204; respectively;(vi) SEQ ID NOs: 184, 190, 193, 197, 198, and 204; respectively;(vii) SEQ ID NOs: 184, 232, 193, 197, 198, and 204; respectively;(viii) SEQ ID NOs: 184, 233, 193, 197, 198, and 204; respectively;(ix) SEQ ID NOs: 184, 234, 193, 197, 198, and 204; respectively;(x) SEQ ID NOs: 184, 342, 193, 197, 198, and 204, respectively;(xi) SEQ ID NOs: 184, 343, 193, 197, 198, and 204, respectively;(xii) SEQ ID NOs: 184, 344, 193, 197, 198, and 204, respectively;(xiii) SEQ ID NOs: 184, 345, 193, 197, 198, and 204, respectively;(xiv) SEQ ID NOs: 184, 346, 193, 197, 198, and 204, respectively;(xv) SEQ ID NOs: 184, 347, 193, 197, 198, and 204, respectively;(xvi) SEQ ID NOs: 184, 348, 193, 197, 198, and 204, respectively;(xvii) SEQ ID NOs: 184, 190, 193, 360, 198, and 204, respectively;(xviii) SEQ ID NOs: 184, 190, 193, 361, 198, and 204, respectively;(xix) SEQ ID NOs: 184, 190, 193, 362, 198, and 204, respectively;(xx) SEQ ID NOs: 184, 190, 193, 363, 198, and 204, respectively;(xxi) SEQ ID NOs: 184, 190, 193, 364, 198, and 204, respectively;(xxii) SEQ ID NOs: 184, 190, 193, 365, 198, and 204, respectively;(xxiii) SEQ ID NOs: 184, 190, 193, 366, 198, and 204, respectively;(xxiv) SEQ ID NOs: 184, 344, 193, 362, 198, and 204, respectively;(xxv) SEQ ID NOs: 184, 349, 193, 197, 198, and 204, respectively;(xxvi) SEQ ID NOs: 184, 190, 351, 197, 198, and 204, respectively;(xxvii) SEQ ID NOs: 184, 190, 352, 197, 198, and 204, respectively;(xxviii) SEQ ID NOs: 184, 190, 353, 197, 198, and 204, respectively;(xxix) SEQ ID NOs: 184, 190, 354, 197, 198, and 204, respectively;(xxx) SEQ ID NOs: 184, 190, 355, 197, 198, and 204, respectively;(xxxi) SEQ ID NOs: 184, 190, 356, 197, 198, and 204, respectively;(xxxii) SEQ ID NOs: 184, 190, 357, 197, 198, and 204, respectively;(xxxiii) SEQ ID NOs: 184, 190, 358, 197, 198, and 204, respectively;(xxxiv) SEQ ID NOs: 184, 190, 359, 197, 198, and 204, respectively;(xxxv) SEQ ID NOs: 184, 190, 193, 367, 198, and 204, respectively;(xxxvi) SEQ ID NOs: 184, 190, 193, 438, 198, and 204, respectively;(xxxvii) SEQ ID NOs: 184, 190, 193, 439, 198, and 204, respectively;(xxxviii) SEQ ID NOs: 184, 190, 193, 440, 198, and 204, respectively;(xxxix) SEQ ID NOs: 184, 190, 193, 441, 198, and 204, respectively;(xl) SEQ ID NOs: 184, 190, 193, 442, 198, and 204, respectively;(xli) SEQ ID NOs: 184, 190, 193, 197, 198, and 368, respectively;(xlii) SEQ ID NOs: 184, 190, 193, 197, 198, and 369, respectively;(xliii) SEQ ID NOs: 184, 190, 193, 197, 198, and 370, respectively;(xliv) SEQ ID NOs: 184, 190, 193, 197, 198, and 371, respectively;(xlv) SEQ ID NOs: 184, 190, 193, 197, 198, and 372, respectively;(xlvi) SEQ ID NOs: 184, 190, 193, 197, 198, and 373, respectively;(xlvii) SEQ ID NOs: 184, 190, 193, 197, 198, and 374, respectively;(xlviii) SEQ ID NOs: 184, 190, 193, 197, 198, and 375, respectively;(xlix) SEQ ID NOs: 184, 190, 193, 197, 198, and 376, respectively;(1) SEQ ID NOs: 184, 190, 193, 197, 198, and 377, respectively;(li) SEQ ID NOs: 350, 190, 193, 197, 198, and 204, respectively; or(lii) SEQ ID NOs: 350, 190, 193, 197, 198, and 377, respectively.

96. The multispecific protein of any one of claims 1-90, 94, or 95, wherein the anti-CD98hc antigen-binding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of:(i) SEQ ID NOs: 205 and 211, respectively;(ii) SEQ ID NOs: 206 and 212, respectively;(iii) SEQ ID NOs: 207 and 213, respectively;(iv) SEQ ID NOs: 208 and 214, respectively;(v) SEQ ID NOs: 209 and 215, respectively;(vi) SEQ ID NOs: 210 and 216, respectively;(vii) SEQIDNOs: 235 and 216, respectively;(viii) SEQIDNOs: 236 and 216, respectively;(ix) SEQIDNOs: 237 and 216, respectively;(x) SEQIDNOs: 378 and 216, respectively;(xi) SEQIDNOs: 379 and 216, respectively;(xii) SEQIDNOs: 380 and 216, respectively;(xiii) SEQIDNOs: 381 and 216, respectively;(xiv) SEQIDNOs: 382 and 216, respectively;(xv) SEQIDNOs: 383 and 216, respectively;(xvi) SEQIDNOs: 384 and 216, respectively;(xvii) SEQIDNOs: 210 and 385, respectively;(xviii) SEQIDNOs: 210 and 386, respectively;(xix) SEQIDNOs: 210 and 387, respectively;(xx) SEQIDNOs: 210 and 388, respectively;(xxi) SEQIDNOs: 210 and 389, respectively;(xxii) SEQIDNOs: 210 and 390, respectively;(xxiii) SEQIDNOs: 210 and 391, respectively;(xxiv) SEQIDNOs: 210 and 391, respectively;(xxv) SEQIDNOs: 380 and 387, respectively;(xxvi) SEQIDNOs: 392 and 216, respectively;(xxvii) SEQIDNOs: 393 and 216, respectively;(xxviii) SEQIDNOs: 394 and 216, respectively;(xxix) SEQIDNOs: 395 and 216, respectively;(xxx) SEQ ID NOs: 396 and 216, respectively;(xxxi) SEQIDNOs: 397 and 216, respectively; (xxxii) SEQIDNOs: 398 and 216, respectively; (xxxiii) SEQIDNOs: 399 and 216, respectively; (xxxiv) SEQIDNOs: 400 and 216, respectively; (xxxv) SEQIDNOs: 401 and 216, respectively; (xxxvi) SEQIDNOs: 210 and 402, respectively; (xxxvii) SEQIDNOs: 210 and 403, respectively; (xxxviii) SEQIDNOs: 210 and 404, respectively;(xxxix) SEQ ID NOs: 210 and 405, respectively;(xl) SEQ ID NOs: 210 and 406, respectively;(xli) SEQ ID NOs: 210 and 407, respectively;(xlii) SEQ ID NOs: 210 and 408, respectively;(xliii) SEQ ID NOs: 210 and 409, respectively;(xliv) SEQ ID NOs: 210 and 410, respectively;(xlv) SEQ ID NOs: 210 and 411, respectively;(xlvi) SEQ ID NOs: 210 and 412, respectively;(xlvii) SEQ ID NOs: 210 and 413, respectively;(xlviii) SEQ ID NOs: 210 and 414, respectively;(xlix) SEQ ID NOs: 210 and 415, respectively;(1) SEQ ID NOs: 210 and 416, respectively;(li) SEQ ID NOs: 210 and 417, respectively;(lii) SEQ ID NOs: 418 and 215, respectively; or(liii) SEQ ID NOs: 418 and 417, respectively.

97. The multispecific protein of any one of claims 1-90, wherein the multispecific protein comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of SEQ ID NOs: 263-287, 291- 307, 436, or combinations thereof.

98. The multispecific protein of any one of claims 1-90, wherein the multispecific protein comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of:(i) SEQ ID NOs: 263, 275, and 283;(ii) SEQ ID NOs: 264, 276, and 283;(iii) SEQ ID NOs: 264, 275, and 283;(iv) SEQ ID NOs: 265, 275, and 283;(v) SEQ ID NOs: 266, 277, and 284;(vi) SEQ ID NOs: 267, 278, and 284;(vii) SEQ ID NOs: 267, 277, and 284;(viii) SEQ ID NOs: 268, 277, and 284;(ix) SEQ ID NOs: 269, 279, and 284;(x) SEQ ID NOs: 270, 280, and 284;(xi) SEQ ID NOs: 270, 279, and 284;(xii) SEQ ID NOs: 271, 279, and 284;(xiii) SEQ ID NOs: 272, 281, and 285;(xiv) SEQ ID NOs: 273, 282, and 285;(xv) SEQ ID NOs: 273, 281, and 285;(xvi) SEQ ID NOs: 274, 281, and 285;(xvii) SEQ ID NOs: 272, 281, and 286;(xviii) SEQ ID NOs: 273, 282, and 286;(xix) SEQ ID NOs: 273, 281, and 286;(xx) SEQ ID NOs: 274, 281, and 286;(xxi) SEQ ID NOs: 272, 281, and 287;(xxii) SEQ ID NOs: 273, 282, and 287;(xxiii) SEQ ID NOs: 273, 281, and 287;(xxiv) SEQ ID NOs: 274, 281, and 287; or(xxv) SEQ ID NOs: 436, 279, and 284.

99. The multispecific protein of claim 24-42 and 55-98, wherein the Fc region is fully active.

100. The multispecific protein of claim 24-42 and 55-98, wherein the Fc region is partially active.

101. The multispecific protein of claim 24-42 and 55-98, wherein the Fc region is not silent.

102. A composition comprising the multispecific protein of any one of the previous claims.

103. A host cell comprising the multispecific protein of any one of claims 1-101.

104. A pharmaceutical composition comprising i) the multispecific protein of any one of claims 1-101, and iii) a pharmaceutically acceptable carrier, excipient or stabilizer.

105. A nucleotide sequence encoding the multispecific protein of any one of claims 1-101.

106. A method of administering or transporting compositions comprising administering to the subject any of the multispecific protein of any one of claims 1-101, the composition ofclaim 102, the pharmaceutical composition of claim 104, or the nucleotide sequence of claim 105.

107. A method of treating a lysosomal storage disease or disorder in a subject comprising administering to the subject any of the multispecific protein of any one of claims 1-101, the composition of claim 102, the pharmaceutical composition of claim 104, or the nucleotide sequence of claim 105.

108. The method of claim 107, wherein the lysosomal storage disease or disorder is Gaucher’s disease or Neimann-Pick type C disease.

109. A method of treating a CNS disease or disorder in a subject comprising administering to the subject any of the multispecific protein of any one of claims 1-101, the composition of claim 102, the pharmaceutical composition of claim 104, or the nucleotide sequence of claim 105.

110. The method of claim 109, wherein the CNS disease or disorder is selected from Parkinson’s disease, Alzheimer’s disease, and ALS.

111. The multispecific protein of any one of claims 1-101, the composition of claim 102, the pharmaceutical composition of claim 104, or the nucleotide sequence of claim 105 for use in the method of any one of claims 106-110.

112. Use of the multispecific proteins of claims 1-101, the composition of claim 102, the pharmaceutical composition of claim 104, or the nucleotide sequence of claim 105 in the method of any one of claims 106-110.

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